First report of Stagonosporopsis pogostemonis causing leaf spot on cauliflower in Italy.

First report of Stagonosporopsis pogostemonis causing leaf spot on cauliflower in Italy Wassim Habib1, Mariangela Carlucci2, Roberto Fasano3, Franco Nigro1,2 † 1 Centre of Research, Experimentation and Training in Agriculture (CRSFA) - Basile Caramia, via Cisternino 281, Locorotondo, 70010 Bari, Italy 2 Department of Soil, Plant and Food Sciences, University of Bari - Aldo Moro, via Amendola 165A, 70126 Bari, Italy 3 Bioricerche, Phytopathological Lab, via Pio XI 30, Bellizzi, 84092 Salerno, Italy † Corresponding author: F. Nigro. Email: franco.nigro@uniba.it Keywords: Phoma-like, Multi-locus gene sequencing, Brassica oleracea L. var. botrytis, Campania. In September 2022, a severe foliar disease was observed on 2-3-week-old 'Corsaro' and 'Parthenon' seedlings of cauliflower (Brassica oleracea L. var. botrytis) grown under greenhouses in a nursery in Caserta province, Campania (Italy), with an incidence of 90% on about 150,000 plants. Affected plants showed numerous small, irregular, and depressed lesions bordered by a chlorotic halo on leaves that become necrotic, and later drop out giving a shot-hole effect (Fig. 1). Isolations were performed on leaf portions from 30 symptomatic samples which were surface-sterilised by dipping in 70 % ethanol for 30s, then in a sodium hypochlorite solution (1%) for 30s, and rinsed two times in distilled sterilised water. Tissue fragments were left to dry on sterile filter paper and then seeded on potato dextrose agar (PDA) amended with 0.5 g/L of streptomycin sulphate (Sigma-Aldrich S.R.L, Steinheim, Germany). After 6 to 8 days of incubation at 24 ± 1°C, colonies revealed a consistent growth of a Phoma-like fungus resembling Stagonosporopsis genus. On PDA, one-week-old colonies were dark olivaceous green to brown with white regular margin (Fig. 2B). Pycnidia (32-160 µm × 30-95 µm) were subglobose, and conidia (2.7-4.8 µm × 1-2.7 µm) ellipsoidal, with rounded ends, aseptate, with two polar guttules (Fig. 2C). All isolates had similar morphological features, therefore further analyses were performed on one representative strain (CRSFA.753.22). The multilocus phylogenetic approach using Maximum likelihood method and Tamura-Nei model on rDNA-ITS, TUB and RPB2 sequences (accession numbers: OQ318550, OQ326503, OQ326504) including reference strains of Stagonosporopsis (Dong et al. 2021) demonstrated that CRSFA.753.22 clustered with Stagonosporopsis pogostemonis Luo, Huang & Manawas holotype (ZHKUCC 21-0001) with 91% bootstrap support. ITS and RPB2 sequences had 100% BLAST match to ZHKUCC 21-0001, whereas TUB sequence had 99.69%. Pathogenicity tests were performed on 20 healthy 'Corsaro' plantlets. Leaves were surface sterilized with 70% alcohol and wounded with a sterile tip of a needle. A suspension of conidia and mycelial fragments, obtained from a 10-days-old colony of the strain CRSFA.753.22, was sprayed on the leaf surfaces at a distance of 20 cm. Five controls plants were inoculated with sterile distilled water. Plants were then covered with plastic bags and kept on the shelf in a growth chamber at 25° C, with a 12-h photoperiod. The first symptoms developed on leaves five days post-inoculation as irregular small brown spots, that were gradually expanded. Leaf tissues showed chlorosis which evolved into necrosis (Fig. 2A). Stagonosporopsis pogostemonis was consistently re-isolated from all diseased leaves, but not from control plants, thus fulfilling Koch's postulates. In Italy, Stagonosporopsis species have been reported as severe plant pathogens (Garibaldi et al. 2022; Guarnaccia et al. 2022), and this is the first report of the species S. pogostemonis in the country. References Dong, Z.Y., et al. 2021. Pathogens 10:1093. doi: 10.3390/pathogens10091093 Garibaldi, A., et al. 2022. Plant Pathol. J. 104:1157. doi: 10.1007/s42161-022-01138-7 Guarnaccia, V., et al. 2022. Plant Pathol. J. 104:1491. doi: 10.1007/s42161-022-01197-w.

Non-Targeted Spectranomics for the Early Detection of Xylella fastidiosa Infection in Asymptomatic Olive Trees, cv. Cellina di Nardò.

Olive quick decline syndrome (OQDS) is a disease that has been seriously affecting olive trees in southern Italy since around 2009. During the disease, caused by Xylella fastidiosa subsp. pauca sequence type ST53 (Xf), the flow of water and nutrients within the trees is significantly compromised. Initially, infected trees may not show any symptoms, making early detection challenging. In this study, young artificially infected plants of the susceptible cultivar Cellina di Nardò were grown in a controlled environment and co-inoculated with additional xylem-inhabiting fungi. Asymptomatic leaves of olive plants at an early stage of infection were collected and analyzed using nuclear magnetic resonance (NMR), hyperspectral reflectance (HSR), and chemometrics. The application of a spectranomic approach contributed to shedding light on the relationship between the presence of specific hydrosoluble metabolites and the optical properties of both asymptomatic Xf-infected and non-infected olive leaves. Significant correlations between wavebands located in the range of 530-560 nm and 1380-1470 nm, and the following metabolites were found to be indicative of Xf infection: malic acid, fructose, sucrose, oleuropein derivatives, and formic acid. This information is the key to the development of HSR-based sensors capable of early detection of Xf infections in olive trees.

First report of Ceratocystis ficicola causing wilt disease on common fig (Ficus carica) in Italy.

Common fig (Ficus carica L.) in is one of the most important crops in the Mediterranean area. In Italy, it is grown on a total area of 2118 ha. In Apulia (South-eastern Italy), the annual production of fig exceeds 3200 tons annually and together with olive and grapevine, they characterize the Apulian agricultural panorama. In September 2021, symptoms of a vascular wilt disease and, in severe cases, decline tree mortality were observed in Salento area (Apulia). Symptomatic Affected plants showed symptoms of leaf wilt and different stages of disease expression, which begins with leaf chlorosis on shoots, followed by wilting, extensive defoliation and twig dieback. On the main branches andlower part of the trunk and in some cases on lateral branches, bark cracks and cankers were observed and extended wood discoloration was detected in cross sections. In two orchards located in Salice Salentino (Site 1) and Squinzano (Site 2), where disease incidence exceeded 80%, 3-5 wood discs per tree were gathered from affected tissues from two (Site 1) and four (Site 2) trees. Isolations were performed on malt extract agar 2% amended with 0.5 g L-1 streptomycin sulfate. A Ceratocystis species was recovered from all samples trees with high frequencies (83.3%). Two-week-old colonies on potato dextrose agar showed black ascomata with 300-600 µm wide bases and 1100-2250 µm long necks. On the tips of the necks, ascospores (5-6x4-5 µm) exuded in creamy white sticky masses. Endoconidia (5-9x4.5-7 µm) were abundant, cylindrical, aseptate, and produced in chains. Two monoconidial representative strains CRSFA.Cer.033 (Site 1) and CRSFA.Cer.035 (Site 2) were deposited in the DISSPA Di.S.S.P.A. collection of the University of Bari. Species identification was done through sequence analyses of rDNA internal transcribed spacer region (ITS) using ITS5/ITS4 primers (White et al. 1990), elongation factor 1 alpha gene (TEF) using EF1/EF2 (O'Donnell et al. 1998) and RNA polymerase II gene (RPB2) gene using RPB2-5F/FRPB2fRPB2-7cR (Liu et al. 1999). Sequences were deposited in NCBI GenBank (accession numbers: OQ329983-OQ335969 (ITS), OQ352265-OQ352266 (TEF), OQ352268-OQ352267 (RPB2)). The sequences of both Apulian isolates were identical. BLAST searches revealed high similarity to the sequences of two isolates of Ceratocystis ficicola Kajitani and Masuya from Japan: ex-type CMW38543 and CMW38544, specifically 98.41% identity matching with KY685076 (ITS), 100% with KY685079 (TEF), and 99.87% with KY685083 and KY685082 (RPB2). Pathogenicity tests were conducted, on six common fig 'Dottato' seedlings by inoculating one year-old twigs with mycelium plugs (Bolboli et al. 2022). Control plants were inoculated with PDA plugs without mycelium. After one month, all inoculated twigs showed symptoms of wilt. Forty days post-inoculation, the bark of inoculated twigs was removed, and longitudinal and transverse sections revealed wood discolorations extending above and below the inoculation point. Ceratocystis ficicola was consistently reisolated from symptomatic tissues and identified as described above, thus fulfilling Koch's postulates. Neither symptoms nor positive isolations were observed in control seedlings. The pathogen was first described in 2011 in Japan (Kajitani and Masuya, 2011) where it currently affects all fig-producing areas. In 2018, it was reported for the first time in the EPPO region in Greece (Tsopelas et al., 2021), and to our knowledge, this is the first report on its detection in Italy. Since February 2022, C. ficicola has been included in the EPPO alert list because of its potential to cause tree mortality and the difficulty of its eradication. Regional national surveys are therefore urgently needed to determine its distribution in the fig growing areas of Italy and limit its spread. References Bolboli, Z., et al. 2022. Mycol. Prog. 21:89. doi: 10.1007/s11557-022-01834-9 Kajitani, Y., and Masuya, H., 2011. Mycoscience 52:349. doi: 10.1007/s10267-011-0116-5 Liu, Y. J., et al. 1999. Mol. Biol. and Evol. 16:1799. doi: 10.1093/oxfordjournals.molbev.a026092 O'Donnell, K., et al. 1998. Proc Natl Acad Sci USA 95:2044. doi: 10.1073/pnas.95.5.2044 Tsopelas, P., et al. 2021. Phytopathol. Mediterr. 60:337. doi: 10.36253/phyto-12794 White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego. doi: 10.1016/0307-4412(91)90165-5.

Susceptible and resistant olive cultivars show differential physiological response to Xylella fastidiosa infections.

Olive quick decline syndrome (OQDS) is a severe disease, first described in Italy in late 2013, caused by strains of Xylella fastidiosa subsp. pauca (Xfp) in susceptible olive cultivars. Conversely, resistant olive cultivars do not develop OQDS but present scattered branch dieback, which generally does not evolve to severe canopy decline. In the present study, we assessed the physiological responses of Xfp-infected olive trees of susceptible and resistant cultivars. Periodic measurements of stomatal conductance (gs) and stem water potential (Ψstem) were performed using a set of healthy and Xfp-infected plants of the susceptible "Cellina di Nardò" and resistant "Leccino" and "FS17" cultivars. Strong differences in Δgs and ΔΨstem among Xfp-infected trees of these cultivars were found, with higher values in Cellina di Nardò than in Leccino and FS17, while no differences were found among healthy plants of the different cultivars. Both resistant olive cultivars showed lower water stress upon Xfp infections, compared to the susceptible one, suggesting that measurements of gs and Ψstem may represent discriminating parameters to be exploited in screening programs of olive genotypes for resistance to X. fastidiosa.

Diversity of Colletotrichum Species Associated with Olive Anthracnose Worldwide.

Olive anthracnose caused by Colletotrichum species causes dramatic losses of fruit yield and oil quality worldwide. A total of 185 Colletotrichum isolates obtained from olives and other hosts showing anthracnose symptoms in Spain and other olive-growing countries over the world were characterized. Colony and conidial morphology, benomyl-sensitive, and casein-hydrolysis activity were recorded. Multilocus alignments of ITS, TUB2, ACT, CHS-1, HIS3, and/or GAPDH were conducted for their molecular identification. The pathogenicity of the most representative Colletotrichum species was tested to olive fruits and to other hosts, such as almonds, apples, oleander, sweet oranges, and strawberries. In general, the phenotypic characters recorded were not useful to identify all species, although they allowed the separation of some species or species complexes. ITS and TUB2 were enough to infer Colletotrichum species within C. acutatum and C. boninense complexes, whereas ITS, TUB2, ACT, CHS-1, HIS-3, and GADPH regions were necessary to discriminate within the C. gloesporioides complex. Twelve Colletotrichum species belonging to C. acutatum, C. boninense, and C. gloeosporioides complexes were identified, with C. godetiae being dominant in Spain, Italy, Greece, and Tunisia, C. nymphaeae in Portugal, and C. fioriniae in California. The highest diversity with eight Colletotrichum spp. was found in Australia. Significant differences in virulence to olives were observed between isolates depending on the Colletotrichum species and host origin. When other hosts were inoculated, most of the Colletotrichum isolates tested were pathogenic in all the hosts evaluated, except for C. siamense to apple and sweet orange fruits, and C. godetiae to oleander leaves.

Screening of Olive Biodiversity Defines Genotypes Potentially Resistant to Xylella fastidiosa.

The recent outbreak of the Olive Quick Decline Syndrome (OQDS), caused by Xylella fastidiosa subsp. pauca (Xf), is dramatically altering ecosystem services in the peninsula of Salento (Apulia Region, southeastern Italy). Here we report the accomplishment of several exploratory missions in the Salento area, resulting in the identification of thirty paucisymptomatic or asymptomatic plants in olive orchards severely affected by the OQDS. The genetic profiles of such putatively resistant plants (PRPs), assessed by a selection of ten simple sequence repeat (SSR) markers, were compared with those of 141 Mediterranean cultivars. Most (23) PRPs formed a genetic cluster (K1) with 22 Italian cultivars, including 'Leccino' and 'FS17', previously reported as resistant to Xf. The remaining PRPs displayed relatedness with genetically differentiated germplasm, including a cluster of Tunisian cultivars. Markedly lower colonization levels were observed in PRPs of the cluster K1 with respect to control plants. Field evaluation of four cultivars related to PRPs allowed the definition of partial resistance in the genotypes 'Frantoio' and 'Nocellara Messinese'. Some of the PRPs identified in this study might be exploited in cultivation, or as parental clones of breeding programs. In addition, our results indicate the possibility to characterize resistance to Xf in cultivars genetically related to PRPs.

Assessment of the Hyperspectral Data Analysis as a Tool to Diagnose Xylella fastidiosa in the Asymptomatic Leaves of Olive Plants.

Xylella fastidiosa is a bacterial pathogen affecting many plant species worldwide. Recently, the subspecies pauca (Xfp) has been reported as the causal agent of a devastating disease on olive trees in the Salento area (Apulia region, southeastern Italy), where centenarian and millenarian plants constitute a great agronomic, economic, and landscape trait, as well as an important cultural heritage. It is, therefore, important to develop diagnostic tools able to detect the disease early, even when infected plants are still asymptomatic, to reduce the infection risk for the surrounding plants. The reference analysis is the quantitative real time-Polymerase-Chain-Reaction (qPCR) of the bacterial DNA. The aim of this work was to assess whether the analysis of hyperspectral data, using different statistical methods, was able to select with sufficient accuracy, which plants to analyze with PCR, to save time and economic resources. The study area was selected in the Municipality of Oria (Brindisi). Partial Least Square Regression (PLSR) and Canonical Discriminant Analysis (CDA) indicated that the most important bands were those related to the chlorophyll function, water, lignin content, as can also be seen from the wilting symptoms in Xfp-infected plants. The confusion matrix of CDA showed an overall accuracy of 0.67, but with a better capability to discriminate the infected plants. Finally, an unsupervised classification, using only spectral data, was able to discriminate the infected plants at a very early stage of infection. Then, in phase of testing qPCR should be performed only on the plants predicted as infected from hyperspectral data, thus, saving time and financial resources.

A non-targeted metabolomics study on Xylella fastidiosa infected olive plants grown under controlled conditions.

In the last decade, the bacterial pathogen Xylella fastidiosa has devastated olive trees throughout Apulia region (Southern Italy) in the form of the disease called "Olive Quick Decline Syndrome" (OQDS). This study describes changes in the metabolic profile due to the infection by X. fastidiosa subsp. pauca ST53 in artificially inoculated young olive plants of the susceptible variety Cellina di Nardò. The test plants, grown in a thermo-conditioned greenhouse, were also co-inoculated with some xylem-inhabiting fungi known to largely occur in OQDS-affected trees, in order to partially reproduce field conditions in terms of biotic stress. The investigations were performed by combining NMR spectroscopy and MS spectrometry with a non-targeted approach for the analysis of leaf extracts. Statistical analysis revealed that Xylella-infected plants were characterized by higher amounts of malic acid, formic acid, mannitol, and sucrose than in Xylella-non-infected ones, whereas it revealed slightly lower amounts of oleuropein. Attention was paid to mannitol which may play a central role in sustaining the survival of the olive tree against bacterial infection. This study contributes to describe a set of metabolites playing a possible role as markers in the infections by X. fastidiosa in olive.

A geostatistical fusion approach using UAV data for probabilistic estimation of Xylella fastidiosa subsp. pauca infection in olive trees.

Xylella fastidiosa is one of the most destructive plant pathogenic bacteria worldwide, affecting more than 500 plant species. In Apulia region (southeastern Italy), X. fastidiosa subsp. pauca (Xfp) is responsible for a severe disease, the olive quick decline syndrome (OQDS), spreading epidemically and with dramatic impact on the agriculture, the landscape, the tourism, and the cultural heritage of this region. An early detection of the infected plants would hinder the rapid spread of the disease. The main objective of this paper was to define a geostatistical approach of data fusion, which combines remote (radiometric), and proximal (geophysical) sensor data and visual inspections with plant diagnostic tests, to provide probabilistic maps of Xfp infection risk. The study site was an olive grove located at Oria (province of Brindisi, Italy), where at the time of monitoring (September 2017) only few plants showed initial symptoms of the disease. The measurements included: 1) acquisitions of reflected electromagnetic radiation with UAV (Unmanned Aerial Vehicle) equipped with a multi-spectral camera; 2) geophysical surveys on the trunks of 49 plants with Ground Penetrating Radar (GPR); 3) disease severity rating, by visual inspection of the proportion of canopy with symptoms; 4) qPCR (real time-quantitative Polymerase Chain Reaction) data from tests on 61 plants. The data were submitted to a set of processing techniques to define a "data fusion" procedure, based on non-parametric multivariate geostatistics. The approach allowed marking those areas where the risk of infection was higher, and identifying the possible infection entry routes into the field. The probability map of infection risk could be used as an effective tool for a preventive action and for a better organization of the monitoring plans.

Identification of Arthrinium marii as Causal Agent of Olive Tree Dieback in Apulia (Southern Italy).

Olive (Olea europaea L. var. sativa) is one of the most economically important tree crops grown in the Mediterranean basin. Arthrinium Kunze ex Fr. (teleomorph: Apiospora Sacc.) is a widespread fungal genus, and Arthrinium marii Larrondo & Calvo is a ubiquitous species, found in algae, soil, plants, and agricultural communities. A. marii was isolated from olive trees showing dieback from orchards located in Andria and in Fasano, Brindisi (Apulia, southern Italy) and identified based on morphological features and molecular analysis of four genomic regions (ITS, TUB2, TEF1, and LSU). Two-year-old olive plants artificially inoculated with three representative A. marii isolates showed complete dieback within 6 months, and the fungus was reisolated, satisfying Koch's postulates. This is the first report of A. marii causing dieback on olive trees that could represent an important threat for olive cultivation.

Time-dependent effects of Pochonia chlamydosporia endophytism on gene expression profiles of colonized tomato roots.

A transcriptome analysis was produced from tomato roots inoculated with the hyphomycete Pochonia chlamydosporia at three different times. Gene expression data were also yielded from fungus grown in vitro or endophytic. A next-generation sequencing (NGS) and network analysis approach were applied. We identified 3.676 differentially expressed tomato genes (DEG), highlighting a core of 93 transcripts commonly down- or upregulated at every time point, shedding light on endophytism process. Functional categories related to plant information-processing system, which recognizes, percepts, and transmits signals, were associated with gene upregulated early in time, with higher representations in processes such as plant defense regulation later in time. Network analysis of a DEG subset showed dominance of MAP kinase hubs in the uninoculated control samples, replaced by an increased centrality of WRKY transcription factor and ETR-ethylene response factor genes in the colonized roots. Fungus genes expressed during progression of plant colonization, therefore related to the host colonization process or endophytism persistence, were also identified. Data provided a high-resolution insight on tomato transcriptome changes as induced by endophytism, highlighting a specific modulation of stress-responsive transcripts, related to a selective activation of defense pathways, likely required by the fungus to establish a persistent endophytic lifestyle.

Genetic Diversity of Verticillium dahliae Populations From Olive and Potato in Lebanon.

Verticillium dahliae is widely distributed in potato and olive fields in Lebanon, causing serious economic losses. However, little is known about the inoculum source, population structure, and genetic diversity of the pathogen or the mechanisms of dissemination within Lebanon. To understand the population structure, a total of 203 isolates sampled from olive (n = 78) and potato (n = 125) were characterized for species, mating type, and race, and the genetic relationships were delineated using 13 microsatellite markers. All isolates except one from potato were V. dahliae, with 55.1 and 12.1% race 1, and 43.6 and 83.1% race 2 in olive and potato, respectively. The genetic structure of the studied population was best described by two large and two small clusters. Membership in the two large clusters was determined by the presence or absence of the effector gene Ave1. Furthermore, genetic structure was moderately associated with the host of origin but was weakly associated with the geographic origin. All but four isolates represented by three multilocus haploid genotypes were MAT1-2. This study identified a clear lack of gene flow between virulence genotypes of V. dahliae despite the proximity of these cropping systems and the wide distribution of genetic diversity among hosts and geographic regions in Lebanon.

Molecular characterisation of a novel gemycircularvirus associated with olive trees in Italy.

A new gemycircularvirus sequence was obtained from total DNAs extracted from olive leaves and twigs tissues. Its complete genome consists of a single-stranded circular DNA of 2,145 nt, whose sequence was obtained by rolling circle amplification (RCA). Genome analysis identified three open reading frames, sharing homologies with the coat and replication-associated proteins, these latter in the anti-parallel strand, of known gemycircularvirus species. Search for homologies showed that the circular ssDNA sequence is distantly related to other gemycircularviruses thus originating from a new virus species, for which the name olive-associated gemycircularvirus 1 (OaGV1) is proposed. A survey in two different olive-growing areas of the Apulian region (Southern Italy), showed a limited distribution of OaGV1.

Long-Distance Spread of Verticillium dahliae Through Rivers and Irrigation Systems.

Verticillium dahliae Kleb. is a soilborne pathogen causing Verticillium wilt disease on several hosts. The pathogen survival structure (i.e., microsclerotia) can be efficiently spread by different dispersal methods. In the present study, the medium to long dispersal spread of the pathogen through rivers and irrigation canals was investigated. Samples of sediments (n = 29) were gathered from eight Lebanese rivers and three regional irrigation canals, in addition to samples of soil particles and plant residues (n = 14) from irrigation filters in commercial orchards. Specific conventional and real-time nested polymerase chain reaction assays detected the pathogen in six rivers-Al Kabir, Al Bared, Litani, Al Awali, Ostwan, and Litani South-and in all sampled canals-Ostwan, Al Bared, and Litani Canal 900. Starting DNA quantities ranged from 0.2 pg to 21.318 ng and an inoculum density, determined by a traditional plating method, varied between nondetectable and 0.2 microsclerotia/g. Viable V. dahliae microsclerotia were also found in residues collected from mesh-type irrigation filters of five commercial orchards. This study confirms that water is an important inoculum source of V. dahliae, being involved in the efficient spread of microsclerotia in Lebanese agricultural areas.

Identification of tomato miRNAs responsive to root colonization by endophytic Pochonia chlamydosporia.

The molecular mechanisms active during the endophytic phase of the fungus Pochonia chlamydosporia are still poorly understood. In particular, few data are available on the links between the endophyte and the root response, as modulated by noncoding small RNAs. In this study, we describe the microRNAs (miRNAs) that are differentially expressed (DE) in the roots of tomato, colonized by P. chlamydosporia. A genome-wide NGS expression profiling of small RNAs in roots, either colonized or not by the fungus, showed 26 miRNAs upregulated in inoculated roots. Their predicted target genes are involved in the plant information processing system, which recognizes, percepts, and transmits signals, with higher representations in processes such as apoptosis and plant defense regulation. RNAseq data showed that predicted miRNA target genes were downregulated in tomato roots after 4, 7, 10, and 21 days post P. chlamydosporia inoculation. The differential expression of four miRNAs was further validated using qPCR analysis. The P. chlamydosporia endophytic lifestyle in tomato roots included an intricate network of miRNAs and targets. Data provide a first platform of DE tomato miRNAs after P. chlamydosporia colonization. They indicated that several miRNAs are involved in the host response to the fungus, playing important roles for its recognition as a symbiotic microorganism, allowing endophytism by modulating the host defense reaction. Data also indicated that endophytism affects tRNA fragmentation. This is the first study on miRNAs induced by P. chlamydosporia endophytism and related development regulation effects in Solanum lycopersicum.

Globally invading populations of the fungal plant pathogen Verticillium dahliae are dominated by multiple divergent lineages.

The spread of aggressive fungal pathogens into previously non-endemic regions is a major threat to plant health and food security. Analyses of the spatial and genetic structure of plant pathogens offer valuable insights into their origin, dispersal mechanisms and evolution, and have been useful to develop successful disease management strategies. Here, we elucidated the genetic diversity, population structure and demographic history of worldwide invasion of the ascomycete Verticillium dahliae, a soil-borne pathogen, using a global collection of 1100 isolates from multiple plant hosts and countries. Seven well-differentiated genetic clusters were revealed through discriminant analysis of principal components (DAPC), but no strong associations between these clusters and host/geographic origin of isolates were found. Analyses of clonal evolutionary relationships among multilocus genotypes with the eBURST algorithm and analyses of genetic distances revealed that genetic clusters represented several ancient evolutionary lineages with broad geographic distribution and wide host range. Comparison of different scenarios of demographic history using approximate Bayesian computations revealed the branching order among the different genetic clusters and lineages. The different lineages may represent incipient species, and this raises questions with respect to their evolutionary origin and the factors allowing their maintenance in the same areas and same hosts without evidence of admixture between them. Based on the above findings and the biology of V. dahliae, we conclude that anthropogenic movement has played an important role in spreading V. dahliae lineages. Our findings have implications for the development of management strategies such as quarantine measures and crop resistance breeding.

Gene silencing and gene expression in phytopathogenic fungi using a plant virus vector.

RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.

Genetic responses induced in olive roots upon colonization by the biocontrol endophytic bacterium Pseudomonas fluorescens PICF7.

Knowledge on the genetic basis underlying interactions between beneficial bacteria and woody plants is still very limited, and totally absent in the case of olive. We aimed to elucidate genetic responses taking place during the colonization of olive roots by the native endophyte Pseudomonas fluorescens PICF7, an effective biocontrol agent against Verticillium wilt of olive. Roots of olive plants grown under non-gnotobiotic conditions were collected at different time points after PICF7 inoculation. A Suppression Subtractive Hybridization cDNA library enriched in induced genes was generated. Quantitative real time PCR (qRT-PCR) analysis validated the induction of selected olive genes. Computational analysis of 445 olive ESTs showed that plant defence and response to different stresses represented nearly 45% of genes induced in PICF7-colonized olive roots. Moreover, quantitative real-time PCR (qRT-PCR) analysis confirmed induction of lipoxygenase, phenylpropanoid, terpenoids and plant hormones biosynthesis transcripts. Different classes of transcription factors (i.e., bHLH, WRKYs, GRAS1) were also induced. This work highlights for the first time the ability of an endophytic Pseudomonas spp. strain to mount a wide array of defence responses in an economically-relevant woody crop such as olive, helping to explain its biocontrol activity.

Growth responses of crop and weed species to heavy metals in pot and field experiments.

Greenhouse and field studies were performed to examine the growth responses and possible phytoremediation capacity towards heavy metals of several Brassicaceae (Brassica alba, Brassica carinata, Brassica napus and Brassica nigra) and Poaceae (durum wheat and barley). Soils used featured total concentrations of Cr, Cu, Pb and Zn largely exceeding the maximum levels permitted by the Italian laws. Different organic amendments were tested such as a compost and the plant growth-promoting rhizobacterium Bacillus licheniformis. In the greenhouse experiment, plant length, leaf area index and shoots dry matter were evaluated periodically for the Brassicaceae examined. Whereas plant length, grains production, weight of 1,000 seeds, ear fertility and tiller density were determined under field conditions at the end of the crop cycle for wheat and barley. In general, the species tested appeared to be tolerant to high heavy metal concentrations in soil, and slightly significant differences were found for all parameters considered. A marked growth increase was shown to occur for Brassicaceae cultivated on compost- and bacillus-amended contaminated soils, with respect to non-amended contaminated soils. With some exception, higher growth parameters were measured for wheat and barley plants cropped from contaminated soils in comparison to non-contaminated soils. Further, bacillus amendment enhanced the length of wheat and barley plants in both non-contaminated and contaminated soils, while different effects were observed for the other parameters evaluated.

Long-term fungal inhibitory activity of water-soluble extracts of Phaseolus vulgaris cv. Pinto and sourdough lactic acid bacteria during bread storage.

The antifungal activity of proteinaceous compounds from different food matrices was investigated. In initial experiments, water-soluble extracts of wheat sourdoughs, cheeses, and vegetables were screened by agar diffusion assays with Penicillium roqueforti DPPMAF1 as the indicator fungus. Water-soluble extracts of sourdough fermented with Lactobacillus brevis AM7 and Phaseolus vulgaris cv. Pinto were selected for further study. The crude water-soluble extracts of L. brevis AM7 sourdough and P. vulgaris cv. Pinto had a MIC of 40 mg of peptide/ml and 30.9 mg of protein/ml, respectively. MICs were markedly lower when chemically synthesized peptides or partially purified protein fractions were used. The water-soluble extract of P. vulgaris cv. Pinto showed inhibition toward a large number of fungal species isolated from bakeries. Phaseolin alpha-type precursor, phaseolin, and erythroagglutinating phytohemagglutinin precursor were identified in the water-soluble extract of P. vulgaris cv. Pinto by nano liquid chromatography-electrospray ionization-tandem mass spectrometry. When the antifungal activity was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all three proteins were inhibitory. A mixture of eight peptides was identified from the water-soluble extract of sourdough L. brevis AM7, and five of these exhibited inhibitory activity. Bread was made at the pilot plant scale by sourdough fermentation with L. brevis AM7 and addition of the water-soluble extract (27%, vol/wt; 5 mg of protein/ml) of P. vulgaris cv. Pinto. Slices of bread packed in polyethylene bags did not show contamination by fungi until at least 21 days of storage at room temperature, a level of protection comparable to that afforded by 0.3% (wt/wt) calcium propionate.