We report the research results on the biocontrol of Panama disease using a synthetic microbial community derived from banana rhizosphere. 45 soil samples were taken from Tenerife and used to isolate beneficial microbes. In vitro assays were used to select antagonistic microorganisms against Fusarium oxysporum f. sp. cubense tropical race 4. Effective microorganims composed a synthetic community used for 3 in planta biocontrol trials. Results are presented and discussed.

Virus-induced gene silencing (VIGS) is a well-established reverse genetics technology for assessment of gene functions in plants. VIGS is a transient loss-of-function assay that involves three steps: engineering viral genomes to include fragments of host genes that are targeted to be silenced, infecting the plant hosts and suppressing the target genes expression by post-transcriptionalgene silencing (PTGS).Suppression of specific mRNA accumulation allows correlation between gene silencing and the deriving phenotype, providing clues on gene functions.However, the efficiency of this technology may be scarce. In these cases, weak and/or non-homogeneous distribution of VIGS through the plant may generate results not fully coherent,. This often limits the extensive application of the technique to more permissive plant species such as Nicotiana benthamiana.Aiming at increasing VIGS efficiency in functional studies,particularly in key crop species, we produced and tested new constructs using a Tobacco rattle virus (TRV)-based vector in tomato (Solanum lycopersicum) and other solanaceous crops. This innovative approach consisted in cloning into the TRV vector a short fragment of a host gene containing at its termini mutations designed forthe expression of small interfering RNAs (siRNAs) that mimicked a microRNA(miRNA) structure.The recently developed artificial microRNAs (amiRNAs) technology modifies an endogenous gene silencing mechanism that processes natural miRNA precursors to small silencing RNAs targeting transcripts for degradation. Based on natural miRNA structures, amiRNAs are commonly designed to contain mismatches at specific nucleotides with respect to their target sites.We designed a vector where amiRNA-like small RNAs are generated when viral intermediate dsRNA forms are targeted by the host PTGS machinery in the cytoplasm.In the viral vector, we inserted mutant sequences designed to contain at both their 5' and 3' termini one or two mismatches at selected positions. Mismatched sequences were computed by the WMD3 web tool (wmd3.weigelworld.org), an algorithm that generates all possible amiRNAs using full-length target gene sequences as input.Short (110nt) amiRNA-like containing sequences were compared for their VIGS efficiency with wild-type sequences, shorter- or longer-sized inserts and inverted-repeat constructs. Upon inoculation of our constructs , VIGS established earlier and more extensively than its wild-type counterpart in tomato, N. benthamiana and N. tabacum. For instance, suppression of the tomato reporter gene magnesium chelatase (ChlI or SU) with the VIGS-amiRNA-like construct produced the typical yellow phenotype earlier and more extensively than the standard TRV-PDS (phytoene desaturase) VIGS vector. Quantitative RT-PCR confirmed the efficiency of our VIGSamiRNA-like constructs in terms of post-transcriptional suppression of host target mRNAs. Our results are discussed in the light of their beneficial contribution t

Lo spettro di attività antimicrobica in vitro di tiofanate metile, flutolanil, Streptomyces lydicus ceppo WYEC 108 e Streptomyces sp. ceppo AtB-42 è stato valutato nei confronti di otto funghi fitopatogeni terricoli: Pythium sp., Fusarium solani, Rhizoctonia solani, Sclerotinia sclerotiorum, Verticillium dahliae, Pyrenochaeta lycopersici, Phytophthora sp. e Cylindrocarpon destructans. Tiofanate metile è risultato particolarmente efficace nel ridurre la crescita di C. destructans (CL50=0,05 g/L, CL95=9,5 g/L), F. solani (CL50=0,006 g/L, CL95=10,0g/L), P. lycopersici (CL50=0,01 g/L, CL95=0,1 g/L), S. sclerotiorum (CL50=0,005 g/L, CL95=0,01 g/L) e V. dahliae (CL50=0,006 g/L, CL95=5,8g/L). Flutolanil è risultato, invece, più efficace nei confronti di R. solani (CL50=0,0006 g/L, CL95=0,007 g/L). L'attivitàantimicrobica di tiofanate metile e flutolanil nei confronti di Pythium sp. e Phytophthora sp. è stata inferiore a quella del formulato di riferimento, propamocarb+fosetil Al. I ceppi di streptomiceti saggiati hanno mostrato uno spettro di attività antimicrobica piuttosto ampia. S.lydicus WYEC 108 ha completamente inibito la crescita di tutti i fitopatogeni saggiati.Streptomyces sp. AtB-42 e S. griseoviridis K61, quest'ultimo utilizzato come ceppo microbico di riferimento in questo lavoro, hanno drasticamente ridotto (rispettivamente almeno 65% e 92%), ma solo in pochi casi inibito totalmente, la crescita dei fitopatogeni.

We provide notes about the biology and the ethology of Parahypopta caestrum (Hubner) (Lepidoptera Cossidae), based on field observations conducted in asparagus plantations in Apulia region, Italy. Furthermore, the effect of 6 entomopathogenic nematode (EPN) strains (Steinernematidae and Heterorhabditidae) and 3 entomopathogenic fungal (EPF) isolates (Beauveria bassiana) was evaluated in laboratory assays against III instar larvae of the asparagus moth. The results showed that all the nematodes and fungal strains affected the asparagus moth survival, except the Steinernema affine and Heterorhabditis bacteriophora strains. Steinernema feltiae and B. bassiana showed the best performances, killing on average 90% of the P. caestrum larvae. Considering the lack of effective chemical control means, the microbiological control of the asparagus moth by EPNs and EPFs reveals promising perspectives and needs further investigations.

Verticillium wilt of olive is best managed by resistant cultivars but those currently available show incomplete resistance to the defoliating (D) Verticillium dahliae pathotype. Moreover, these cultivars do not satisfy consumers' demand for high yields and oil quality. Highly resistant rootstocks would be of paramount importance for production of agronomically-adapted and commercially-desirable olive cultivars in D V. dahliae-infested soils. In this work, resistance to D V. dahliae in wild olive clones Ac-13, Ac-18, OutVert and StopVert was assessed by quantifying the fungus DNA along the stem using a highly sensitive real-time qPCR protocol and a stem colonization index (SCI) based on isolation of V. dahliae following artificial inoculations under conditions highly conducive for Verticillium wilt. 'Ac-13?, 'Ac-18?, 'OutVert' and 'StopVert' showed a symptomless reaction to D V. dahliae. The mean amount of D V. dahliae DNA quantified in stem of the four clones ranged from 3.64 to 28.89 pg /100 ng olive DNA, which was 249 to 1,537 times lower than that in susceptible 'Picual' olive. The reduction in the quantitative stem colonization of wild olive clones by D V. dahliae was also indicated by a sharp decrease in the SCI. Overall, there was a pattern of decreasing SCI in acropetal progression along the plant axis, as well as correlation between positive re-isolation and quantification of pathogen DNA. The results of this research show that wild olive clones 'Ac-13?, 'Ac-18?, 'OutVert' and 'StopVert' have a valuable potential as rootstocks for the management of Verticillium wilt in olive.

Virus-induced gene silencing (VIGS) is a well-established reverse genetics technology for assessment of gene functions in plants. VIGS is a transient loss-of-function assay that involves three steps: engineering viral genomes to include fragments of host genes that are targeted to be silenced, infecting the plant hosts and suppressing the target genes expression by post-transcriptional gene silencing (PTGS), the defense mechanism deployed by plants against virus infections. Suppression of specific mRNA accumulation allows correlation between gene silencing and the deriving phenotype, providing clues on gene functions. However, the efficiency of this technology may be affected by various factors, including virus vector properties and susceptibility of plant host species. In several cases, weak and/or non-homogeneous distribution in the plant (or in the single leaf) of VIGS may generate results not fully coherent, particularly in terms of correlation between phenotype and accumulation levels of the specifically suppressed RNA. This often limits the extensive application of the technique to more permissive plant species such as Nicotiana benthamiana. Aiming at increasing VIGS efficiency in functional studies, particularly in key crop species, we produced and tested new constructs using a Tobacco rattle virus (TRV)-based vector in tomato (Solanum lycopersicum) and other solanaceous crops. This innovative approach consisted in cloning into the TRV vector a short fragment of a host gene containing at its termini mutations designed for the expression of small interfering RNAs (siRNAs) that mimicked a microRNA (miRNA) structure. The recently developed artificial microRNAs (amiRNAs) technology modifies an endogenous gene silencing mechanism that processes natural miRNA precursors to small silencing RNAs targeting transcripts for degradation. Based on natural miRNA structures, amiRNAs are commonly designed to contain mismatches at specific nucleotides with respect to their target sites. Unlike the conventional amiRNA strategy, where target-specific 21nt small silencing RNAs derive from longer double-stranded RNA (dsRNA) precursors that are processed in the nucleus by DCL1, we designed a vector where amiRNA-like small RNAs are generated when viral intermediate dsRNA forms are targeted by the host PTGS machinery in the cytoplasm. In the viral vector, we inserted mutant sequences designed to contain at both their 5' and 3' termini one or two mismatches at selected positions. Mismatched sequences were computed by the WMD3 web tool (wmd3.weigelworld.org), an algorithm that generates all possible amiRNAs using full-length target gene sequences as input. Short (110nt) amiRNA-like containing sequences were compared for their VIGS efficiency with wild-type sequences, shorter- or longer-sized inserts and inverted-repeat constructs. Upon inoculation of our constructs , VIGS established earlier and more extensively than its wild-type counterpart in tomato, N.

The streptomycete strain AtB-42 was previously selected from a consortium of 300 rhizospherecompetentisolates. It exerted a strong antagonism in vitro against phytopathogenic fungi and canreduce by 30% the severity of corky root of tomato (Pyrenochaeta lycopersici) in the field. Otherisolates, such as StB-8, resulted ineffective in vitro and in greenhouse assays. Multilocus sequenceanalysis using five genes (atpD, gyrB, rpoB, recA and trpB) revealed Streptomyces fulvissimus DSM40593 as the strain closest to AtB-42, and S. globisporus C-1027 the closest to StB-8. In order to unveilthe antagonism mechanism, we subjected AtB-42 and StB-8 to genome sequencing (Illumina) andmetabolomics analysis (LC-MS qTOF). The 7866 and 7086 protein-coding genes predicted in AtB-42and StB-8, respectively, formed 4838 orthologous clusters (shared between the strains), while 166clusters were unique to AtB-42 and 101 unique to StB-8. However, gene ontology enrichment analysiswas not informative in explaining the mechanism of antagonism. Furthermore, 19 secondary metabolitebiosynthetic gene clusters were found exclusively in AtB-42, other 18 solely in StB-8, while 19 wereshared between them. Hence, the 19 AtB-42-specific secondary metabolites could be putativelyinvolved in its antifungal activity, and include antibiotics such as oxazolomycin, macrotetrolide,sporolide, meridamycin, desotamide, platencin, fredericamycin, albachelin, kirromycin, the NRPSbleomycin and pactamycin, alkylresorcinol (type III PKS), nanchangmycin (saccharide), auricin (type IIPKS), roseoflavin, the biofilm inhibitor cahuitamycins and the siderophore griseobactin. Surprisingly,these substances were not detected by LC-MS qTOF, but deoxyvalidamine, which is the componentpseudo-amino-sugar of validamycin, was found uniquely in AtB-42.

The potential of grafting susceptible cultivars onto resistant rootstocks was evaluated for the control of Verticillium wilt in olive.

Ozone is widely used as a disinfectant, and ozonated water has been known to confer some protection of plants against several biotic stresses. By applying four foliar spray treatments of ozonated water (10 ppm ozone) on tomato seedlings, i.e. two pre- and two post-inoculation with Tomato spotted wilt virus (TSWV), we observed reduction of disease incidence and severity by 20%, as well as a virus titrrreduction by 80% at 19 days post-inoculation. The same treatments also reduced the number of galls induced by root knot nematode (RKN; Meloidogyne incognita) by 29%. Soil drenching with ozonated water for four consecutive days before inoculation reduced RKN gall formation by 60%, but not TSWV infection. Overall, in mock-inoculated plants, foliar sprays induced PR1b1 expression in leaves, though other salicylate- (PAL and PR-5x) or jasmonate-dependent genes (LoxD, AOS and PinI) were substantially unaffected. Soil drenching promptly enhancedtranscription of PAL and PR1b1 in roots and leaves, down-regulated PR-5x and did not affect expression of LoxD and AOS. PinI was significantly down-regulated only in leaves. The impact of ozonated water applications on the expression of these genes did not correlate with that of benzothiadiazole, a known inducer of systemic acquired resistance. This demonstrates that ozonated water may protect tomato fromtwo very different biotic stresses, especially when applied at the sites of their infection, and modulates salicylate and jasmonate pathways differently from benzothiadiazole. This research was supported by the Fondazione Cassa di Risparmio di Puglia, Italy, within the Project 'Risposte di difesa contro nematodi e virus indotte da trattamenti di ozono in pomodoro'.

An amplicon-metagenomic approach was used to investigate Phytophthora spp. diversity in waterscollected from four Scottish natural habitats, i.e. Invergowrie, Glensaugh Research Station, CairngormsNational Park and Sourhope Research Station. Sequencing of the rDNA ITS1 barcode-region on anIllumina MiSeq platform with genus-specific primers produced around 700,000 sequences that wereclustered into 4,671 OTUs using a 99% similarity threshold. Assigning taxonomy to the representativesequences of the OTUs was done using the BLAST algorithm and a manually curatedOomycetesdatabase. The results showed that majority of the sequences were associated to the genusPhytophthora. Although few OTUs were associated to other Oomycetes, the use of genus-specific ITS1primers allowed understanding well the diversity of Phytophthora population. Moreover, the resultsrevealed a clear site-to-site variation, where the lowest species diversity was found in GSB Glensaughand the highest diversity at Invergowrie. The analysis of ?-diversity, based on Bray Curtis matrices,confirmed differences (P<0.001) in populations associated to the investigated environments, with onlyfew OTUs shared among samples. This approach proved effective in the study of Phytophthora spp.diversity and the use of genus-specific primers contributed to an unprecedented level of accuracy,compared to traditional techniques based on direct isolation, while preserving sensitivity of identificationand relative quantification of Phytophthora species. This study demonstrated that Illumina sequencingof the ITS1 region may serve to study in depth diversity and ecological niches of Phytophthora species.

We analyzed the transcriptome (RNA-Seq) of leaf samples collected from a field crop of tomato cv. Docet (Sw5 resistance gene) in Apulia, southern Italy, with different symptom severity and accumulation levels of a resistance-breaking strain of Tomato spotted wilt virus (TSWV). Four groups of samples were assumed to be different stages of plant tissue colonization by the virus: plants without symptoms anda virus titre (group A) or 1 ×?102 TSWV reads per million (rpm; B), and plants with symptoms and 1 ×?104 rpm (C) or 2 ×?105 rpm (D). Transcriptome sequencing revealed that plant response to TSWV infection is profoundly related to its accumulation level in the tissues. At an early stage of infection (B vs. A comparison), genes related to photosystem I were down-regulated, and oxidoreductase activityincreased. Considerable virus colonization (C vs. B) activated defense-related mechanisms such as cell surface receptor signalling, phenylpropanoid biosynthesis and transcription factor activity. In contrast, photosynthesis, transmembrane transporter activity, and biosynthesis of monosaccharides and peptides were down-regulated. This scenario increased at an advanced stage of colonization (D vs.C), with attenuation of response to stimuli (e.g., surface receptor signaling and protein kinase activity) and an increase of catalytic activities such as ubiquitin- protein transferase and ribonuclease. TSWV infection constantly injured tomato cell metabolism(e.g., photosynthesis, monosaccharide and peptide biosynthesis, ion transporter activity) while plant defense (e.g., cell surface receptor signaling, phenylpropanoid pathway), clearly ineffective in such compatible plant-virus interaction, occurred late and disappeared soon after.

The effects of a previously selected entomopathogenic fungus, Beauveria bassiana AL1 strain, and the B. bassiana based mycoinsecticide Naturalis (ATCC 74040 Beauveria bassiana strain) were evaluated on the system Ceratitis capitata - Psyttalia concolor in laboratory assays. First, the entomopathogenic fungal strains were tested for their virulence against 2, 4, and 6-days old puparia of C. capitata. Subsequently, P. concolor emergence from C. capitata puparia treated or not with the fungal strains was evaluated at three different time points (2, 4 and 6 days) from the parasitization and the following pupation. Results showed that the entomopathogenic fungal applications affected the medfly survival. The effect of fungal treatments was higher on 2-day puparia (49.16 and 51.33% of mycosed puparia for ATCC 74040 and AL1 strain respectively) while the rate of mycoses was lower and ranged between 39 and 27.16% when fungal treatments were performed on 4 and 6-day puparia. Furthermore, fungal treatments affected the P. concolor emergence (c.a. 80% in the untreated control) particularly when applied 2 days after the parasitization and the C. capitata pupation (43.16 and 47.83% for the ATCC 74040 and the AL1 strains respectively), while when treatments were performed on older puparia, the P.concolor emergence ranged from 63.33 to 68.66%. Results suggest that the entomopathogenic B. bassiana strains are effective against C. capitata puparia but they may be detrimental against its endoparasitoid P. concolor, particularly when applied in the earlier stages of the parasitization process.

The virulence of three strains of Beauveria bassiana and one of Metarhizium anisopliae was tested against Trialeurodes vaporariorum and its parasitoid Encarsia formosa in laboratory assays. These strains were previously selected for their virulence against Galleria mellonella and Tenebrio molitor. The commercial B. bassiana strain ATCC 74040, both as pure fungal culture and formulated myco-insecticide (Naturalis), was included in the assays as positive control. First, the entomopathogenic fungal strains were tested for their virulence against T. vaporariorum nymphs on tomato leaf disks. Then, the E. formosa development was evaluated under treatment with the entomopathogenic fungal strains at five different time points from the parasitization of T. vaporariorum nymphs. The virulence of our entomopathogenic fungal strains was superior to that of ATCC 74040, although not significantly, resulting in a cumulative mortality (CM) of T. vaporariorum nymphs 7 days after inoculation (DAI) greater than 86 %. Our M. anisopliae strain CIST8 was the most effective (96.6 % CM 7 DAI), even superior to the myco-insecticide Naturalis (94.2 %), which was more effective than the ATCC 74040 pure strain (85.6 %). The entomopathogenic fungal strains, and especially Naturalis, negatively affected E. formosa development and its parasitization activity of T. vaporariorum nymphs. This effect was more pronounced when the fungal strains were applied before parasitization. Results suggest that the application of entomopathogenic fungi is incompatible with E. formosa release on crops.

The effects of a previously selected entomopathogenic fungus, Beauveria bassiana AL1 strain, and the B. bassiana-based mycoinsecticide Naturalis were evaluated on the Trialeurodes vaporariorum-Encarsia formosa system. The parasitoid's ability to recognize uninfected and infected hosts was examined by evaluation of E. formosa tropism in 'choice' conditions and its activity (residence time on leaf, searching time, handling hosts time and hosts acceptance) in no-choice conditions. Finally, the role of E. formosa in transmitting the mycoses from infected to uninfected host populations was estimated. E. formosa showed no differential tropism in 'choice' conditions. It was able neither to locate the host at distance nor to discriminate between infected and uninfected hosts. E. formosa was able to vector fungal propagules from contaminated to uncontaminated hosts during its activity. The results of these laboratory experiments have clarified some biological and behavioral aspects of pathogen-host-parasitoid interactions.

The effects of Streptomyces spp. isolates in the biological control of corky root of tomato and Verticilliumwilt of eggplant was determined in in vitro, greenhouse and field trials. Twenty-six Streptomyces spp.isolates were obtained from the rhizospheres of different vegetable crops in southern Italy. In in vitrodual culture tests, mycelial radial growth of Pyrenochaeta lycopersici and Verticillium dahliae was reducedup to 18.6% and 30.1%, respectively. Radial growth of seven other fungal pathogens was variably reducedas well. The isolates StB-3, StB-6, StB-11 and StB-12 showed a good antagonistic effect against bothP. lycopersici and V. dahliae, while the rest of isolates eventually showed antagonism against only onepathogen.In pot-experiments in the greenhouse three of the four above-mentioned Streptomyces spp. isolatessignificantly reduced corky root up to 64.9% (StB-11), and all four isolates reduced foliar symptoms ofVerticillium wilt (AUDPC) up to 48.3%, but none of them reduced the severity of vascular browning. Innaturally infested field trials, StB-11 significantly reduced corky root severity in tomato by 48.2%, StB-12 by 35% and StB-6 by 32.6%, but none of the isolates were effective in controlling Verticillium wiltof eggplant. The effectiveness of the streptomycete AtB-42, successfully used in previous researches,was here confirmed as it reduced corky root of tomato in the field by 33.6%. In conclusion, our researchdemonstrates that under field conditions corky root of tomato, but not Verticillium wilt of eggplant, canbe effectively controlled by the Streptomyces spp. isolates used in this study.

The reactions to shot-hole disease (Stigmina carpophila) of nine plum cultivars and seed-propagated myrobalan trees were evaluated during three years in three commercial orchards located at Nova Sin, Matera, Italy. In July 2006, May 2007, June 2007 and June 2008, shot-hole severity and twig defoliation were assessed. Pathogen pressure was higher in 2007 and 2008 than in 2006. In the three years of study, cultivar Golden Plumza was the most susceptible to the shot-hole measured by leaf severity, however it showed variable defoliation percentages in different years. The seed-propagated myrobalan trees were the most resistant, although they showed a slight degree of defoliation in 2007 and 2008. Intermediate reactions to the disease occurred in cultivars Angeleno, Autumn Giant, Fortune, Green Sun, October Sun. Santa Rosa, Sorriso di Primavera and T.C. Sun. A significant correlation between shot-hole leaf severity and twig defoliation was detected. To our knowledge, this is the first report on screening for resistance of plum cultivars to shot-hole disease. (C) 2010 Elsevier Ltd. All rights reserved.

A severe soft rot disease was observed in some commercial artichoke crops (F1-hybrid cultivars) during 2014 in the Bari,Foggia (Apulia,Southern Italy) and Matera (Basilicata,Southern Italy) provinces. Symptoms were chlorosis and wilting of the older leaves accompanied by dark-green to dark-brown soft rotting of the pith. Several bacterial isolates were obtained from affected plants by culturing on semi-selective crystal violet pectate agar medium. Biochemical,physiological and molecular analyses and pathogenicity tests,performed using five selected isolates,identified three of them as Pectobacterium carotovorum subsp. brasiliense. To the best of our knowledge,this is the first report of a bacterial soft rot caused by P. carotovorum subsp. brasiliense in Italy and the first finding of this bacterial subspecies on artichoke.

Gerbera jamesonii, an economically crop in the Italian cutflower market is reported to be affected by phytoplasmas oftwo 16S ribosomal groups from different parts of the world:16SrII from Australia (Siddique, 2005), and 16SrI from Italy(Bertaccini and Bellardi, 1998). Gerbera plants with virescence,phyllody and abnormal flower colour, indicative ofphytoplasmal infection, were observed in Apulia (southernItaly) in January 2010. Disease incidence was near 100% incv. Maxima while no apparent symptom were seen in cv.Fuego. For phytoplasma detection, total DNA was extractedfrom the leaves of three symptomatic and one symptomlessplant of each cultivar and used in nested PCR with primersP1/P7 (Schneider et al., 1995) and R16F2/R2 (Lee et al.,1993). DNA fragments of 1.2 kb, corresponding to 16SrDNA, were amplified only from DNA of the three symptomaticsamples. When digested with MseI, amplicons showedidentical RFLP patterns, which were indistinguishable fromthose produced by the European aster yellows strain of the"Candidatus Phytoplasma asteris". The 1,803 bp ampliconfrom P1/P7 PCR amplification was cloned into pGEM vector,and plasmid DNAs from two transformed clones weresequenced (BioFab Research, Italy). Both clones showed thesame sequence (GenBank accession No. JF795864) andClustalW2 alignment confirmed that 16S rDNA of gerberaphytoplasma shared 99.9% identity with Oenothera phytoplasma86-7 [GenBank accession No. M30790; Lim andSears (1989)], and 99.0% identity to several "Ca. P. asteris"isolates of different ribosomal subgroups (A, B, D, E, F, K,and P. GenBank accession Nos. HQ589187, EF634457,AY265206, AY265213, AY265211, U96616, AF503568, respectively)."Ca. P. asteris" is widely spread in Italian cutflower crops but this is its first report from gerbera in Apulia.

During October 2015, severe symptoms of collar rot wereobserved in a commercial field of two month-old cauliflower(Brassica oleracea L. var. botrytis) in BAT province (Apulia,southern Italy). Among several cultivars grown in the samefield (6 ha), only cultivar Tipoff F1 (Bejo) showed diseasesymptoms with ca. 5% of plants affected. First symptoms appearedon collar tissues and consisted of wide water-soakedlesions which progressively darkened, rotted and extendedto the midrib of basal leaves.Pythium-like colonies were consistently isolated fromdecayed plants on potato dextrose agar (PDA), and onerepresentative isolate, designated DiSSPA P8, showed morphologicalcharacters consistent with those described by vander Plaats-Niterink (1981) for the type specimen CBS 398.51of Pythium ultimum Trow var. ultimum, recently renamedGlobisporangium ultimum (Trow) Uzuhashi, Tojo & Kakish.(Uzuhashi et al., 2010).The coxII gene as well as ITS and D1/D2 regions of rDNAwere sequenced and deposited in GenBank under accessionNos. KY753869, KY392755 and KY753868, respectively. Oncethese sequences were included in the phylogenetic trees constructedby Uzuhashi et al. (2010), DiSSPA P8 clustered withG. ultimum strain UZ056 with a 99% sequence identity to thecorresponding sequences from this isolate.Furthermore, the representative isolate induced collarrot symptoms, similar to those observed in the field, on sixweek-old cauliflower plants cv. Tipoff F1 after two days at26°C from inoculation with colonized PDA discs on superficiallywounded stem.To the best of our knowledge, this is the first report ofcollar rot caused by G. ultimum on cauliflower in Italy.

In June 2015, crown galls were observed in a 30 day-old greenhouse crop of Aster sp. in Lecce province (southern Italy). Symptoms occurred on ca. 20% of plants and consisted of stunting and, below the soil line, white-cream spherical galls which progressively darkened. Two representative isolates, producing 5 mm-circular convex colonies with reddish center and whitish border on selective agar medium 1A, were designated DiSSPA Ag26 and DiSSPA Ag27 and subjected to biochemical assays according to Schaad et al. (2001). They were Gram-negative, oxidase positive, able to grow in 2% NaCl and in ferric ammonium citrate and produced 3-ketolactose, acidity from melezitose but not from erythritol, alkalinity from litmus milk but no alkali from malonic, L-tartaric and mucic acids and utilized citrate but not L-tyrosine. Morphological and biochemical characters of our isolates were similar to those of Agrobacterium tumefaciens species complex, formerly called A. tumefaciens biovar 1 (Costechareyre et al., 2010).In a phylogenetic tree based on the sequence of DNA gyrase subunit B gene (gyrB), constructed as per Paulawska and Ka?u?na (2012), DiSSPA Ag26 (accession no. MF000330) and DiSSPA Ag27 (MF000331) clustered within the genomovar G1 of A. tumefaciens species complex, with the best match (99%) with Ch3 strain (HQ438217).Moreover, our isolates induced gall formation on stem of five week-old tomato cv. Marmande, tobacco cv. Samsun and on carrot disks after three weeks at room temperature from needle-inoculation of a bacterial cell suspension (108 CFU/mL). Bacteria re-isolated exhibited the same traits of the inoculated isolates. To the best of our knowledge, this is the first report of crown gall caused by A. tumefaciens species complex genomovar G1 on Aster sp. in Italy.

In Apulia (southern Italy), dill (Anethum graveolens) is cultivated in a few greenhouses and fields, mainly located between Bari and Brindisi. In December-January of 2010 to 2014, leaf blight was observed in greenhouse-grown dill crops, when they were near the harvest stage. Symptoms differed from those induced by some known foliar pathogens of dill, i.e. Sclerotinia sclerotiorum and Erysiphe heraclei, but recalled those caused by Botrytis cinerea. In fact, leaves that were first discolored, turned light to dark brown, and finally wilted, rendering the affected plants unsuitable for harvest.In the presence of high relative humidity, a whitish-cream mycelium developed on symptomatic leaves. Light microscope observations revealed the presence of hyaline hyphae with clamp connections and lunate ballistospores [16.4 ± 1.7 (standard deviation) . 11.0 ± 1.3 ?m]. A 609 bp product amplified by PCR from fungal DNA using the ITS1/ITS4 primer pair was sequenced (BMR Genomics, Italy) and the sequence of an isolate designated IPSP-GB520 was deposited in GenBank under the accession No. KP890654. BLAST alignment revealed a 99% homology at the nucleotide level with several accessions of Itersonilia perplexans Derx (Basidiomycota, Cystofilobasidiaceae), including two isolates (DQ667163 and NR_077117) conserved at the CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands.Koch's postulates were fulfilled with a successful detachedleaf assay. Based on morphological characteristics and nucleotide sequence homology, the fungus was identified as I. perplexans. In Italy, an early report ascribed leaf blight of dill to I. pastinacae, based on morphometric observations and pathogenicity tests (Matta and Garibaldi, 1968). To the best of my knowledge, this is the first report of I. perplexans on dill in Italy.

In July 2014, a white fungal efflorescence was observed on the stems and both surfaces of mature and young leaves of a caper plant (Capparis spinosa) growing in the Campus of the University of Bari. Light microscope observations revealed the presence of simple or branched conidiophores emerging through leaf stomata, and bearing conidia singly or in short chains. Primary (pyriform) and secondary (cylindrical) conidia, typical of the anamorphic stage of Leveillula taurica (Lév.) G. Arnaud, causal agent of powdery mildew (Palti, 1988), measured on average 62.2×20.2 ?m (±7.23×±2.18 ?m standard deviation). The teleomorphic stage was not observed during threemonths of observation. A 630-bp PCR amplicon obtained with the ITS1/ITS4 primer pair was sequenced (BMR Genomics, Italy) and deposited in GenBank under the accession No. KP164030 (isolate designated CSP-PUG1). The sequence shared up to 99% homology with several accessions of L. taurica, including the one previously reported on caper (AB045002), and other species of the genus. The Koch's postulates were met with a successful pathogenicity test on caper. It is worthnoting that, besides this plant, I have never observed powdery mildew on spontaneous caper plants in different Apulian (southern Italy) sites, even when they were growing close to highly susceptible plant species such as Convolvulus arvensis. Therefore, the sporadic pathogen occurrence on this plant might be due to microclimatic conditions unusual for caper. Indeed, the diseased plant had grown under a prolonged shading near a building, but caper normally grows in sun-drenched placeslike stone walls and rocky cliffs. To the best of my knowledge, this is the first report of L. taurica on caper in Italy.

In October 2013, unusual chlorotic patches were observed on the middle leaves of a few tomato plants cv. Lotty grown in a greenhouse located in the countryside of Fasano (Apulia, Southern Italy). Younger leaves and fruits were symptomless and no aggravation of symptoms was detected as time went by. Electron microscope observations of leaf dips revealed the presence of filamentous virus particles ca. 520 nm in length. Mechanical inoculations with leaf extracts from symptomatic tomatoes elicited a mosaic reaction in Nicotiana benthamiana, but not in tomato plants cv. UC82, which, however, were systemically infected, as shown by RT-PCR. A RT-PCR product of the expected size (ca. 700 bp) was obtained using the degenerate broad-spectrumpotexvirus primers Potex5/Potex1RC (van der Vlugt and Berendsen, 2002). The amplicon was custom sequenced (BMR Genomics, Italy) and the sequence deposited in GenBank under the accession No. KM923762. BLAST alignment showed that the 700 bp amplicon shared 97-99% homology with the RNA-dependent RNA polymerase (RdRp) gene of several isolates of Pepino mosaic virus (PepMV) genotype CH2, 82% homology with the LP and EU genotypes, and 79-80% with the US genotype. A RT-PCR-restriction fragment length polymorphism (RFLP) analysis of the RdRp amplicon with EcoRI and BglII restriction endonucleases confirmed that our isolate, designated PUG1, belongs to the CH2 genotype (Hanssen, 2010). Our observations and assays are consistent with the presence of PepMV in the tomato plants tested, and the fact that PUG1 is a mild isolate of the virus. PepMV has previously been recorded from tomato in three Italian regions, i.e. Sardinia, Sicily and Campania, but this the first report from Apulia.

Virus-induced gene silencing (VIGS) is a transient loss-of-function assay that involves threesteps: engineering the genome of a viral vector to include a fragment of host gene that is targeted tobe silenced, infecting the plant hosts and suppressing the target gene expression by posttranscriptionalgene silencing (PTGS). VIGS is a well-established reverse genetics technology forassessment of gene functions in plants. However, the efficiency of this technology may be low insome plant species, and this often limits the application of the technique to more permissive modelhosts. Aiming at increasing VIGS efficiency in functional studies, particularly in key crop speciessuch as tomato (Solanum lycopersicum), we tested an innovative approach that consisted in: a)enhancement of the target gene cleavage efficiency by exploiting the artificial microRNA(amiRNA) technology; and b) validation of a bioinformatic method for selecting the most suitablegene fragments for induction of gene silencing.The recently developed amiRNA technology modifies an endogenous gene silencingmechanism that processes natural miRNA precursors to small silencing RNAs targeting specifictranscripts for degradation. Based on natural miRNA structures, amiRNAs are commonly designedto contain mismatches at specific nucleotides with respect to their target sites, thus increasingeffectiveness of target gene cleavage as compared to RNA silencing processes guided by otherperfectly matching small RNAs.The WMD3 software (wmd3.weigelworld.org) was used for both identification of putativeamiRNA sequences and selection of suitable gene regions. In WMD3, an algorithm generates insilico all possible amiRNAs putatively able to anneal to full-length target mRNA. We selected andcompared cDNA fragments (110-120 nt) from gene regions with either high or low content ofputative amiRNAs, inserted point mutations to express amiRNA-like small RNAs from the viralvectors, and cloned these cDNAs into tobacco rattle virus (TRV)-based VIGS vectors. The variableVIGS effects of such vectors were analyzed on two tomato reporter genes, phytoene desaturase(PDS) and magnesium chelatase (ChlI or SU), whose VIGS phenotypes consist in leaf bleachingand yellowing, respectively, and therefore could be visually assessed. VIGS efficiency by severaldifferent inserts was compared by evaluating intensity of VIGS phenotype, target mRNA levels andaccumulation of VIGS-target specific small interfering RNAs. Overall, our results clearly indicatedthat: i) VIGS efficiency increased when gene sequences inserted in TRV vectors included amiRNAlikepoint mutations; ii) VIGS efficiency was significantly reduced when cDNA fragments fromgene regions with low amiRNA content were expressed in TRV vectors; and iii) WMD3 wasproved an effective bioinformatic tool to select proper target gene sequences in VIGS experiment.Our results are discussed in the light of their beneficial contri

Leaf blight caused by Itersonilia perplexans Derx is commonly observed on dill (Anethum graveolens L.) at the crop maturity stage. In the fields of southern Italy, I. perplexans attacks begin in late autumn, when the weather is cool and humid. In laboratory, the fungal growth rate was maximum at 20 °C, the ballistoconidia production was at temperatures of 25 °C or higher, and growth was interrupted at 30 °C or above. It is difficult to manage the disease because very few fungicides are admitted for use on dill. Therefore, we screened in vitro several fungicides for their toxicity against I. perplexans, and discovered the high effectiveness of difenoconazole, with EC50 and EC95 of 0.5 and 0.95 ?g mL-1, respectively, both for mycelium growth and ballistoconidia production. Ciprodinil+fludioxonil, azoxystrobin (both with EC50 = 0.69 ?g mL-1) and boscalid (EC50 = 7.32 ?g mL-1) were toxic to I. perplexans, but did not suppress completely the pathogen even at doses of 10,000 ?g mL-1. Mancozeb+metalaxil showed EC50 of 15.5 ?g mL-1 and completely suppressed the fungus at 100 ?g mL-1. Copper oxychloride was toxic only at doses higher than 100 ?g mL-1, and sulphur did not affect the pathogen growth. Therefore, difenoconazole is worthy of further evaluations in the field for the control of leaf blight of dill caused by I. perplexans.

Our objective was to evaluate if natural recovery may be exploited in disease control of Verticillium wilt in olive. Therefore, we evaluated the following: the incidence of natural recovery; the Verticillium dahliae viability within olive tissues over time and the effectiveness of soil solarization, calcium cyanamide and pollarding of trees at soil level in promoting natural recovery. Methods: Three different experiments (A, B and C) were performed in commercial olive orchards planted with the highly susceptible cv. 'Bella di Cerignola' and infested with the non-defoliating V. dahliae pathotype. Results: In experiment A, in the period 2010-2012, natural recovery occurred on 35 of 138 diseased trees (25 %); however, this recovery was transient and lasted between 3 months for 11 trees (8 %) and 21 months for one tree (0.7 %). V. dahliae tended to be inactivated in twigs within 1 or 2 years after symptom onset (experiment A). However, it was evident that V. dahliae was more abundant in larger (trunk and first- or second-order branches) versus thinner woody parts of olive trees (roots; experiment B). In the attempt to explore whether natural recovery could be further stimulated artificially, it was observed that soil solarization and soil application of calcium cyanamide were ineffective in promoting its occurrence. Tree pollarding at soil level induced a transient recovery, which lasted only 1 year (experiment C). Conclusions: Based on our observations, natural recovery of susceptible olive from Verticillium wilt has a low impact on the disease epidemiology in the short-term only and cannot be effectively stimulated in practice by soil solarization, calcium cyanamide or tree pollarding. © 2014 Springer International Publishing Switzerland.

The pathogenicity of 23 isolates of Beauveria bassiana (Ascomycota, Hypocreales: Cordycipitaceae) and four of Metarhizium anisopliae (Ascomycota, Hypocreales: Clavicipitaceae) was tested against Galleria mellonella (Lepidoptera: Galleriidae) and Tenebrio molitor (Coleoptera: Tenebrionidae) larvae in laboratory assays, using 2106 conidia mL-1 fungal suspensions. The commercial myco-insecticide Naturalis (Intrachem Bio Italia, Italy), containing the ATCC 74040 B. bassiana strain, was included in the assays for comparison. Mycosed larvae were counted 1, 2, 3, 7, 10, 14 and 17 days after inoculation and the cumulative mortality data were used to calculate mean survival time (MST) and lethal times (LT50 and LT95). No difference between B. bassiana and M. anisopliae were detected in the pathogenicity against the two insect species. However, a wide variability occurred among fungal isolates within species. The two B. bassiana isolates AL1 and ALB55 killed G. mellonella larvae within the shortest time (MST of 2.2 and 2.3 days, respectively), as well as the ALB55 did against T. molitor larvae (MST of 2.8 days). Naturalis was superior to these two B. bassiana isolates, causing a MST of 1.1 day or shorter on the insect larvae. Overall, G. mellonella resulted more sensitive than T. molitor, as showed also by the non-inoculated controls, for which MSTs were 7.7 and 8.4 days, respectively. Due to the rapid and effective insecticide action, the ALB55 B. bassiana isolate can be considered as a new promising candidate for the microbial pest control.

Different bacterial groups in irrigation well water are strongly implicated in soil health and plant development. Herein, 48 bacterial strains were isolated from agricultural well water in northern Algeria. Among them, four strains were selected based on their antifungal potential and their ability to express Plant Growth Promoting traits such as Indole Acetic Acid (IAA), hydrolytic enzymes, siderophores etc. The isolates were identified as Pseudomonas sp. (B, D and N strains) and Serratia sp. (C strain) by 16S rRNA gene sequencing. Mycelial growth inhibition against Botrytis cinerea and Aspergillus niger ranged from 60 to 90% for the four strains. Moreover, volatiles compounds emission by the isolates resulted in Plant Growth Inhibition values ranging from 13 to 50%, specifically against B. cinerea. Impressively, the strains' antifungal activity showed high inducibility as it was obtained only by the filtered supernatants from bacterial cultures previously in contact with the fungus. Finally, a greenhouse assay, carried out to determine the strains' efficacy in promoting plant growth and protecting seedlings under Pythium aphanidermatum-infected soil, revealed that the strain N markedly enhanced pea germination (+250%) and fresh weight (+43%) and tomato fresh weigh (+10%). The results constitute an attempt for better use of the bacterial functional diversity from irrigation wells in sustainable agriculture.

Systemin is a plant signal peptide hormone involved in the responses to wounding and insect damage in the Solanaceae family. It works in the same signaling pathway of jasmonic acid (JA) and enhances the expression of proteinase inhibitors. With the aim of studying a role for systemin in plant antiviral responses, a tomato (Solanum lycopersicum) transgenic line overexpressing the prosystemin cDNA, i.e. the systemin precursor, was inoculated with Cucumber mosaic virus (CMV) strain Fny supporting either a necrogenic or a non-necrogenic satellite RNA (satRNA) variant. Transgenic plants showed reduced susceptibility to both CMV/satRNA combinations. While symptoms of the non-necrogenic inoculum were completely suppressed, a delayed onset of lethal disease occurred in about half of plants challenged with the necrogenic inoculum. RT-qPCR analysis showed a correlation between the systemin-mediated reduced susceptibility and the JA biosynthetic and signaling pathways (e.g. transcriptional alteration of lipoxygenase D and proteinase inhibitor II). Moreover, transgenically overexpressed systemin modulated the expression of a selected set of receptor- like protein kinase (RLK) genes, including some playing a known role in plant innate immunity. A significant correlation was found between the expression profiles of some RLKs and the systemin-mediated reduced susceptibility to CMV/satRNA. These results show that systemin can increase plant defenses against CMV/satRNA through transcriptional reprogramming of diverse signaling pathways.

In tomato, resistance to Tomato spotted wilt virus (TSWV) is conferred by the dominant gene, designated Sw-5. Virulent Sw-5 resistance breaking (SRB) mutants of TSWV have been reported on Sw-5 tomato cultivars. Two different PCR-based allelic discrimination techniques, namely Custom TaqMan(TM) SNP Genotyping and high-resolution melting (HRM) assays, were developed and compared for their ability to distinguish between avirulent (Sw-5 non-infecting, SNI) and SRB biotypes. TaqMan assays proved to be more sensitive (threshold of detection in a range of 50-70 TSWV RNA copies) and more reliable than HRM, assigning 25 TSWV isolates to their correct genotype with an accuracy of 100%. Moreover, the TaqMan SNP assays were further improved developing a rapid and simple protocol that included crude leaf extraction for RNA template preparations. On the other hand, HRM assays showed higher levels of sensitivity than TaqMan when used to co-detect both biotypes in different artificial mixtures. These diagnostic assays contributed to gain preliminary information on the epidemiology of TSWV isolates in open field conditions. In fact, the presented data suggest that SRB isolates are present as stable populations established year round, persisting on both winter (globe artichoke) and summer (tomato) crops, in the same cultivated areas of Southern Italy.

Rootstocks highly resistant to the highly virulent Verticillium dahliae defoliating (D)pathotype would be of much interest for the management of Verticillium wilt in olive andgrowing of Verticillium-susceptible olive cultivars in geographic areas where D V. dahliaeprevails. Recently, research done at the University of Bari, Italy, and University of Córdoba,Spain, have led to the identification of some wild olive genotypes that could be of use asresistant rootstocks, including the currently patented clones STOPVERT and OUTVERT,AC13 and AC18. To further characterize the resistance reaction shown by those genotypesin previous studies, we have carried out a series of experiments using standardizedprotocols and controlled conditions optimal for development of Verticillium wilt. Own-rootedplants of a range of ages were inoculated with a range of high inoculum concentrations ofselected, highly virulent D isolate V138I by root dipping and/or transplanting in an artificiallyinfested soil mixture. Plants were inoculated once or twice in a sequence, and incubated inthe growth chamber for 3 to 4 months under optimal conditions for disease development.Disease reaction was assessed by the development of foliar symptoms, isolation of thepathogen from the lower, middle, and upper main stem, and molecular quantification of thefungus in the sampled tissues using a real-time quantitative PCR (qPCR) protocol with adetection limit of 18 fg of V. dahliae DNA in infected, symptomless tissues. ClonesSTOPVERT and OUTVERT showed a symptomless reaction to inoculation compared with100 % dead plants in susceptible 'Picual' olive and mild disease reaction in tolerant'Frantoio'. V. dahliae was isolated from middle stem parts of STOPVERT and OUTVERTplants to a lesser extent than from the lower stem, but isolations from 'Frantoio' plantsyielded the fungus from all stem parts at similar proportions. On average, the concentrationof V. dahliae DNA per 100 ng of stem DNA ranged from 5.6 to 41.1 pg in STOPVERTplants, from 13.7 to 80.9 pg in OUTVERT plants, and from 94.6 to 141.6 pg in 'Frantoio'plants. The larger of those concentrations is 120 times lower than that found in susceptible'Picual' olive. Extending the time of incubation of infected STOPVERT and OUTVERTplants reduced the frequency of successful isolations and quantification from previouslyinfected tissues. Clones AC13 and AC18 also showed a highly resistant reaction to root-dipinoculation with V. dahliae 138I, though slight symptoms developed in some 'AC18' plants.Average V. dahliae DNA concentration per 100 ng of stem DNA was 10,9 and 86,7 pg in'AC13' and 'AC18' plants, respectively, compared with 42,9 pg and 16,6 ng in 'Frantoio' and'Picual' olives. Histopathological assessment of the plants reaction is in progress. Also,experiments are being conducted to determine the influence of genetic and geographicdiversity of D V. dahliae isolates on the resistant

Streptomycetes are the largest taxon of antibiotic producers in the microbial world. Nevertheless,they are less studied than other biocontrol agents against Fusarium diseases and, perhaps, plantdiseases in general. Plant diseases incited by Fusarium species are notably challenging. Four speciescomplexes in the genus are pathogenic: Fusarium fujikuroi, F. graminearum, F. solani and F. oxysporum.Being a vascular pathogen, F. oxysporum is particularly difficult to control by using microbialantagonists. Most research has remained at early experimental stages, and only few Streptomyces spp.strains have been assayed under diverse conditions. Few commercial products consisting ofStreptomyces spp. have often provided fluctuating results across trials. Five biocontrol trialsconducted in the field using streptomycetes report reductions of diverse Fusarium wilts by 0-55%,with one case (cucumber) of yield increase by 1.3-fold. Among 38 articles dealing with potexperiments,16 report a disease control level above 50%, seven of which with a level above 70%. Thechitinolytic activity of Streptomyces spp. strains plays an important role in the biocontrol of Fusariumdiseases, and the plant growth promotion trait is a cherished outcome. More attention is being paidto strains endophytic, producing volatile organic compounds, or to the identification of antibioticsand metabolites responsible for the biocontrol. Bioformulation is a critical point in the use ofbiocontrol agents, but it has been still poorly considered in the experiments. The biocontrol efficacyof well-designed streptomycete consortia or the so-called microbial synthetic communities, possiblyin proper bioformulations, and the role of streptomycetes in the reduction of mycotoxins in thegrains are aspects that would merit further investigation in the future.

In prove di lotta contro fitopatogeni terricoli del pomodoro (Fusarium solani, Rhizoctonia solani e Pythium sp.), l'efficacia di tiofanate metile è stata messa a confronto con altri fungicidi chimici (propamocarb+fosetil Al e flutolanil) e antagonisti microbici (Streptomyces griseoviridis ceppo K61, S. lydicus ceppo WYEC 108 e Streptomyces sp. ceppo AtB-42).Inoltre, tiofanate metile è stato valutato in diversi protocolli applicativi contro la verticilliosi del carciofo (Verticillium dahliae). Su pomodoro, tiofanate metile è risultato costantemente il mezzo di lotta più efficace, riducendo del 66% la moria delle piante causata da Pythium sp. e del 44-73% la gravità delle infezioni dei tre patogeni, con variabili incrementi di biomassa delle piante. Tali risultati sono stati talvolta superiori, sebbene non significativamente, rispetto a quelli ottenuti con propamocarb+fosetil Al. Streptomyces sp. AtB-42 ha ridotto le infezioni di F. solani e R. solani rispettivamente del 58% e 54%, livelli superiori a quelli ottenuti con S. griseoviridis K61. Su carciofo, tre somministrazioni post-trapianto con 0,65 L/ha di tiofanate metile, precedute dall'immersione delle radici nella sospensione del fungicida al trapianto, hanno costituito il protocollo applicativo più valido, determinando il 35-40% di riduzione della verticilliosi e il 10% di incremento di biomassa fogliare.

Plant viruses are obligate parasites that exploit host components for replication and spread inside the host. Transport of the viral genome is enabled by movement proteins (MPs) targeting the cell periphery to mediate passage throughout plasmodesmata (PD). Pectin methylesterase (PME) is one of the critical host factors facilitating MPs in PD gating, and a direct interaction of PME with Tobacco mosaic virus (TMV) MP is required for viral movement and in turn for virus viability. PME is a critical enzyme for host development and defence, acting via complex mechanisms involving multigenic and tissue specific isoforms and endogenous inhibitors. This composite activity of PME suggests that level and timing of protein accumulation, with respect to virus inoculation and MP expression, can be critical for the functional outcome of the PME-MP interaction and in turn for the success of a viral infection. Based on this notion, we tested different experimental conditions to evaluate the beneficial effect of the downregulation of PME gene expression on the development of TMV-induced disease and on plant protection. We used virus induced gene silencing technology (VIGS) to downregulate PME gene expression, which resulted in a 30-45 % reduction of TMV symptom severity and, correspondingly, to a 60 % reduction of TMV RNA accumulation in systemic leaves. VIGS proved to be a rapid and effective technology for PME gene silencing in functional assays and for plant defence from viral infection. Our findings indicate that N. benthamiana plants with hindered expression of PME survive a TMV infection, which kills non-silenced plants within a week.