Revealing Cues for Fungal Interplay in the Plant-Air Interface in Vineyards.

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.

A new high-resolution melting assay for genotyping Alternaria species causing citrus brown spot.

BACKGROUND: Alternaria brown spot is one of the most important diseases of tangerines and their hybrids worldwide. To set up effective control strategy, the accurate detection and identification of the species responsible for the diseases is crucial. However, characterization based on morphology and/or multilocus genetic approaches is time consuming, requires great expertise and sometimes is not conclusive. Therefore, the set-up of a rapid and efficient DNA-based assay might be of paramount importance. High-resolution melting (HRM) analysis represents an interesting tool for the uncovering of nucleotide variations as small as one base difference and, as such, relevant to species characterization. RESULTS: In the present investigation, an HRM assay based on the Alternaria barcoding region OPA1-3 was set up. Specimen strains of the main citrus-associated Alternaria species and morphotypes generated distinct and normalized profiles, allowing their differentiation when HRM-tested. Moreover, when the assay was used to screen an Alternaria collection from citrus fruit and leaves, it distributed the 180 isolates in three independent clusters, readily and consistently resolved. Isolates were identified as belonging to the species Alternaria alternata and the species complex A. arborescens. Within A. alternata, the morphotypes alternata (77% of the collection) and limoniasperae (17% of the collection) were present. CONCLUSIONS: Although further validation experiments will be performed to optimize the assay for a diagnostic use, this HRM approach might represent a rapid, sensitive and specific method for the detection and identification of Alternaria spp. responsible for citrus brown spot disease. © 2018 Society of Chemical Industry.

Occurrence of Sheraphelenchus sucus (Nematoda: Aphelenchoidinae) and Panagrellus sp. (Rhabditida: Panagrolaimidae) Associated with Decaying Pomegranate Fruit in Italy.

Two different nematode species were recovered from pomegranate decaying fruit in two localities in Southern Italy: the mycetophagus nematode Sheraphelenchus sucus and a bacterial feeder nematode belonging to the Panagrolaimidae (Rhabditida) family. Morphometrics of the Italian population of S. sucus closely resemble that of the type population, whereas some differences were found when compared with another population from Iran. Molecular characterization of the Italian S. sucus using the 18S rRNA gene, D2-D3 expansion domains of the 28S rDNA, the ITS region, and the partial mitochondrial COI were carried out. Sequences of the 18S rRNA gene, the D2-D3 domains, and the ITS were analyzed using several methods for inferring phylogeny to reconstruct the relationships among Sheraphelenchus and Bursaphelenchus species. The bacterial feeder Panagrellus sp. was characterized at the molecular level only. The D2-D3 expansion domains and ITS sequences of this Italian panagrolaimid were determined. The D2-D3 sequences of the Italian panagrolaimid showed 99% similarity with the corresponding sequence of Panagrellus sp. associated with Rhynchophorus ferrugineus. This is the first report on the tritrophic association of S. sucus and Rhabditida that uses both insects and pomegranate fruit as hosts.

Metabarcoding Analysis of Phytophthora Diversity Using Genus-Specific Primers and 454 Pyrosequencing.

A metabarcoding method based on genus-specific primers and 454 pyrosequencing was utilized to investigate the genetic diversity of Phytophthora spp. in soil and root samples of potted plants, from eight nurseries. Pyrosequencing enabled the detection of 25 Phytophthora phylotypes distributed in seven different clades and provided a much higher resolution than a corresponding cloning/Sanger sequencing approach. Eleven of these phylotypes, including P. cactorum, P. citricola s.str., P. palmivora, P. palmivora-like, P. megasperma or P. gonapodyides, P. ramorum, and five putative new Phytophthora species phylogenetically related to clades 1, 2, 4, 6, and 7 were detected only with the 454 pyrosequencing approach. We also found an additional 18 novel records of a phylotype in a particular nursery that were not detected with cloning/Sanger sequencing. Several aspects confirmed the reliability of the method: (i) many identical sequence types were identified independently in different nurseries, (ii) most sequence types identified with 454 pyrosequencing were identical to those from the cloning/Sanger sequencing approach and/or perfectly matched GenBank deposited sequences, and (iii) the divergence noted between sequence types of putative new Phytophthora species and all other detected sequences was sufficient to rule out sequencing errors. The proposed method represents a powerful tool to study Phytophthora diversity providing that particular attention is paid to the analysis of 454 pyrosequencing raw read sequences and to the identification of sequence types.

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.

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.

Soybean and casein hydrolysates induce grapevine immune responses and resistance against Plasmopara viticola.

Plasmopara viticola, the causal agent of grapevine downy mildew, is one of the most devastating grape pathogen in Europe and North America. Although phytochemicals are used to control pathogen infections, the appearance of resistant strains and the concern for possible adverse effects on environment and human health are increasing the search for alternative strategies. In the present investigation, we successfully tested two protein hydrolysates from soybean (soy) and casein (cas) to trigger grapevine resistance against P. viticola. On Vitis vinifera cv. Marselan plants, the application of soy and cas reduced the infected leaf surface by 76 and 63%, as compared to the control, respectively. Since both hydrolysates might trigger the plant immunity, we investigated their ability to elicit grapevine defense responses. On grapevine cell suspensions, a different free cytosolic calcium signature was recorded for each hydrolysate, whereas a similar transient phosphorylation of two MAP kinases of 45 and 49 kDa was observed. These signaling events were followed by transcriptome reprogramming, including the up-regulation of defense genes encoding pathogenesis-related (PR) proteins and the stilbene synthase enzyme responsible for the biosynthesis of resveratrol, the main grapevine phytoalexin. Liquid chromatography analyses confirmed the production of resveratrol and its dimer metabolites, δ- and ε-viniferins. Overall, soy effects were more pronounced as compared to the cas ones. Both hydrolysates proved to act as elicitors to enhance grapevine immunity against pathogen attack.