Alessandro Passera

Competition assays revealed Paenibacillus pasadenensis strain R16 as a novel antifungal agent

The development of new sustainable containment strategies of plant diseases is very important to guarantee food security while reducing the environmental impact of agriculture. Research of new biocontrol agents is a long and difficult process that involves several steps that start from the identification of possible candidates which, for example, show antibiotic activities, and ends with in field, large scale trials. In this study, the plant growth promoting potential and antifungal effect exerted by a novel, putative candidate biocontrol agent, strain R16, identified as Paenibacillus pasadenensis by sequence analysis of 16S rRNA and rpoB genes, against three important plant pathogenic fungi (Botrytis cinerea, Fusarium verticillioides, and Phomopsis viticola), were assessed. Biochemical assays to determine plant growth promoting potential gave negative results for siderophore production and phosphate solubilization, and positive results for ACC-deamination and IAA production. Further biochemical assays for endophytic lifestyle and antifungal activity gave positive results for catalase and chitinase activity, respectively. In vitro antagonism assays showed that strain R16 is effective against B. cinerea, reducing mycelial growth both in dual-culture and through volatile substances, characterized to be mostly composed by farnesol, and inhibiting conidia germination. Good antagonistic potential was also observed in vitro towards P. viticola, but not towards F. verticillioides. Moreover, in vivo assays confirmed the strain R16 activity reduced the infection rate on B. cinerea-inoculated berries. The obtained results firstly proved that P. pasadenesis strain R16 is a putative plant growth promoter and effective against phytopathogenic fungi. Further studies will be needed to investigate the possible application of this strain as a biocontrol agent.

New insights on Flavescence dorée phytoplasma ecology in the vineyard agro-ecosystem in southern Switzerland

Phytoplasmas associated with Flavescence doree (FDp) grapevine disease are quarantine pathogens controlled through mandatory measures including the prompt eradication and destruction of diseased plants, and the insecticide treatments against the insect vector, the ampelophagous leafhopper Scaphoideus titanus. In the present study, a multidisciplinary approach has been applied to investigate the FDp ecological cycle in a test vineyard agro-ecosystem in Canton Ticino, south Switzerland. Despite the scarce population density of S. titanus, a regular trend of new infections (3.4% of the total vines) through the years was observed. The leafhopper Orientus ishidae was found as the most abundant among the captured insect species known as phytoplasma vectors (245 out of 315 specimens). The population of O. ishidae was evidenced prevalently (167 specimens) in the south-western side of the vineyard and within the neighbouring forest constituted mainly by hazel (Corylus avellana) and willow (Salix spp.). These plant species were found infected by FDp related strains (30% of analysed trees) for the first time in this study. Interestingly, O. ishidae was found to harbour FDp related strains in high percentage (26% of the analysed pools). In addition, 16SrV phytoplasma group was detected for the first time in the insect Hyalesthes obsoletus and a FDp related strain in Thamnotettix dilutior, present in low populations within the test vineyard. Molecular characterisation and phylogenetic analyses of methionine aminopeptidase (map) gene sequences of FDp and related strains, here identified, revealed the great prevalence of the map-type FD2 in grapevines (97%) and in O. ishidae pools (72%). Such a map-type was found also in hazel and in T. dilutior, but not in S. titanus. Moreover, map-types FD1 and FD3 were identified for the first time in Switzerland in several host plants and phytoplasma vectors, including grapevine (FD1), S. titanus (FD1) and O. ishidae (FD1 and FD3). Based on the data obtained in this study, it is reasonable to hypothesise that the ecological cycle of FDp could be related not exclusively to the grapevine-specific feeding diet of S. titanus, but it could include other insect vector(s) and/or plant host(s). Further studies will be needed to prove the role of O. ishidae as vector able to transmit FDp from wild plants (e.g. hazel) to grapevine.

Functional characterization of Lactobacillus plantarum ITEM 17215: A potential biocontrol agent of fungi with plant growth promoting traits, able to enhance the nutritional value of cereal products

In this work, we explored the potential of 25 Lactobacillus plantarum strains isolated from cereals and milk-based products, testing characteristics related to antifungal activity and to nutritional quality. The tested strains demonstrated interesting beneficial traits, such as the ability to utilize fructo-oligosaccharides, prebiotic substances that help probiotic microorganisms to grow in the human gut, and to reduce phytate, an antinutrient present in cereal sector. Regarding mould inhibition, we highlighted the ability of the strains to inhibit Penicillium roqueforti, Mucor circinelloides and mycotoxinogenic moulds associated with cereal grains as Aspergillus flavus, A. niger, Fusarium verticillioides. Moreover, a moderate reduction of the bioavailability of aflatoxin AFB1 was detected. The selected L. plantarum strain ITEM 17215, showed a strong inhibitory ability towards fungal growth and was able to produce 1,2-dihydroxybenzene, benzoic acid, p-hydroxyphenyllactic acid and 3-phenyllactic acid. The latter compound, already described as efficient antifungal inhibitor, was the most abundant and its concentration was further increased by adding phenylalanine and phenylpyruvic acid in the growth medium. The metabolites produced by strain ITEM 17215 could also be related to the ability of the strain to induce cereal germination and promote plant growth. This aspect, not yet investigated in L. plantarum, could have interesting applications in the agro-food sector.

FIRST REPORT OF FUSARIUM TEMPERATUM CAUSING EAR ROT ON MAIZE IN ITALY

A two-year survey (2011-2012) was conducted to determine the population composition of Fusarium species on maize (Zea mays L.) in Lombardy (northern Italy). Maize ears exhibiting typical symptoms of Fusarium ear rot (FER) (about 30%), such as white to pink colored mycelia on the tip or scattered all over the ear, were collected at harvest. Rotted kernels were surface sterilized, rinsed in sterile water, dried and placed on potato dextrose agar (PDA) amended with dichloran and antibiotics. Developed Fusarium colonies were single-spore purified and grown on Spezieller Nahrstoffarmer agar (Leslie and Summerell, 2006). Based on morphological characteristics, 15 Fusarium spp. strains were identified as Fusarium subglutinans. Colonies on PDA showed a cottony aerial mycelium, initially white, becoming pinkish white. No microconidia chains were detected, conidiophores were erect and terminated in one to three phialides. Macroconidia were falcate, three to five septate, with a slightly beaked apical cell and a barely or distinct footlike basal cell. The translation elongation factor-1α (EF-1α) gene was partially sequenced for all the F. subglutinans strains using primers EF1 and EF2 (Scauflaire et al., 2011). BLASTn analysis showed that the nucleotide sequence (673 bp) of one strain (isolate GV2188, GenBank Accession No. KX156836) shared 100% sequence identity with F. temperatum (HM067689), a recently described species closely related to F. subglutinans (Scauflaire et al., 2011). Using silk channel inoculation method, F. temperatum pathogenicity was assessed on 10 maize ears (LG 32.85 hybrid) under greenhouse conditions. After 30 days, FER symptoms, i.e. whitish pink to lavender fungal growth on kernels, were observed only on inoculated ears and not on water controls. Kochs postulates were fulfilled by re-isolating the fungus from infected kernels. To our knowledge, this is the first report of F. temperatum in Italy associated with FER.

Unique resistance traits against downy mildew from the center of origin of grapevine ( Vitis vinifera )

The Eurasian grapevine (Vitis vinifera), an Old World species now cultivated worldwide for high-quality wine production, is extremely susceptible to the agent of downy mildew, Plasmopara viticola. The cultivation of resistant V. vinifera varieties would be a sustainable way to reduce the damage caused by the pathogen and the impact of disease management, which involves the economic, health and environmental costs of frequent fungicide application. We report the finding of unique downy mildew resistance traits in a winemaking cultivar from the domestication center of V. vinifera, and characterize the expression of a range of genes associated with the resistance mechanism. Based on comparative experimental inoculations, confocal microscopy and transcriptomics analyses, our study shows that V. vinifera cv. Mgaloblishvili, native to Georgia (South Caucasus), exhibits unique resistance traits against P. viticola. Its defense response, leading to a limitation of P. viticola growth and sporulation, is determined by the overexpression of genes related to pathogen recognition, the ethylene signaling pathway, synthesis of antimicrobial compounds and enzymes, and the development of structural barriers. The unique resistant traits found in Mgaloblishvili highlight the presence of a rare defense system in V. vinifera against P. viticola which promises fresh opportunities for grapevine genetic improvement.

Hybrid genome assembly and annotation of Paenibacillus pasadenensis strain R16 reveals insights on endophytic life style and antifungal activity.

Bacteria of the Paenibacillus genus are becoming important in many fields of science, including agriculture, for their positive effects on the health of plants. However, there are little information available on this genus compared to other bacteria (such as Bacillus or Pseudomonas), especially when considering genomic information. Sequencing the genomes of plant-beneficial bacteria is a crucial step to identify the genetic elements underlying the adaptation to life inside a plant host and, in particular, which of these features determine the differences between a helpful microorganism and a pathogenic one. In this study, we have characterized the genome of Paenibacillus pasadenensis, strain R16, recently investigated for its antifungal activities and plant-associated features. An hybrid assembly approach was used integrating the very precise reads obtained by Illumina technology and long fragments acquired with Oxford Nanopore Technology (ONT) sequencing. De novo genome assembly based solely on Illumina reads generated a relatively fragmented assembly of 5.72 Mbp in 99 ungapped sequences with an N50 length of 544 Kbp; hybrid assembly, integrating Illumina and ONT reads, improved the assembly quality, generating a genome of 5.75 Mbp, organized in 6 contigs with an N50 length of 3.4 Mbp. Annotation of the latter genome identified 4987 coding sequences, of which 1610 are hypothetical proteins. Enrichment analysis identified pathways of particular interest for the endophyte biology, including the chitin-utilization pathway and the incomplete siderophore pathway which hints at siderophore parasitism. In addition the analysis led to the identification of genes for the production of terpenes, as for example farnesol, that was hypothesized as the main antifungal molecule produced by the strain. The functional analysis on the genome confirmed several plant-associated, plant-growth promotion, and biocontrol traits of strain R16, thus adding insights in the genetic bases of these complex features, and of the Paenibacillus genus in general.

Studies of Microbiota Dynamics Reveals Association of “Candidatus Liberibacter Asiaticus” Infection with Citrus (Citrus sinensis) Decline in South of Iran

Citrus Decline Disease was recently reported to affect several citrus species in Iran when grafted on a local rootstock variety, Bakraee. Preliminary studies found “Candidatus Phytoplasma aurantifoliae” and “Candidatus Liberibacter asiaticus” as putative etiological agents, but were not ultimately able to determine which one, or if an association of both, were causing the disease. The current study has the aim of characterizing the microbiota of citrus plants that are either asymptomatic, showing early symptoms, or showing late symptoms through amplification of the V1–V3 region of 16S rRNA gene using an Illumina sequencer in order to (i) clarify the etiology of the disease, and (ii) describe the microbiota associated to different symptom stages. Our results suggest that liberibacter may be the main pathogen causing Citrus Decline Disease, but cannot rule out the possibility of phytoplasma being involved as well. The characterization of microbiota shows that the leaves show only two kinds of communities, either symptomatic or asymptomatic, while roots show clear distinction between early and late symptoms. These results could lead to the identification of bacteria that are related to successful plant defense response and, therefore, to immunity to the Citrus Decline Disease.

Prevalence of a ‘Candidatus Phytoplasma solani’ strain, so far associated only with other hosts, in Bois Noir-affected grapevines within Tuscan vineyards

Due to its complex epidemiological cycle, including several polyphagous insect vectors and host plants, and the absence of efficient control strategies, Bois Noir (BN) disease of grapevine is encroaching wider territories in the main viticultural areas worldwide. Molecular approaches allowed to increase the knowledge about its etiological agent (Bois Noir phytoplasma, BNp; ‘Candidatus Phytoplasma solani’ species), revealing interesting features concerning BNp population structure and dynamics and transmission routes in vineyard agro‐ecosystems. In the present study, a multilocus sequence typing approach (vmp1 and stamp genes) was utilised for describing the genetic diversity among BNp strain populations in 17 vineyards localised in two distinct geographic areas in Tuscany (central Italy). The results confirmed that BNp ecology in Tuscan vineyards is mainly associated to the bindweed‐related host system, and allowed the identification of 14 BNp vmp1/stamp genotypes. Interestingly, the prevalent genotype (Vm43/St10) was never found in grapevines outside of Tuscany. Moreover, statistical analyses showed significant differences between the composition of BNp strain populations identified in grapevines from north‐western and central‐eastern Tuscany. These results reinforce the hypothesis that distinct geographic areas, probably associated with different ecological niches, can drive the selection of BNp strains, also favouring the entrance of unusual ‘Ca. Phytoplasma solani’ genotypes in vineyards.

Epidemiological role of spontaneous weeds in the spreading of "bois noir" phytoplasma

Survival of Hyalesthes obsoletus on some weeds was investigated to verify their possible role in “bois noir” epidemiology. Data evidenced that H. obsoletus survived after 14 days of forced feeding on Urtica dioica, Convolvolus arvensis, Artemisia vulgaris, Plantago lanceolata and Taraxacum officinale. This evidence reinforced the hypothesis of a possible involvement of such weeds, carrying the same “bois noir” phytoplasma tuf-type identified in grapevines and insects, in “bois noir” spread in north-eastern Italy.