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Olive anthracnose (OA) caused by Colletotrichum spp. and olive leaf spot (OLS) caused by Ventuaria oleaginea are two of the most important diseases affecting olive trees. OA mainly affects mature olive drupes while OLS is typically known as a defoliating pathogen. Field treatments with a pomegranate peel extract (PGE) at 3, 6 or 12 g/l seven days before the expected full bloom prevented the colonization of flowers by Colletotrichum spp. and reduced latent infections in small olive drupes over the summer period by 53-100%. The same treatments significantly reduced or completely prevented the latent infections of V. oleaginea in olive leaves. Interestingly, PGE at the higher concentration (12 g/l) proved always more effective that Flint Max, a chemical formulate recently registered in Europe on olive. On plants sprayed with PGE, the lower quantity of over-summering inoculum of Colletotrichum spp. effectively delayed the disease outbreak in autumn of approximately ten days. In particular, PGE at 12 g/l reduced the incidence of anthracnose by 84.5% as compared to the untreated control. Treatments also reduced the premature defoliation of plants. Similar results to those described for a single PGE treatment made before the full bloom where achieved with two applications made before and after flowering. Overall, the present study highlighted the importance of controlling the establishment of latent infections of both Colletotrichum spp. and V.oleaginea to effectively protect olive trees. These results together with previous reports suggest the possible use of PGE as a key natural antifungal preparation to control the olive diseases affecting aerial plant organs.

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.

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. European Institute of Innovation and Technology; Italian Ministry for Education, University and ResearchMinistry of Education, Universities and Research (MIUR); Fondazione Cassa di Risparmio di Cuneo Published version The Authors wish to thank the European Institute of Innovation and Technology for funding the project "CLEANFRUIT-Standardization of innovative pest control strategies to produce zero residue fruit for baby food and other fruit produce" (EITFood initiative), the Italian Ministry for Education, University and Research for funding the project "A gnotobiotic-based approach to unravel the role of the plant microbiome and develop synthetic communities increasing plant growth and stress tolerance-NATURE" (PRIN2017 programme) and Fondazione Cassa di Risparmio di Cuneo for funding the project, "SMART APPLE-Innovative and SMART technologies for sustainable APPLE production".

Black spot is a major foliar disease of sweet basil (Ocimum basilicum) present in a typical cultivation area of northern Italy, including the Liguria and southern Piedmont regions, where this aromatic herb is an economically important crop. In this study, 15 Colletotrichum isolates obtained from sweet basil plants with symptoms of black spot sampled in this area were characterized morphologically and by nuclear DNA analysis using internal transcribed spacers (ITS) and intervening 5.8S nrDNA as well as part of the &beta, tubulin gene (TUB2) regions as barcode markers. Analysis revealed all but one isolate belonged to the recently described species C. ocimi of the C. destructivum species complex. Only one isolate was identified as C. destructivum sensu stricto (s.s.). In pathogenicity tests on sweet basil, both C. ocimi and C. destructivum s.s. isolates incited typical symptoms of black spot, showing that although C. ocimi prevails in this basil production area, it is not the sole causal agent of black spot in northern Italy. While no other hosts of C. ocimi are known worldwide, the close related species C. destructivum has a broad host range, suggesting a speciation process of C. ocimi within this species complex driven by adaptation to the host.

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 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.

Microorganisms are the main drivers shaping the functioning and equilibrium of all ecosystems, contributing to nutrient cycling, primary production, litter decomposition, and multi-trophic interactions. Knowledge about the microbial assemblies in specific ecological niches is integral to understanding the assemblages interact and function the function, and becomes essential when the microbiota intersects with human activities, such as protecting crops against pests and diseases. Metabarcoding has proven to be a valuable tool and has been widely used for characterizing the microbial diversity of different environments and has been utilized in many research endeavors. Here we summarize the current status of metabarcoding technologies, the advantages and challenges in utilizing this technique, and how this pioneer approach is being applied to studying plant diseases and pests, with a focus on plant protection and biological control. Current and future developments in this technology will foster a more comprehensive understanding of microbial ecology, and the development of new, innovative pest control strategies.

A total of 161 Phytophthora infestans isolates, collected from infected potato and tomato plants during 2008–2014, were characterized based on mating type, metalaxyl sensitivity and polymorphism at 12 simple sequence repeat (SSR) loci, in order to investigate the population of P. infestans in the north-west of Algeria, an emerging potato production region. The majority of isolates were of A2 mating type (112 isolates). A high percentage (89 %) of resistance to metalaxyl among isolates was detected. The metalaxyl resistant phenotype was present in both mating types with a higher percentage in A2 mating type isolates. SSR-based genotypic analysis of P. infestans population showed a low diversity. Genotype 13_A2 was the predominant in the population with a frequency of 67 % followed by 2_A1 (21 %) and 23_A1 (5 %). Genotype 23_A1 was detected only in tomato and potato isolates collected in 2013 and 2014.

Plant-associated microorganisms play a crucial role in plant health and productivity. Belowground microbial diversity is widely reported as a major factor in determining the composition of the plant microbiome. In contrast, much less is known about the role of the atmosphere in relation to the plant microbiome. The current study examined the hypothesis that the atmospheric microbiome influences the composition of fungal communities of the aboveground organs (flowers, fruit, and leaves) of table grape and vice versa. The atmosphere surrounding grape plantings exhibited a significantly higher level of fungal diversity relative to the nearby plant organs and shared a higher number of phylotypes (5,536 OTUs, 40.3%) with the plant than between organs of the same plant. Using a Bayesian source tracking approach, plant organs were determined to be the major source of the atmospheric fungal community (92%). In contrast, airborne microbiota had only a minor contribution to the grape microbiome, representing the source of 15, 4, and 35% of the fungal communities of leaves, flowers, and fruits, respectively. Moreover, data indicate that plant organs and the surrounding atmosphere shared a fraction of each other’s fungal communities, and this shared pool of fungal taxa serves as a two-way reservoir of microorganisms. Microbial association analysis highlighted more positive than negative interactions between fungal phylotypes. Positive interactions were more common within the same environment, while negative interactions appeared to occur more frequently between different environments, i.e., atmosphere, leaf, flower, and fruit. The current study revealed the interplay between the fungal communities of the grape phyllosphere with the surrounding air. Plants were identified as a major source of recruitment for the atmospheric microbiome, while the surrounding atmosphere contributed only a small fraction of the plant fungal community. The results of the study suggested that the plant–air interface modulates the plant recruitment of atmospheric fungi, taking a step forward in understanding the plant holobiont assembly and how the atmosphere surrounding plants plays a role in this process. The impact of plants on the atmospheric microbiota has several biological and epidemiological implications for plants and humans.

The fungal community composition and structure of two ancient tetraploid wheat varieties, native to the Sicilian territory of Italy, Perciasacchi (winter wheat) and Tumminia (spring wheat) were investigated using High Throughput Sequencing (HTS). This showed a predominance of Ascomycetes and Basidiomycetes including Alternaria, Fusarium, Mycosphaerella, Filobasidium, Cystofilobasidium, Cryptococcus, Leucosporidium, Dioszegia, Puccinia, Sporobolomyces, Aureobasidium, Cladosporium, Holtermanniella and Gibberella. Principal Coordinates Analysis (PCoA) and Linear discriminant analysis Effect Size (LEfSe) showed that Aureobasidium, Leucosporidium and Puccinia differentiated between the two wheat varieties. In addition, the microbial association analysis suggested that some endophytic taxa play important roles within the wheat fungal community. Genera such as Cryptococcus and Cystofilobasidium were shown to have consistent antagonistic activity against Gibberella spp., while, Acremonium and a group of unidentified ascomycetes had mutual exclusion relationships with Puccinia. Since both Gibberella and Puccinia contain several economically important pathogens of wheat, the detected fungal interactions may indicate microbial-mediated resistance in these wheat varieties.

The aim of this study was to investigate the occurrence, diversity, and distribution of Phytophthora species in Protected Natural Areas (PNAs), including forest stands, rivers, and riparian ecosystems, in Sicily (Italy), and assessing correlations with natural vegetation and host plants. Fifteen forest stands and 14 rivers in 10 Sicilian PNAs were studied. Phytophthora isolations from soil and stream water were performed using leaf baitings. Isolates were identified using both morphological characters and sequence analysis of the internal transcribed spacer (ITS) region. A rich community of 20 Phytophthora species from eight phylogenetic clades, including three new Phytophthora taxa, was recovered (17 species in rhizosphere soil from forest stands and 12 species in rivers). New knowledge about the distribution, host associations, and ecology of several Phytophthora species was provided.

A pomegranate peel extract (PGE) was evaluated as a natural antifungal preparation for the control of postharvest rots. In vitro trials revealed a strong fungicidal activity against germination of conidia of Botrytis cinerea , Penicillium digitatum and Penicillium expansum . Almost complete inhibition of all fungal spore germination was achieved after 20 h of incubation with PGE. PGE was very effective in inhibiting decay following artificial inoculations of lemons by P. digitatum and Penicillium italicum , grapefruits by P. italicum and apples by P. expansum . At concentrations of 1.2 and 12 g/l complete inhibition of infection was achieved in the majority of host pathogen combinations. Furthermore, it was also effective in reducing natural rots under semi-commercial conditions on both sweet cherries and lemons: on cherries Monilinia laxa and B. cinerea rots were reduced by 61.0% ( cv . Bigarreau Moreau) and 95.6% ( cv . Giorgia), respectively, and on lemons 87.8% reduction of total rot was achieved. PGE treatment showed residual effect as it was effective in inhibiting infections made at 6, 12, and 24 h after the application of the extract in fruit wounds. Additionally, PGE exhibited curative activity and reduced the incidence of rots when it was applied 6 and 24 h after inoculation. Considering that PGE was extracted and stabilized using safe chemicals (food grade ethanol and citric acid) and that it did not have any apparent phytotoxic effect on treated fruit, PGE proved to be effectively eco-friendly and safe control mean for postharvest rots of fruit.

The olive fruit fly (OFF), Bactrocera oleae is the most devastating pest affecting olive fruit worldwide. Previous investigations have addressed the fungal microbiome associated with olive drupes or B. oleae, but the impact of the insect on fungal communities of olive fruit remains undescribed. In the present work, the fungal microbiome of olive drupes, infested and non-infested by the OFF, was investigated in four different localities and cultivars. Olive fruit fly infestations caused a general reduction of the fungal diversity, a higher quantity of the total DNA and an increase in taxa that remained unidentified or had unknown roles. The infestations led to imbalanced fungal communities with the growth of taxa that are usually outcompeted. While it was difficult to establish a cause-effect link between fly infestation and specific fungi, it is clear that the fly alters the natural microbial balance, especially the low abundant taxa. On the other hand, the most abundant ones, were not significantly influenced by the insect. In fact, despite the slight variation between the sampling locations, Aureobasidium, Cladosporium, and Alternaria, were the dominant genera, suggesting the existence of a typical olive fungal microbiome.

A homothallic Phytophthora species was found to be consistently associated with a rot of mature fruits of two local cultivars of olive (Olea europaea) in Calabria, southern Italy. The phylogenetic analysis of sequences of the ITS1‐5.8S‐ITS2 region and cox1 gene enabled its identification as a new species of clade 2, with a basal position compared to previously described subclades. The new species is described formally with the epithet Phytophthora oleae, referring to the natural matrix from which it was isolated. A unique combination of molecular and morphological characters clearly separates P. oleae from other already described Phytophthora species. This new species produced semipapillate, occasionally bipapillate, persistent sporangia on simple sympodially branching sporangiophores as well as globose and smooth‐walled oogonia, paragynous antheridia and spherical, plerotic oospores. The pathogenicity of P. oleae was confirmed in inoculation trials on fruits of three olive cultivars, including the two local cultivars from which the pathogen had been isolated.

A large number of SSR loci were screened in the genomic assemblies of 14 different isolates of Phytophthora nicotianae and primers were developed for amplification of 17 markers distributed among different contigs. These loci were highly polymorphic and amplified from genetically distant isolates of the pathogen. Among these, nine were further validated using a multiplexed genotyping assay with differentially labeled primers (FAM or HEX) to allow for duplex PCR amplification. The use of reverse primers with a 5′ PIG tail was important to increase the quality and reliability of the analyses. A total of 46 alleles were detected in 5 tester isolates of P. nicotianae representing the breadth of diversity in the species. Furthermore, a high incidence of heterozygosity was determined with two alleles detected in 67% of the primer/isolate combinations. Three different alleles where detected for a single locus/isolate combination, indicating variation in ploidy. These markers represent a valuable new tool for the characterization of populations of P. nicotianae .

During various surveys of Phytophthora diversity in Europe, Chile and Vietnam slow growing oomycete isolates were obtained from rhizosphere soil samples and small streams in natural and planted forest stands. Phylogenetic analyses of sequences from the nuclear ITS, LSU, β-tubulin and HSP90 loci and the mitochondrial cox1 and NADH1 genes revealed they belong to six new species of a new genus, officially described here as Nothophytophthora gen. nov., which clustered as sister group to Phytophthora. Nothophytophthora species share numerous morphological characters with Phytophthora: persistent (all Nothophytophthora spp.) and caducous (N. caduca, N. chlamydospora, N. valdiviana, N. vietnamensis) sporangia with variable shapes, internal differentiation of zoospores and internal, nested and extended (N. caduca, N. chlamydospora) and external (all Nothophytophthora spp.) sporangial proliferation; smooth-walled oogonia with amphigynous (N. amphigynosa) and paragynous (N. amphigynosa, N. intricata, N. vietnamensis) attachment of the antheridia; chlamydospores (N. chlamydospora) and hyphal swellings. Main differing features of the new genus are the presence of a conspicuous, opaque plug inside the sporangiophore close to the base of most mature sporangia in all known Nothophytophthora species and intraspecific co-occurrence of caducity and non-papillate sporangia with internal nested and extended proliferation in several Nothophytophthora species. Comparisons of morphological structures of both genera allow hypotheses about the morphology and ecology of their common ancestor which are discussed. Production of caducous sporangia by N. caduca, N. chlamydospora and N. valdiviana from Valdivian rainforests and N. vietnamensis from a mountain forest in Vietnam suggests a partially aerial lifestyle as adaptation to these humid habitats. Presence of tree dieback in all forests from which Nothophytophthora spp. were recovered and partial sporangial caducity of several Nothophytophthora species indicate a pathogenic rather than a saprophytic lifestyle. Isolation tests from symptomatic plant tissues in these forests and pathogenicity tests are urgently required to clarify the lifestyle of the six Nothophytophthora species. info:eu-repo/semantics/publishedVersion

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.

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.

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.

Alternaria brown spot is one of the most important diseases of tangerines and their hybrids worldwide. Recently, outbreaks in Mediterranean areas related to susceptible cultivars, refocused attention on the disease. Twenty representatives were selected from a collection of 180 isolates of Alternaria spp. from citrus leaves and fruit. They were characterized along with reference strains of Alternaria spp. Micro- and macroscopic characteristics separated most Alternaria isolates into six morphotypes referable to A. alternata (5) and A. arborescens (1). Phylogenetic analyses, based on endopolygalacturonase (endopg) and internal transcribed spacer (ITS), confirmed this finding. Moreover, a five-gene phylogeny including two anonymous genomics regions (OPA 1-3 and OPA 2-1), and the beta-tubulin gene (ß-tub), produced a further clustering of A. alternata into three clades. This analysis suggested the existence of intra-species molecular variability. Investigated isolates showed different levels of virulence on leaves and fruit. In particular, the pathogenicity on fruit seemed to be correlated with the tissue of isolation and the clade. The toxigenic behavior of Alternaria isolates was also investigated, with tenuazonic acid (TeA) being the most abundant mycotoxin (0.2-20 mg/L). Isolates also synthesized the mycotoxins alternariol (AOH), its derivate alternariol monomethyl ether (AME), and altenuene (ALT), although to a lesser extent. AME production significantly varied among the six morphotypes. The expression of pksJ/pksH, biosynthetic genes of AOH/AME, was not correlated with actual toxin production, but it was significantly different between the two genotypes and among the four clades. Finally, ten isolates proved to express the biosynthetic genes of ACTT1 phytotoxin, and thus to be included in the Alternaria pathotype tangerine. A significant correlation between pathogenicity on leaves and ACTT1 gene expression was recorded. The latter was significantly dependent on geographical origin. The widespread occurrence of Alternaria spp. on citrus fruit and their ability to produce mycotoxins might represent a serious concern for producers and consumers.

An amplicon metagenomic approach based on the ITS2 region of fungal rDNA was used to identify the composition of fungal communities associated with different strawberry organs (leaves, flowers, immature and mature fruits), grown on a farm using management practices that entailed the routine use of various chemical pesticides. ITS2 sequences clustered into 316 OTUs and Ascomycota was the dominant phyla (95.6%) followed by Basidiomycota (3.9%). Strawberry plants supported a high diversity of microbial organisms, but two genera, Botrytis and Cladosporium, were the most abundant, representing 70–99% of the relative abundance (RA) of all detected sequences. According to alpha and beta diversity analyses, strawberry organs displayed significantly different fungal communities with leaves having the most diverse fungal community, followed by flowers, and fruit. The interruption of chemical treatments for one month resulted in a significant modification in the structure of the fungal community of leaves and flowers while immature and mature fruit were not significantly affected. Several plant pathogens of other plant species, that would not be intuitively expected to be present on strawberry plants such as Erysiphe, were detected, while some common strawberry pathogens, such as Rhizoctonia, were less evident or absent.

Penicillium polonicum, Alternaria alternata, and Macrophomina phaseolina were identified as causal agents of three different postharvest diseases of cactus pear fruit in southern Italy. P. polonicum was responsible for blue mould and was identified by sequencing part of the ?-tubulin gene DNA region. A. alternata and M. phaseolina were the causal agents of a dry rot on fruit stored at 5°C and a basal soft rot on fruit stored at 20°C, respectively. The last two fungal species were identified by sequencing the GAPDH and TEF1-alpha gene regions.

Detection of Botrytis cinerea latent infections on grapes before storage is essential for effective control strategies. In the present study, a molecular detection method was developed to detect and quantify B. cinerea in grape tissues. Preliminary investigations, conducted on local varieties by fruit freezing, identified the ‘Red Globe’ variety as the less contaminated one and confirmed the preferential localization of latent infections in the berry-pedicel attachment zone (berry calottes) and pathogen presence on stamens. A quantitative real-time PCR (qPCR) detection method, based on a probe designed on B. cinerea intergenic spacer (IGS) regions and a reported probe for Vitis vinifera as internal control, was utilized to reveal the presence of symptomless infections on bunches. The system proved to be highly specific and sensitive, enabling quantification of as little as 10 fg of B. cinerea DNA and detection of single conidia in artificially inoculated grape berries; moreover, it allowed reliable detection of the pathogen in naturally infected asymptomatic tissues. In particular, the qPCR assay revealed the presence of B. cinerea in 80 and 65% of apparently healthy calottes and stamens, respectively, with an efficiency higher than that obtained from freezing and plating techniques. Furthermore, significant correlations (R2 = 0.89 and 0.94) were found between qPCR results and the actual disease incidence on bunches from which calottes and stamens were sampled.

The fungal diversity associated with leaves, flowers and fruits of olive (Olea europaea) was investigated in different phenological stages (May, June, October and December) using an implemented metabarcoding approach. It consisted of the 454 pyrosequencing of the fungal ITS2 region and the subsequent phylogenetic analysis of relevant genera along with validated reference sequences. Most sequences were identified up to the species level or were associated with a restricted number of related taxa enabling supported speculations regarding their biological role. Analyses revealed a rich fungal community with 195 different OTUs. Ascomycota was the dominating phyla representing 93.6% of the total number of detected sequences followed by unidentified fungi (3.6%) and Basidiomycota (2.8%). A higher level of diversity was revealed for leaves compared to flowers and fruits. Among plant pathogens the genus Colletotrichum represented by three species (C. godetiae syn. C. clavatum, C. acutatum s.s and C. karstii) was the most abundant on ripe fruits but it was also detected in other organs. Pseudocercospora cladosporioides was detected with a high frequency in all leaf samples and to a less extent in ripe fruits. A much lower relative frequency was revealed for Spilocaea oleagina and for other putative pathogens including Fusarium spp., Neofusicoccum spp., and Alternaria spp. Among non-pathogen taxa, Aureobasidium pullulans, the species complex of Cladosporium cladosporioides and Devriesia spp. were the most represented. This study highlights the existence of a complex fungal consortium including both phytopathogenic and potentially antagonistic microorganisms that can have a significant impact on olive productions.

The activity of 19 inorganic and organic salts to control table grape storage rots was preliminarily assessed by in vitro and in vivo tests. Several salts reduced the growth of Botrytis cinerea Pers. on amended (0.1–2%, w/v) glucose–agar; however, only calcium chloride (CC), potassium carbonate (PC), sodium bicarbonate (SB) and sodium carbonate (SC) significantly reduced the incidence of grey mould on small table grape bunches (cv. Italia). PC, SB and SC showed a similar effect in the in vitro (inhibition of mycelial growth and conidia germination of B. cinerea) and in vivo tests (reduction of incidence of grey mould on table grape), whereas CC was effective only in vivo. Efficacy of salts applied before harvest was extensively evaluated in small-scale tests (CC, PC, SB, and SC), and in large-scale tests (CC, SB, SC). In both, small- and large-scale tests, field applications of salts resulted in a significant reduction of botrytis storage rots. Field rots (mainly sour rot) were significantly reduced when vines were sprayed at least 21 days before harvest. In large-scale tests, simulating the practical commercial conditions in Southern Italy, two salt applications (30 and 90 days before harvest) of CC, SC, or SB significantly reduced postharvest grey mould from 63.8% among untreated controls to 22.5, 31.2, and 29.5%, respectively. In the same conditions, field rots were 23.4% (untreated control), 9.5% (CC), 11.9% (SC) and 12.0% (SB). On the whole, salts showed an activity higher or similar to that of conventional chemical treatments. Finally, results from specific tests suggested that pH and inhibition of polygalacturonase activity of B. cinerea seem to play a role in the mode of action of SB, SC, PC and CC.

Postharvest applications of calcium chloride (CC) and sodium bicarbonate (SB) were the most effective of 17 salts to control rots caused by Botrytis cinerea on wounded sweet cherries. The combination of CC and SB with a known antagonist (Aureobasidium pullulans, strain L47) reduced Botrytis rot by 98 and 94%, respectively. Tests with pre- and postharvest treatments were conducted in 2000 and 2001 using CC, SB, and L47, alone or in combination. In both years, postharvest treatments significantly reduced rot incidence compared to the controls. The combinations of L47 + CC and L47 + SB reduced total rots ranging from 62 to 75% and were the most effective. The application of Limpel's formula indicated a significant synergistic improvement in effectiveness when postharvest treatments with the biocontrol agent and either salt were combined. Compared to the untreated control, preharvest applications of the antagonist and salts alone resulted in a significant reduction of rots ranging from 24 to 58%; however, their combined application did not improve the level of control. CC and SB did not inhibit A. pullulans in vitro and had no influence on natural epiphytic populations of yeasts, yeast-like fungi, and filamentous fungi on fruit surfaces. In postharvest application, the population of the antagonist was not reduced by the presence of salts, whereas on fruit treated before harvest, its population was lower than that observed on fruit treated with L47 alone.

Among 17 salts, calcium chloride (CC) and sodium bicarbonate (SB) were selected as the most effective against Botrytis cinerea in trials on wounded sweet cherries. The combination of CC and SB with Aureobasidium pullulans, strain L47, reduced Botrytis rot by 98 and 94%, respectively. Tests with postharvest treatments were conducted in 2000 and 2001 using CC, SB, and L47, alone or in combination. In both years, postharvest treatments gave significant reduction of rot incidence compared to the control. The combinations L47+CC and L47+SB were the most effective with a reduction of total rots ranging from 62 to 75%. CC and SB did not show any in vitro toxic effect on A. pullulans and did not modify the natural epiphytic population of yeasts, yeast-like fungi, and filamentous fungi on the fruit surface. In postharvest application, the population of the antagonist was not influenced by the presence of salts.

The polymerase chain reaction (PCR) was used for the specific detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils. Primers were based on the nucleotide sequences of the internal transcribed space regions (ITS1 and ITS2) of 16 different species of Phytophthora. Two primer pairs, Pn5B–Pn6 and Pc2B–Pc7, were designed specifically to amplify DNA from P. nicotianae and P. citrophthora, respectively. Another primer pair (Ph2–ITS4) was designed to amplify DNA from many Phytophthora species. All primer pairs were assessed for specificity and absence of cross-reactivity, using DNA from 118 isolates of Phytophthora and 82 of other common soil fungi. In conventional PCR, with a 10-fold dilution series of template DNA, the limit of detection was of 1pgμl−1 DNA for all the primer pairs (Ph2–ITS4, Pn5B–Pn6, and Pc2B–Pc7). In nested PCR, with primers Ph2–ITS4 in the first round, the detection limit was of 1fgμl−1 for both the primer sets (Pn5B–Pn6 and Pc2B–Pc7). Simple, inexpensive and rapid procedures for direct extraction of DNA from soil and roots were developed. The method yielded DNA of a purity and quality suitable for PCR within 2–3h. DNA extracted from soil and roots was amplified by nested PCR utilizing primers Ph2–ITS4 in the first round. In the second round the primer pairs Pn5B–Pn6 and Pc2B–Pc7 were utilized to detect P. nicotianae and P. citrophthora, respectively. Comparison between the molecular method and pathogen isolation by means of a selective medium did not show any significant differences in sensitivity.

A metagenomic approach based on the use of genus specific primers was developed and utilized to characterize Colletotrichum species associated with the olive phyllosphere and carposphere. Selected markers enabled the specific amplification of almost the entire ITS1-5.8S-ITS2 region of the rDNA and its use as barcode gene. The analysis of different olive samples (green and senescent leaves, floral residues, symptomatic and asymptomatic fruits, and litter leaves and mummies) in three different phenological phases (June, October and December) enabled the detection of 12 genotypes associated with 4 phylotypes identified as C. godetiae, C. acutatum s.s., C. gloeosporioides s.s. and C. kahawae. Another three genotypes were not identified at the level of species but were associated with the species complexes of C. acutatum, C. gloeosporioides and C. boninense sensu lato. Colletotrichum godetiae and C. acutatum s.s. were by far the most abundant while C. gloeosporioides s.s. was detected in a limited number of samples whereas ther phylotypes were rarely found. The high incidence of C. acutatum s.s. represents a novelty for Italy and more generally for the Mediterranean basin since it had been previously reported only in Portugal. As regards to the phenological phase, Colletotrichum species were found in a few samples in June and were diffused on all assessed samples in December. According to data new infections on olive tissues mainly occur in the late fall. Furthermore, Colletotrichum species seem to have a saprophytic behavior on floral olive residues. The method developed in the present study proved to be valuable and its future application may contribute to the study of cycle and aetiology of diseases caused by Colletotrichum species in many different pathosystems.

Nineteen yeast isolates obtained from the surface of several fruits and vegetables grown in Southern Italy and Israel were compared by molecular analysis using arbitrarily primed polymerase chain reaction (AP-PCR) and random amplified polymorphic DNA technique (RAPD-PCR). Genetic analysis made it possible to distinguish between closely-related genitically different strains which had the same morphological characteristic, and to discard isolates which were genetically identical. Following PCR characterisation, 6 isolates were selected and tested for their biocontrol activity against major postharvest pathogens (Penicillium digitatum on grapefruit, Botrytis cinerea, Rhizopus stolonifer and Aspergillus niger on table grape and B. cinerea and R. stolonifer on cherry tomato). All the isolates showed a good biocontrol efficacy on both wounded and non-wounded fruits. Furthermore, the preharvest application of the most effective antagonist (LS15) on table grape resulted in a significant reduction in grey mold ranging from 28.3% to 38.2% compared to the untreated control. The RAPD-PCR technique was also useful for identifying and monitoring the survival of the antagonist after field application.

Aureobasidium pullulans, a cosmopolitan yeast-like fungus, colonises leaf surfaces and is a potential biocontrol agent for plant pathogens. Forty-one isolates of the fungus from the surface of several fruits and vegetables cultivated in Southern Italy were compared by molecular analysis and biocontrol activity. Characterisation of the isolates by using arbitrarily primed PCR (ap-PCR) confirmed the presence of a high genetic variability within the species. All the isolates were evaluated for their ability to control postharvest grey mold of apples, and two of them (SL250 and SL236), plus a proven antagonist (isolate L47), were able to control Penicillium digitatum on grapefruit, Botrytis cinerea, Rhizopus stolonifer and Aspergillus niger on table grape and B. cinerea and R. stolonifer on cherry tomato. Furthermore, preharvest application of isolate L47 on table grape resulted in a significant reduction of grey mold ranging from 27.1 to 49.5% compared to the untreated control. Random amplified polymorphic DNA technique (RAPD) was a useful method for the identification and evaluation of the survival rate of the applied antagonist. © 1999 Elsevier Science B.V. All rights reserved.

Mammella, M. A., Martin, F. N., Cacciola, S. O., Coffey, M. D., Faedda, R., and Schena, L. 2013. Analyses of the population structure in a global collection of Phytophthora nicotianae isolates inferred from mitochondrial and nuclear DNA sequences. Phytopathology 103:610-622. 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 β-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.

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.

Molecular analyses of a large population of isolates, previously identified as group B or genetic group A4 of the Colletotrichum acutatum species complex, mainly of Italian origin from olive, but also from other hosts collected since 1992, confirmed a well-resolved phylogenetic lineage with distinctive phenotypic characters which can be recognized as a separate species. Based on RAPD genomic fingerprinting, ITS and β-tubulin DNA sequences, this species was clearly distinct from C. acutatum sensu stricto, C. fioriniae and C. simmondsii as well as from the genetic groups A1, A6, A7 and A8, all previously referred to as C. acutatum sensu lato. Group A4 is widespread in Europe, being responsible for olive anthracnose epidemics in some Mediterranean countries, including Greece, Italy, Montenegro, Portugal and Spain; moreover, it causes anthracnose diseases on a wide range of other hosts including about 20 different genera of woody and herbaceous plants, ornamentals and fruit trees. This new anamorphic taxon is described as Colletotrichum clavatum sp. nov.

A new method based on the analysis of mitochondrial intergenic regions characterized by intraspecific variation in DNA sequences was developed and applied to the study of the plant pathogen Phytophthora nicotianae. Two regions flanked by genes trnY and rns and trnW and cox2 were identified by comparing the whole mitochondrial genomes of Phytophthora infestans, Phytophthora ramorum, and Phytophthora sojae and amplified using primers designed from the flanking conserved genes. These regions were sequenced from 51 isolates of P. nicotianae of both A1 and A2 mating type recovered from different hosts and geographic regions. Amplicon length varied from 429 bp to 443 bp (trnY/rns) and 322 bp to 373 bp (trnW/cox2) with intraspecific variation due to single nucleotide polymorphisms and indels. Seventeen, seven and 20 different haplotypes were detected by individually analyzing regions trnY-rns, trnW-cox2 and the combined data set of sequences from both regions, respectively. Phylogenetic analysis inferred with three different methods enabled the grouping of isolates in five clades, each containing different mitochondrial haplotypes and revealed diversity in the mitochondrial genome of P. nicotianae. The majority of isolates from citrus grouped in a single clade indicating either movement of isolates on planting stock or an association of particular isolates with this host. Phylogenetic groups were not correlated with the radial growth rate of the isolates or the rapidity of apple flesh colonization. The method developed in the present study represents an innovative molecular tool for the characterization of natural populations of P. nicotianae and should be easily expanded to other species of Phytophthora as well as other plant pathogens. It can be used to track specific haplotypes and, thanks to its high genetic resolution, it could be standardized and applied in a DNA barcoding like strategy for the precise identification of sub-specific taxa. Compared to alternative molecular methods, a major advantage is that results are unbiased (a list of nucleotides) and highly reproducible, thus enabling the comparison of data from different laboratories and time periods. Furthermore, the method could be further enhanced by the identification of additional variable mitochondrial and/or nuclear genomic regions.

A survey on Phytophthora spp. in the soils and roots of citrus groves was carried out in the main Syrian growing areas of Lattakia and Tartous. Traditional assays (selective medium with soil dilution plates) were used for pathogen detection, and molecular (PCR) assays were used for unanmbiguous identification of P. nicotianae and P. citrophtora in 38.5% of the collected samples. In both locations, P. citrophthora was the predominant species.

The effectiveness of seven phenolic compounds (esculetin, ferulic acid, quercetin, resveratrol, scopoletin, scoparone and umbelliferone) in controlling Penicillium expansum growth and patulin accumulation was evaluated by both in vitro and in vivo studies. The in vitro screening showed that quercetin and umbelliferone were the most effective compounds in controlling Penicillium growth and patulin accumulation, respectively. Quercetin and umbelliferone, resulted to be effective also in the in vivo screening, were further tested (alone and in combination) on Granny Smith and Golden Delicious apples. The efficacy in controlling Penicillium growth was better expressed on Golden Delicious than on Granny Smith apples, with quercetin providing a better control of both incidence of decay and disease severity as compared to umbelliferone. Both compounds exerted a considerable control of patulin accumulation on the two apple cultivars. Quercetin and umbelliferone can be considered as natural compounds to be used as alternative strategy to chemical fungicides in postharvest control of P. expansum infections on apples.

Rosellinia necatrix is a soil borne pathogen causing a disease commonly named “white root rot”. The pathogen, widely distributed throughout temperate and tropical climates, recently showed an increasing trend of attacks on a number of different host species. Economic losses are particularly serious in the nurseries and on orchard trees, although field crops and weeds can also be severely damaged. The pathogen is mainly disseminated by propagating materials and can survive in soil for many years. Control strategies, which include cultural practices, soil disinfestations, chemical treatments, soil solarization and biological control are expensive and not always resolutive. Therefore, white root rot control largely depends on attempts to exclude the pathogen through the use of R. necatrix-free propagating material and planting in non-infested soils. In this context a fundamental role is played by specific rules, promoting the commercialisation of healthy propagating materials and the availability of new molecular detection methods to exclude presence of the pathogen in soil and host tissues.

Background Microsatellites or single sequence repeats (SSRs) are a powerful choice of marker in the study of Phytophthora population biology, epidemiology, ecology, genetics and evolution. A strategy was tested in which the publicly available unigene datasets extracted from genome sequences of P. infestans, P. sojae and P. ramorum were mined for candidate SSR markers that could be applied to a wide range of Phytophthora species. Results A first approach, aimed at the identification of polymorphic SSR loci common to many Phytophthora species, yielded 171 reliable sequences containing 211 SSRs. Microsatellites were identified from 16 target species representing the breadth of diversity across the genus. Repeat number ranged from 3 to 16 with most having seven repeats or less and four being the most commonly found. Trinucleotide repeats such as (AAG)n, (AGG)n and (AGC)n were the most common followed by pentanucleotide, tetranucleotide and dinucleotide repeats. A second approach was aimed at the identification of useful loci common to a restricted number of species more closely related to P. sojae (P. alni, P. cambivora, P. europaea and P. fragariae). This analysis yielded 10 trinucleotide and 2 tetranucleotide SSRs which were repeated 4, 5 or 6 times. Conclusion Key studies on inter- and intra-specific variation of selected microsatellites remain. Despite the screening of conserved gene coding regions, the sequence diversity between species was high and the identification of useful SSR loci applicable to anything other than the most closely related pairs of Phytophthora species was challenging. That said, many novel SSR loci for species other than the three 'source species' (P. infestans, P. sojae and P. ramorum) are reported, offering great potential for the investigation of Phytophthora populations. In addition to the presence of microsatellites, many of the amplified regions may represent useful molecular marker regions for other studies as they are highly variable and easily amplifiable from different Phytophthora species.

The natural resistance of fruits, vegetables and flowers to diseasegenerally declines after harvest due to ripening and senescence,leading to decay development and physiological impairment, with severe economical loss. Fungal and bacterial diseases, starting either as latent infections established in the field, or as wound infections during harvesting and subsequent handling, are the major causes of losses during packaging, trading and distribution to the consumers. In cut flowers, blockage of the xylem vessels by microorganisms that accumulate in the vase solution or in the vessels themselves, is another cause of postharvest deterioration (van Doom et al , 1990). Several methods are currently available to preserve perishable commodities. However, some of these methods are now being questioned. The use of synthetic fungicides to control postharvest diseases is progressively restricted by legislation, social rejection, and development of resistance in disease-causing microorganisms. Furthermore, since the market for postharvest fungicides is relatively small, it has become difficult to sustain the costs of new registration or to support previous ones.

Background While the environment is considered the primary origin for establishing the microbiome of newly developing plants, 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 at least partially inherited through vertical transmission. We investigated where microbes reside within a seed, and how microbes are subsequently transmitted from the seed to seedling’s belowground and aboveground plant tissues. To this aim, an experimental culturing device was constructed to grow oak seedlings in a microbe-free environment while keeping the belowground and aboveground tissues separated.Results Amplicon sequencing of fungal ITS2 and bacterial 16S rDNA revealed that the seed microbiome is diverse and non-randomly distributed within an acorn. The large majority (> 95%) of fungi and bacteria present in the acorn were transmitted to the seedling. The microbial composition of the phyllosphere strongly resembled the composition found in the embryo, whereas the roots and pericarp each had a distinct microbial community. Similar relationships were observed for both fungi and bacteria.Conclusion 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 in oak seedlings. The study provides new information on the importance of the maternal environment as a source of the seedling microbiome, with significant implications for our understanding of the continuity of the plant microbiome across generations.