Chitosan and postharvest decay of fresh fruit: Meta-analysis of disease control and antimicrobial and eliciting activities.

Consumers are increasingly aware of the importance of regular consumption of fresh fruit in their diet. Since fresh fruit are highly sensitive to postharvest decay, several investigations focused on the study natural compounds alternative to synthetic fungicides, to extend their shelf life. A long list of studies reported the effectiveness of the natural biopolymer chitosan in control of postharvest diseases of fresh fruit. However, these findings remain controversial, with many mixed claims in the literature. In this work, we used random-effects meta-analysis to investigate the effects of 1% chitosan on (a) postharvest decay incidence; (b) mycelium growth of fungal pathogens Botrytis cinerea, Penicillium spp., Colletotrichum spp. and Alternaria spp.; and (c) phenylalanine ammonia-lyase, chitinase and ß-1,3-glucanase activities. Chitosan significantly reduced postharvest disease incidence (mean difference [MD], -30.22; p < 0.00001) and in vitro mycelium growth (MD, -54.32; p  < 0.00001). For host defense responses, there were significantly increased activities of ß-1,3-glucanase (MD, 115.06; p = 0.003) and chitinase (MD, 75.95; p < 0.0002). This systematic review contributes to confirm the multiple mechanisms of mechanisms of action of chitosan, which has unique properties in the natural compound panorama. Chitosan thus represents a model plant protection biopolymer for sustainable control of postharvest decay of fresh fruit.

Draft Genomic Resources for the Brown Rot Fungal Pathogen Monilinia laxa.

Monilinia laxa is the causal agent of brown rot on stone fruit, and it can cause heavy yield losses during field production and postharvest storage. This article reports the draft genome assembly of the M. laxa Mlax316 strain, obtained using a hybrid genome assembly with both Illumina short-reads and PacBio long-reads sequencing technologies. The complete draft genome consists of 49 scaffolds with total size of 42.81 Mb, and scaffold N50 of 2,449.4 kb. Annotation of the M. laxa assembly identified 11,163 genes and 12,424 proteins which were functionally annotated. This new genome draft improves current genomic resources available for M. laxa and represents a useful tool for further research into its interactions with host plants and into evolution in the Monilinia genus.

Genetic Variability of Stolbur Phytoplasma in Hyalesthes obsoletus (Hemiptera: Cixiidae) and its Main Host Plants in Vineyard Agroecosystems.

Bois noir is an economically important grapevine yellows that is induced by 'Candidatus Phytoplasma solani' and principally vectored by the planthopper Hyalesthes obsoletus Signoret (Hemiptera: Cixiidae). This study explores the 'Ca. P. solani' genetic variability associated to the nettle-H. obsoletus and bindweed-H. obsoletus systems in vineyard agroecosystems of the central-eastern Italy. Molecular characterization of 'Ca. P. solani' isolates was carried out using polymerase chain reaction/restriction fragment length polymorphism to investigate the nonribosomal vmp1 gene. Seven phytoplasma vmp-types were detected among the host plants- and insect-associated field-collected samples. The vmp1 gene showed the highest polymorphism in the bindweed-H. obsoletus system, according to restriction fragment length polymorphism analysis, which is in agreement with nucleotide sequence analysis. Five vmp-types were associated with H. obsoletus from bindweed, of which one was solely restricted to planthoppers, with one genotype also in planthoppers from nettle. Type V12 was the most prevalent in both planthoppers and bindweed. H. obsoletus from nettle harbored three vmp-types, of which V3 was predominant. V3 was the only type detected for nettle. Our data demonstrate that planthoppers might have acquired some 'Ca. P. solani' profiles from other plant hosts before landing on nettle or bindweed. Overall, the different vmp1 gene rearrangements observed in these two plant hosts-H. obsoletus systems might represent different adaptations of the pathogen to the two host plants. Molecular information about the complex of vmp-types provides useful data for better understanding of Bois noir epidemiology in vineyard agroecosystem.

Effect of Bacteria Inoculation on Colonization of Roots by Tuber melanosporum and Growth of Quercus ilex Seedlings.

Tuber melanosporum is an ascomycete that forms ectomycorrhizal (ECM) symbioses with a wide range of host plants, producing edible fruiting bodies with high economic value. The quality of seedlings in the early symbiotic stage is important for successful truffle cultivation. Numerous bacterial species have been reported to take part in the truffle biological cycle and influence the establishment of roots symbiosis in plant hosts and the development of the carpophore. In this work, three different bacteria formulations were co-inoculated in Quercus ilex L. seedlings two months after T. melanosporum inoculation. At four months of bacterial application, the T. melanosporum ECM root tip rate of colonization and bacterial presence were assessed using both morphological and molecular techniques. A 2.5-fold increase in ECM colonization rate was found in the presence of Pseudomonas sp. compared to the seedlings inoculated only with T. melanosporum. The same treatment caused reduced plant growth either for the aerial and root part. Meanwhile, the ECM colonization combined with Bradyrhizobium sp. and Pseudomonas sp. + Bradyrhizobium sp. reduced the relative density of fibrous roots (nutrient absorption). Our work suggests that the role of bacteria in the early symbiotic stages of ECM colonization involves both the mycorrhizal symbiosis rate and plant root development processes, both essential for improve the quality of truffle-inoculated seedlings produced in commercial nurseries.

Colonization of Vitis spp. wood by sGFP-transformed Phaeomoniella chlamydospora, a tracheomycotic fungus involved in Esca disease.

To evaluate wood colonization and interactions with Vitis spp. of Phaeomoniella chlamydospora, a fungal agent involved in Esca disease, isolate CBS 229.95 was transformed using a pCT74 construct which contained the genetic markers for synthetic green fluorescent protein (sGFP) and hygromycin B phosphotransferase. Nine stable P. chlamydospora fungal transformants (Pch-sGFP lines) were obtained using polyethylene-glycol-mediated transformation of protoplasts. These were characterized for sgfp and hygromycin B phosphotransferase (hph) genome insertions and for sGFP fluorescence emission, using quantitative polymerase chain reaction and fluorimetric systems, respectively. No correlation was observed between sgfp copy number genome insertion and sGFP fluorescence expression. Cuttings of Vitis vinifera 'Montepulciano', 'Verdicchio', 'Sangiovese', 'Biancame', and 'Cabernet Sauvignon'; and the grapevine rootstocks 'Kober 5BB', 'SO4', '420A', '1103P', and V. rupestris were inoculated by immersion in a conidial suspension of the selected fungal Pch-sGFP71 line and incubated at 4 ± 1 and 25 ± 1°C. Wood colonization was estimated through epifluorescence microscopy and was affected by incubation temperature. After 6 months at 4 ± 1°C, the fungal growth was completely inhibited. At 25 ± 1°C, the highest extent of wood colonization was recorded in Montepulciano and Verdicchio, with the lowest in the rootstocks SO4 and V. rupestris. The expression of the Pch-sGFP71 transformed line was localized in the xylem area, primarily around the vessels. The use of sGFP-transformed P. chlamydospora helped to clarify different aspects associated with the location of this pathogen in grapevine tissue, before disease symptom expression.

Tracking of Diversity and Evolution in the Brown Rot Fungi Monilinia fructicola, Monilinia fructigena, and Monilinia laxa.

Monilinia species are among the most devastating fungi worldwide as they cause brown rot and blossom blight on fruit trees. To understand the molecular bases of their pathogenic lifestyles, we compared the newly assembled genomes of single strains of Monilinia fructicola, M. fructigena and M. laxa, with those of Botrytis cinerea and Sclerotinia sclerotiorum, as the closest species within Sclerotiniaceae. Phylogenomic analysis of orthologous proteins and syntenic investigation suggest that M. laxa is closer to M. fructigena than M. fructicola, and is closest to the other investigated Sclerotiniaceae species. This indicates that M. laxa was the earliest result of the speciation process. Distinct evolutionary profiles were observed for transposable elements (TEs). M. fructicola and M. laxa showed older bursts of TE insertions, which were affected (mainly in M. fructicola) by repeat-induced point (RIP) mutation gene silencing mechanisms. These suggested frequent occurrence of the sexual process in M. fructicola. More recent TE expansion linked with low RIP action was observed in M. fructigena, with very little in S. sclerotiorum and B. cinerea. The detection of active non-syntenic TEs is indicative of horizontal gene transfer and has resulted in alterations in specific gene functions. Analysis of candidate effectors, biosynthetic gene clusters for secondary metabolites and carbohydrate-active enzymes, indicated that Monilinia genus has multiple virulence mechanisms to infect host plants, including toxins, cell-death elicitor, putative virulence factors and cell-wall-degrading enzymes. Some species-specific pathogenic factors might explain differences in terms of host plant and organ preferences between M. fructigena and the other two Monilinia species.

The Mycorrizal Status in Vineyards Affected by Esca.

In this work we analyzed the relationship among native arbuscular mycorrhizal fungi (AMF) and vine roots affected by esca, a serious grapevine trunk disease. The AMF symbiosis was analyzed on the roots of neighboring plants (symptomatic and asymptomatic to esca) in 14 sites of three vineyards in Marche region (central-eastern Italy). The AMF colonization intensity, identified by non-vital staining, showed higher value in all esca symptomatic plants (ranging from 24.6% to 61.3%) than neighboring asymptomatic plants (from 17.4% to 57.6%). The same trend of Glomeromycota phylum abundance was detected by analyzing fungal operational taxonomic units (OTUs) linked to the AMF community, obtained by amplicon high throughput analysis of ITS 1 region. Overall, the highest amount of OTUs was detected on roots from symptomatic plants (0.42%), compared to asymptomatic roots (0.29%). Specific primer pairs for native Rhizophagus irregularis and Funneliformis mosseae AMF species, were designed in 28S rRNA and large subunit (LSU) ribosomal RNA, respectively, and droplet digital PCR protocol for absolute quantification was set up. A higher number of DNA copies of both fungal species were detected more frequently in symptomatic than asymptomatic vines. Our study suggests a relationship between esca and native AMF in grapevine. These results underline the importance of native rhizosphere microbial communities for a better knowledge of grapevine esca disease.

Chitosan Coating Enriched With Ruta graveolens L. Essential Oil Reduces Postharvest Anthracnose of Papaya (Carica papaya L.) and Modulates Defense-Related Gene Expression.

Anthracnose of papaya (Carica papaya L.) caused by the fungus Colletotrichum spp. is one of the most economically important postharvest diseases. Coating with chitosan (CS) and Ruta graveolens essential oil (REO) might represent a novel eco-friendly method to prevent postharvest anthracnose infection. These compounds show both antimicrobial and eliciting activities, although the molecular mechanisms in papaya have not been investigated to date. In this study, the effectiveness of CS and REO alone and combined (CS-REO) on postharvest anthracnose of papaya fruit during storage were investigated, along with the expression of selected genes involved in plant defense mechanisms. Anthracnose incidence was reduced with CS, REO, and CS-REO emulsions after 9 days storage at 25°C, by 8, 21, and 37%, respectively, with disease severity reduced by 22, 29, and 44%, respectively. Thus, McKinney's decay index was reduced by 22, 30, and 44%, respectively. A protocol based on reverse transcription quantitative real-time PCR (RT-qPCR) was validated for 17 papaya target genes linked to signaling pathways that regulate plant defense, pathogenesis-related protein, cell wall-degrading enzymes, oxidative stress, abiotic stress, and the phenylpropanoid pathway. CS induced gene upregulation mainly at 6 h posttreatment (hpt) and 48 hpt, while REO induced the highest upregulation at 0.5 hpt, which then decreased over time. Furthermore, CS-REO treatment delayed gene upregulation by REO alone, from 0.5 to 6 hpt, and kept that longer over time. This study suggests that CS stabilizes the volatile and/or hydrophobic substances of highly reactive essential oils. The additive effects of CS and REO were able to reduce postharvest decay and affect gene expression in papaya fruit.

Detection and Quantification of Stagonosporopsis cucurbitacearum in Seeds of Cucurbita maxima Using Droplet Digital Polymerase Chain Reaction.

Stagonosporopsis cucurbitacearum is an important seedborne pathogen of squash (Cucurbita maxima). The aim of our work was to develop a rapid and sensitive diagnostic tool for detection and quantification of S. cucurbitacearum in squash seed samples, to be compared with blotter analysis, that is the current official seed test. In blotter analysis, 29 of 31 seed samples were identified as infected, with contamination from 1.5 to 65.4%. A new set of primers (DB1F/R) was validated in silico and in conventional, quantitative real-time PCR (qPCR) and droplet digital (dd) PCR. The limit of detection of S. cucurbitacearum DNA for conventional PCR was ∼1.82 × 10-2 ng, with 17 of 19 seed samples positive. The limit of detection for ddPCR was 3.6 × 10-3 ng, which corresponded to 0.2 copies/µl. Detection carried out with artificial samples revealed no interference in the absolute quantification when the seed samples were diluted to 20 ng. All seed samples that showed S. cucurbitacearum contamination in the blotter analysis were highly correlated with the absolute quantification of S. cucurbitacearum DNA (copies/µl) in ddPCR (R 2 = 0.986; p ≤ 0.01). Our ddPCR protocol provided rapid detection and absolute quantification of S. cucurbitacearum, offering a useful support to the standard procedure.

Detection of 'Candidatus Phytoplasma solani' in roots from Bois noir symptomatic and recovered grapevines.

'Candidatus Phytoplasma solani' is the causal agent of Bois noir (BN) in grapevine (Vitis vinifera). It is usually detected in leaves, where typical disease symptoms are seen. However, little information is available on the presence of this phytoplasma in grapevine roots. Here, we investigated 'Ca. P. solani' in roots collected from 28 symptomatic, 27 recovered and eight asymptomatic grapevine plants. Protocols based on high-resolution melting (HRM) combined with real-time quantitative PCR (qPCR-HRM) and nested-qPCR-HRM were developed to identify 'Ca. P. solani' tuf-type variants with single nucleotide polymorphisms. In all, 21.4% of roots from symptomatic plants were positive to 'Ca. P. solani' using qPCR-HRM, and 60.7% with nested-qPCR HRM. Also, 7.4% of roots from recovered plants were positive using qPCR-HRM, which reached 44.4% using nested-qPCR HRM. These analyses identified tuf-type b1 on 88.2% of the positive samples from symptomatic grapevines, and 66.6% from recovered grapevines, with all other samples identified as tuf-type a. This study reports the presence of 'Ca. P. solani' in the roots of both symptomatic and recovered grapevines. These qPCR-HRM and nested-qPCR-HRM protocols can be applied to increase the sensitivity of detection of, and to simplify and speed up the screening for, 'Ca. P. solani' tuf-types.