The widespread emergence of Phaeomoniella chlamydospora and Phaeoacremonium minimum in grapevine nurseries over the past two decades has raised concerns about the future sustainability of the viticulture sector. Managing these pathogens is challenging due to the lack of chemicals and the limited effectiveness of biocontrol agents. This study aimed to identify cultivation practices in nurseries to mitigate infection risks in propagation material. By coupling nested PCR with quantitative PCR, pathogen distribution and concentration of prevailing P. chlamydospora along the length of rootstock canes were investigated. Detection frequency of both pathogens decreased from the basal end to tip sections. Therefore, using tip sections highlights the potential of producing pathogen-free propagation material and retaining healthier rootstock mother fields. P. chlamydospora biomass was reduced by 92.5% from the base to the tip in canes harvested in 2020, and by 100% in 2021. Furthermore, the occurrence of both pathogens and the biomass of P. chlamydospora were found to be overall lower in the rootstock mother field after the elimination of esca-affected vines. The incidence of P. chlamydospora at the basal end was reduced by 90.6% (2020-2022), while of P. minimum by 23.0%. Likewise, P. chlamydospora biomass was reduced (2020-2021), ranging from 98.5% at the base to 100% at the tip. These findings emphasise targeted harvesting, tip utilisation, and elimination of esca-affected mother vines as effective strategies for minimising pathogen infections in grapevine nurseries. Integrating these cultivation practices into the existing Petri disease management protocols could enhance the production of high-quality propagation material.

A comprehensive study was conducted on the prevalence of grapevine trunk diseases in Greece, focusing specifically on contamination of grapevine propagation material by Phaeomoniella chlamydospora and black foot disease-related species. Additionally, detection of esca pathogen Fomitiporia mediterranea, causing white rot in grapevine and other woody hosts, was assessed using a new PCR-based assay that distinguishes F. mediterranea from F. punctata. Development of a nested PCR assay, combined with a cost-effective DNA extraction protocol, revealed a high percentage of infection by P. chlamydospora (51%) and Ilyonectria species associated with black foot disease (28%) in different types of grapevine propagation material (dormant cuttings, field-rooted benchgrafts and green-growing plants). Interestingly, black foot disease-related pathogens were more prevalent in nursery plants grown in the field for 6 months (57%) than in other types of propagation material and compared with P. chlamydospora (43%), indicating increased infection of propagation material during growth in the nursery field. The cost-effective molecular method developed in this study could be used in mass inspections of propagation material for phytosanitary purposes. Finally, using primers specific for F. mediterranea and F. puncata developed in this study, combined with the universal ITS4 primer, a collection of Fomitiporia isolates from mature grapevine and other woody hosts from Greece and Italy (southern Europe) were characterized as F. mediterranea, whereas German and Swedish isolates from forest plants (central Europe) were classified as F. punctata. The developed primers can discriminate between the two Fomitiporia species, which are indistinguishable based on culture and morphological characteristics alone.

Verticillium dahliae is a xylem-invading fungal pathogen that causes vascular wilt in a wide range of angiosperms. The pathogen uses a variety of virulence factors to invade and colonize its hosts. Here, we report that VdNEP, an NLP (Necrosis and ethylene inducing peptide 1-Like Protein), functions as one such factor in multiple hosts. Eggplant leaves treated with VdNEP developed necrotic symptoms. Overexpression of VdNEP by incorporating extra copies of the VdNEP gene increased virulence to cotton, eggplant and tomato plants, suggesting its role as a virulence factor in diverse plants. Increased expression of VdNEP among the transformants did not correlate with the number of VdNEP inserts, suggesting that its expression was affected by the genomic context of the insertion sites. Interestingly, a transformant derived from a defoliating strain with high VdNEP transcript levels caused disease symptoms in tomato plants, whereas the corresponding wild-type strain did not cause visible symptoms. The amount of V. dahliae DNA in plants infected with this VdNEP-overexpressing transformant was 22 times higher than that in plants infected with the wild-type isolate, further supporting the critical role of VdNEP in infection. A VdNEP-EGFP fusion was constructed to follow its localization in fungal cells and during infection.

Verticilium dahliae is the most important wilt pathogen of olive trees with a broad host range causing devastating diseases currently without any effective chemical control. Traditional detection methodologies are based on symptoms-observation or lab-detection using time consuming culturing or molecular techniques. Therefore, there is an increasing need for portable tools that can detect rapidly V. dahliae in the field. In this work, we report the development of a novel method for the rapid, reliable and on-site detection of V. dahliae using a newly designed isothermal LAMP assay and crude extracts of olive wood. For the detection of the fungus, LAMP primers were designed targeting the internal transcribed spacer (ITS) region of the rRNA gene. The above assay was combined with a purpose-built prototype portable device which allowed real time quantitative colorimetric detection of V. dahliae in 35 min. The limit of detection of our assay was found to be 0.8 fg/μl reaction and the specificity 100

A three-year survey was conducted to estimate the incidence of grapevine trunk diseases (GTDs) in Greece and identify fungi associated with the disease complex. In total, 310 vineyards in different geographical regions in northern, central, and southern Greece were surveyed, and 533 fungal strains were isolated from diseased vines. Morphological, physiological and molecular (5.8S rRNA gene-ITS sequencing) analyses revealed that isolates belonged to 35 distinct fungal genera, including well-known (e.g., Botryosphaeria sp., Diaporthe spp., Eutypa sp., Diplodia sp., Fomitiporia sp., Phaeoacremonium spp., Phaeomoniella sp.) and lesser-known (e.g., Neosetophoma sp., Seimatosporium sp., Didymosphaeria sp., Kalmusia sp.) grapevine wood inhabitants. The GTDs-inducing population structure differed significantly among the discrete geographical zones. Phaeomoniella chlamydospora (26.62%, n = 70), Diaporthe spp. (18.25%, n = 48) and F. mediterranea (10.27%, n = 27) were the most prevalent in Heraklion, whereas D. seriata, Alternaria spp., P. chlamydospora and Fusarium spp. were predominant in Nemea (central Greece). In Amyntaio and Kavala (northern Greece), D. seriata was the most frequently isolated species (>50% frequency). Multi-genes (rDNA-ITS, LSU, tef1-α, tub2, act) sequencing of selected isolates, followed by pathogenicity tests, revealed that Neosetophoma italica, Seimatosporium vitis, Didymosphaeria variabile and Kalmusia variispora caused wood infection, with the former being the most virulent. To the best of our knowledge, this is the first report of N. italica associated with GTDs worldwide. This is also the first record of K. variispora, S. vitis and D. variabile associated with wood infection of grapevine in Greece. The potential associations of disease indices with vine age, cultivar, GTD-associated population structure and the prevailing meteorological conditions in different viticultural zones in Greece are presented and discussed.

Botryosphaeriaceous fungi are widespread, and cause serious diseases in many economically important crops. Botryosphaeria dothidea, Neofusicoccum mediterraneum and N. parvum are the most important members of this family in the Mediterranean region. These fungi are frequently isolated from the same host, which together with their extensive and increasing host range necessitates development of rapid and reliable diagnostic tools. Species boundaries within the Botryosphaeriaceae have been defined based on phylogenetic analyses of multiple gene sequences, including those of mating type genes. The MAT1-2-1 gene displayed high sequence variability between Botryosphaeriaceous species, so was selected as the target for development of a definitive diagnostic tool. This paper outlines a new and robust molecular tool, composed of three TaqMan assays based on polymorphisms located in the MAT1-2-1 gene of B. dothidea, N. mediterraneum and N. parvum. Each assay differentiated the target species from other Botryosphaeriaceae, and from non-target fungi.

In a previous study, tobacco plants, transformed with a sense construct of the 57K domain of the replicase gene of tobacco rattle virus (TRV), provided resistance against genetically distant isolates of the virus. In this work, 57K-specific siRNAs were detected with RT-qPCR solely in the resistant line verifying the RNA-silencing base of the resistance. The integration sites of the transgene into the plant genome were identified with inverse-PCR. Moreover, the resistance against TRV was practically unaffected by low temperature conditions and the presence of heterologous viruses. The mechanism of the resistance was further examined by a gene expression analysis that showed increased transcript levels of genes with a key-role in the RNA silencing pathway and the basal antiviral defence. This work provides a comprehensive characterization of the robust virus resistance obtained by a sense transgene and underlines the usefulness of transgenic plants obtained by such a strategy.

Verticillium dahliae is a soilborne fungus that causes Verticillium wilt disease in a plethora of crops. Based on symptoms that develop on cotton, olive and okra, V. dahliae isolates are categorized into two pathotypes, namely defoliating and nondefoliating, with the former showing increased virulence and causing severe defoliation. Reliable differentiation between V. dahliae pathotypes is crucial for the management of Verticillium wilt in cotton and olive. In the present study, a polymorphism was detected among isolates of defoliating and nondefoliating pathotypes in Southern blots using the VdNEP gene as a probe. The regions flanking this gene were isolated by inverse PCR and sequence differences in the 3 ' untranslated region (3 '-UTR) of the VdNEP gene were detected between the two pathotypes. Based on these sequences, primers were designed and assessed to develop a multiplex PCR detection assay. Using this assay, a collection of cotton and olive V. dahliae isolates from Greece and Cyprus was screened, revealing that the defoliating pathotype is present in several regional units of Greece. Thus, this work presents a new, sensitive molecular marker for the differentiation between V. dahliae pathotypes based on the VdNEP gene. Because the 3 '-UTR is involved in the phenotypes displayed by the pathotypes, an expression experiment was conducted under conditions simulating the xylem of a host plant. Expression of the VdNEP gene was elevated at all time points in the defoliating compared to the nondefoliating strain, suggesting a possible involvement of VdNEP expression in the defoliation process.

Abstract Background Grapevine trunk diseases (GTDs) is a disease complex caused by wood pathogenic fungi belonging to genera like Phaeomoniella, Phaeoacremonium, Fomitiporia, Eutypa and members of the family Botryosphaeriaceae. However, the co-occurrence of these fungi in symptomatic and asymptomatic vines at equivalent abundances has questioned their role in GTDs. Hence, we still lack a good understanding of the fungi involved in GTDs, their interactions and the factors controlling their assemblage in vines. We determined the fungal and bacterial microbiome in wood tissues of asymptomatic and symptomatic vines of three main Greek cultivars (Agiorgitiko, Xinomavro, Vidiano), each cultivated in geographically distinct viticultural zones, using amplicon sequencing. Results We noted that cultivar/biogeography (lumped factor) was the strongest determinant of the wood fungal microbiome (p < 0.001, 22.7%), while GTD symptoms condition had a weaker but still significant effect (p < 0.001, 3.5%), being prominent only in the cultivar Xinomavro. Several fungal Amplicon Sequence Variants (ASVs), reported as GTD-associated pathogens like Kalmusia variispora, Fomitiporia spp., and Phaemoniella chlamydosporα (most dominant in our study), were positively correlated with symptomatic vines in a cultivar/viticultural zone dependent manner. Random Forest analysis pointed to P. chlamydosporα, K. variispora, A. alternata and Cladosporium sp., as highly accurate predictors of symptomatic vines (0% error rate). The wood bacterial microbiome showed similar patterns, with biogeography/cultivar being the main determinant (p < 0.001, 25.5%) of its composition, followed by the GTD status of vines (p < 0.001, 5.2%). Differential abundance analysis revealed a universal positive correlation (p < 0.001) of Bacillus and Streptomyces ASVs with asymptomatic vines. Network analysis identified a significant negative co-occurrence network between these bacterial genera and Phaemoniella, Phaeoacrominum and Seimatosporium. These results point to a plant beneficial interaction between Bacillus/Streptomyces and GTD pathogens. Conclusions Our study (a) provides evidence that GTD symptomatic plants support a wood fungal microbiome, showing cultivar and biogeography-dependent patterns, that could be used as a proxy to distinguish between healthy and diseased vines, (b) points to strong interactions between the bacterial and fungal wood microbiome in asymptomatic vines that should be further pursued in the quest for discovery of novel biocontrol agents.

Summary Fungi belonging to the Botryosphaeriaceae family are widespread pathogens of many angiosperms, causing disease on various high value crops. The most important members of the family for the Greek region and other Mediterranean countries are Botryosphaeria dothidea, Neofusicoccum hellenicum, Neofusicoccum mediterraneum and Neofusicoccum parvum. The frequently concurrent isolation of Botryosphaeriaceae species from the same host, as well as the extensive host range of B. dothidea, necessitate the development of rapid and reliable detection methods. This study presents a new and robust molecular diagnostic tool, in the form of a PCR method based on primers designed on an SNP (single nucleotide polymorphism) located in the ITS region (Internal Transcribed Region) of B. dothidea and Neofusicoccum species. SNP primers constructed with or without added mismatch nucleotides were combined with the same upstream universal primer to generate distinct amplicons. When evaluated in PCR assays, mismatched primers were found to have the highest differentiation capability. The potential for further development of SNP assays in order to differentiate between species is being evaluated.

Bacterial biological control agents (BCAs) have been increasingly used against plant diseases. The traditional approach to manufacturing such commercial products was based on the selection of bacterial species able to produce secondary metabolites that inhibit mainly fungal growth in optimal media. Such species are required to be massively produced and sustain long-term self-storage. The endpoint of this pipeline is large-scale field tests in which BCAs are handled as any other pesticide. Despite recent knowledge of the importance of BCA-host-microbiome interactions to trigger plant defenses and allow colonization, holistic approaches to maximize their potential are still in their infancy. There is a gap in scientific knowledge between experiments in controlled conditions for optimal BCA and pathogen growth and the nutrient-limited field conditions in which they face niche microbiota competition. Moreover, BCAs are considered to be safe by competent authorities and the public, with no side effects to the environment; the OneHealth impact of their application is understudied. This review summarizes the state of the art in BCA research and how current knowledge and new biotechnological tools have impacted BCA development and application. Future challenges, such as their combinational use and ability to ameliorate plant stress are also discussed. Addressing such challenges would establish their long-term use as centerfold agricultural pesticides and plant growth promoters.

Bacillus amyloliquefaciens is considered the most successful biological control agent due to its ability to colonize the plant rhizosphere and phyllosphere where it outgrows plant pathogens by competition, antibiosis, and inducing plant defense. Its antimicrobial function is thought to depend on a diverse spectrum of secondary metabolites, including peptides, cyclic lipopeptides, and polyketides, which have been shown to target mostly fungal pathogens. In this study, we isolated and characterized the catecholate siderophore bacillibactin by B. amyloliquefaciens MBI600 under iron-limiting conditions and we further identified its potential antibiotic activity against plant pathogens. Our data show that bacillibactin production restrained in vitro and in planta growth of the nonsusceptible (to MBI600) pathogen Pseudomonas syringae pv. tomato. Notably, it was also related to increased antifungal activity of MBI600. In addition to bacillibactin biosynthesis, iron starvation led to upregulation of specific genes involved in microbial fitness and competition. IMPORTANCE Siderophores have mostly been studied concerning their contribution to the fitness and virulence of bacterial pathogens. In the present work, we isolated and characterized for the first time the siderophore bacillibactin from a commercial bacterial biocontrol agent. We proved that its presence in the culture broth has significant biocontrol activity against nonsusceptible bacterial and fungal phytopathogens. In addition, we suggest that its activity is due to a new mechanism of action, that of direct antibiosis, rather than by competition through iron scavenging. Furthermore, we showed that bacillibactin biosynthesis is coregulated with the transcription of antimicrobial metabolite synthases and fitness regulatory genes that maximize competition capability. Finally, this work highlights that the efficiency and range of existing bacterial biocontrol agents can be improved and broadened via the rational modification of the growth conditions of biocontrol organisms.

In plant pathogenic fungi, different signalling pathways operate to control responses to nutrient availability during plant infection. A candidate from the cAMP-PKA signalling pathway, the cAMP-dependent protein kinase A gene, pkaC1, and the beta-1,6-endoglucanase gene, vegB, involved in cell wall degradation, were studied in V. dahliae. Double mutants of the fungus were constructed, with insertional inactivation in the pkaC1 and vegB genes. Different developmental traits and virulence towards eggplant were evaluated in single and double disruption mutants. In all media tested, double mutants showed better radial growth but less conidia and microsclerotia than the wild type. An interaction between vegB and pkaC1 in controlling virulence on eggplants was recorded, as double mutants were slightly less virulent than the single mutant vegB(-), but more virulent than the single mutant pkaC1(-). Concomitant or independent function of the two genes and the signaling pathways they operate in for the different growth parameters and virulence are discussed.

It has been suggested that some microorganisms, including plant growth-promoting rhizobacteria, manipulate the level of ethylene in plants by degrading 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene precursor, into α-ketobutyrate and ammonia, using ACC deaminase (ACCd). Here, we investigated whether ACCd of Verticillium dahliae, a soil-borne fungal pathogen of many important crops, is involved in causing vascular wilt disease. Overexpression of the V. dahliae gene encoding this enzyme, labeled as ACCd, significantly increased virulence in both tomato and eggplant, while disruption of ACCd reduced virulence. Both types of mutant produced more ethylene than a wild-type (70V-WT) strain, although they significantly differed in ACC content. Overexpression strains lowered ACC levels in the roots of infected plants, while the amount of ACC in the roots of plants infected with deletion mutants increased. To test the hypothesis that ACC acts as a signal for controlling defense, roots of WT and Never-ripe (Nr) tomato plants were treated with ACC before V. dahliae inoculation. Plants pretreated with ACC displayed less severe symptoms than untreated controls. Collectively, our results suggest a novel role of ACC as a regulator of both plant defense and pathogen virulence.

HomePlant DiseaseVol. 101, No. 6First Report of Phytophthora palmivora Causing Fruit Rot on Pomegranate in Greece PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Phytophthora palmivora Causing Fruit Rot on Pomegranate in GreeceE. A. Markakis, A. K. Tzima, S. C. Palavouzis, P. P. Antoniou, E. J. Paplomatas, and E. C. TjamosE. A. Markakis, A. K. Tzima, S. C. Palavouzis, P. P. Antoniou, E. J. Paplomatas, and E. C. TjamosAffiliationsAuthors and Affiliations E. A. Markakis A. K. Tzima S. C. Palavouzis P. P. Antoniou E. J. Paplomatas E. C. Tjamos , Laboratory of Plant Pathology, Agricultural University of Athens, Iera Odos 75, Votanikos 11855, Athens, Greece. Published Online:31 Mar 2017https://doi.org/10.1094/PDIS-11-16-1691-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Pomegranate (Punica granatum) is a dynamically increasing alternative crop for Greece grown on more than 2,000 ha. In September 2016, a severe fruit rot disease of pomegranate cv. Wonderful was observed in fields of Lamia, Fthiotida, Greece. Symptoms appeared after heavy early-autumn rainfalls, on fruits that were mainly at the lower part of the trees. Initially, small, circular, light brown, water-soaked lesions covered by white Phytophthora-like spores appeared on the fruit surface. The lesions rapidly enlarged superficially and internally, causing partial or entire fruit rot, whereas no other part of the tree was affected. Disease incidence in orchards was estimated at 10 to 30%. A Phytophthora sp. was consistently and readily isolated from the edges of symptomatic fleshy mesocarp tissue, previously surface-sterilized with 95% ethanol, on potato dextrose agar (PDA). To obtain single hyphal isolates, hyphal tips were transferred into new PDA and the growth rate of the oomycete was 9.0 mm/day at 25°C in the dark. Microscopic observations revealed papillated, ovoid and ellipsoid sporangia measuring 27.6 to 72.0 × 23.0 to 36.8 μm (avg. 47.7 × 30.4 μm) with short pedicels (1.0 to 9.0 μm, avg. 3.4 μm) developing on sympodial sporangiophores after 10 days of growth on PDA. Terminal or intercalary chlamydospores were spherical, thick-walled, ranging from 18.5 to 45.9 μm (avg. 32.7 μm) in diameter. DNA from a representative single-hyphae isolate (code PH5ROD) was extracted and the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) was amplified using the universal primers ITS5 and ITS4 (White et al. 1990). The PCR product was sequenced and deposited in GenBank (accession no. KY242491). On the basis of morphological characteristics (Erwin and Ribeiro 1996) and a BLAST search with 100% identity to published ITS sequences of P. palmivora isolates in GenBank (KT148926, KT148928), the oomycete was identified as P. palmivora. For pathogenicity tests, 20 pomegranate fruits cv. Wonderful were artificially inoculated with the isolate PH5ROD by removing a 4.0 × 2.0 mm disc of the leathery fruit exocarp, inserting a 4.0 mm-diameter mycelial plug of a 10-day-old PDA culture, and covering the hole with the detached exocarp disc. Fruits were sprayed with sterilized distilled water, enclosed in plastic bags, and kept at 25°C with a 12-h photoperiod. Control fruits were inoculated with sterilized PDA plugs. Five days post inoculation, all inoculated fruits exhibited symptoms similar to those observed in orchards, whereas P. palmivora was consistently reisolated, thus confirming Koch's postulates. Neither symptoms nor positive isolations were observed in control plants. Pathogenicity tests were repeated twice. P. palmivora has been reported as the causal agent of pomegranate fruit rot in India (Erwin and Ribeiro 1996) and crown and root rot on pomegranate trees in Turkey (Türkölmez et al. 2016). This is the first report of P. palmivora causing fruit rot on pomegranate in Greece. This disease could result in destructive epidemics after severe rainfalls and cause heavy losses to pomegranate production. Therefore, effective management practices should be investigated and applied.References:Erwin, D. C., and Ribeiro, O. K. 1996. Phytophthora Diseases Worldwide. APS Press, St. Paul, MN. Google ScholarTürkölmez, Ş., et al. 2016. Plant Dis. 100:227. https://doi.org/10.1094/PDIS-04-15-0396-PDN Link, ISI, Google ScholarWhite, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego. Crossref, Google ScholarDetailsFiguresLiterature CitedRelated Vol. 101, No. 6 June 2017SubscribeISSN:0191-2917e-ISSN:1943-7692 Metrics Article History Issue Date: 17 May 2017Published: 31 Mar 2017First Look: 27 Feb 2017Accepted: 20 Feb 2017 Pages: 1060-1060 Information© 2017 The American Phytopathological SocietyCited byPhytophthora palmivora (coconut budrot)CABI Compendium, Vol. CABI CompendiumMorphological and molecular characterization of Phytophthora species associated with root and crown rot of pomegranate in Iran5 December 2020 | Plant Pathology, Vol. 70, No. 3Phytophthora Species Causing Root and Collar Rot of Pomegranate in Turkey29 May 2020 | European Journal of Plant Pathology, Vol. 157, No. 3Phytophthora oleae , a new root pathogen of wild olives12 April 2019 | Plant Pathology, Vol. 68, No. 5

Quinone outside inhibitors (QoI) are powerful fungicides, which have been reported, additionally to their fungicide activity, to increase plant capacity to activate cellular defense responses and to promote plant growth. In this work, the effect of the QoI class fungicide pyraclostrobin was examined against Cucumber mosaic virus (CMV), Potato virus Y (PVY) and Pseudomonas syringae pv. tomato in tomato plants following artificial inoculation of the plants with the pathogens. Under controlled environmental conditions, pyraclostrobin delayed viral and bacterial disease development, even if P. syringae pv. tomato internal population levels were not affected significantly. In contrast, under field conditions in commercial greenhouses, a reduced CMV disease incidence throughout the tomato cultivation period was recorded. Gene expression analysis indicated an effect of pyraclostrobin application on tomato MAPKs transcript levels and a possible interference with plant stress responses.

In spite of their widespread occurrence, only few host jumps by plant viruses have been evidenced and the molecular bases of even fewer have been determined. A combination of three independent approaches, 1) experimental evolution followed by reverse genetics analysis, 2) positive selection analysis, and 3) locus-by-locus analysis of molecular variance (AMOVA) allowed reconstructing the Potato virus Y (PVY; genus Potyvirus, family Potyviridae) jump to pepper (Capsicum annuum), probably from other solanaceous plants. Synthetic chimeras between infectious cDNA clones of two PVY isolates with contrasted levels of adaptation to C. annuum showed that the P3 and, to a lower extent, the CI cistron played important roles in infectivity toward C. annuum. The three analytical approaches pinpointed a single nonsynonymous substitution in the P3 and P3N-PIPO cistrons that evolved several times independently and conferred adaptation to C. annuum. In addition to increasing our knowledge of host jumps in plant viruses, this study illustrates also the efficiency of locus-by-locus AMOVA and combined approaches to identify adaptive mutations in the genome of RNA viruses.

Summary The fungus Ceratocystis platani was detected in various localities of the Gjirokastër prefecture in southern Albania, where it was causing widespread mortality on Platanus orientalis trees. The identification of the fungus was based on both morphological characteristics in culture and DNA sequencing. The pathogenicity of C. platani was confirmed in inoculations on seedlings of P. orientalis . To our knowledge, this is the first report of C. platani in Albania.

Thielaviopsis basicola is a hemibiotrophic root pathogen causing black root rot in a wide range of economically important crops. Our initial attempts to transform T. basicola using standard Agrobacterium tumefaciens-mediated transformation (ATMT) protocols were unsuccessful. Successful transformation required the addition of V8 juice (to induce germination of T. basicola chlamydospores) and higher concentrations of acetosyringone in the co-cultivation medium, and of chlamydospores/endoconidia, A. tumefaciens cells during co-cultivation. With these modifications, two T. basicola strains were successfully transformed with the green (egfp) or red (AsRed) fluorescent protein genes. Chlamydospores/endoconidia transformed with the egfp gene exhibited strong green fluorescence, but their fluorescence became weaker as the germ tubes emerged. Transformants harbouring the AsRed gene displayed strong red fluorescence in both chlamydospores/endoconidia and germ tubes. Fluorescent microscopic observations of an AsRed-labelled strain colonizing roots of transgenic Nicotiana benthamiana plants, which express the actin filaments labelled with EGFP, at 24 hours post inoculation showed varying levels of fungal germination and penetration. At this stage, the infection appeared to be biotrophic with the EGFP-labelled host actin filaments not being visibly degraded, even in host root cells in close contact with the hyphae. This is the first report of ATMT of T. basicola, and the use of an AsRed-labelled strain to directly observe the root infection process.

The β-1,6-endoglucanase gene (vegB) of Verticillium dahliae was isolated using a genome walking technique. Nucleotide and deduced amino acid sequences of the gene showed high identity with the PAN1 sequence deposited at the Verticillium genome database (Broad Institute), but significant differences in intron numbers and sites of insertion. Detailed in silico analysis, accompanied by sequencing of both genomic and cDNA, as well as RT-PCR experiments, provided the correct size of the gene and the exact number, length and positions of introns. The putative protein of this gene was compared with corresponding β-1,6-endoglucanases from other fungi, and sequences were used to construct a phylogenetic tree. A clear differentiation between enzymes derived from plant pathogenic and mycoparasitic fungi was observed, fully supported by bootstrap data. An internal fragment (1.2kb) of vegB was used to disrupt the wild-type gene of a V. dahliae tomato race 2 strain, and the mutant strain, vegB-, was tested for pathogenicity on tomato plants. Results showed a small but constant reduction in disease symptoms only on eggplants for the vegB- strain in comparison with the wild type. Growth on minimal medium supplemented with different carbon sources showed reduced ability of the mutant to breakdown cellulose, whereas growth on glucose, pectin and sucrose was similar to the wild type.