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The genus Diaporthe consists of saprobes, endophytes, and important plant pathogens. Members of this genus are widely distributed and have a broad host range, including grapevines. This study aimed to establish a baseline survey to assess the diversity of Diaporthe species infecting propagation material and to explore their dynamics in disease development. Initially, a survey was conducted in a nursery field, and isolations were carried out from 2-month-old symptomatic grafted vines of cv. Agiorgitiko grafted onto rootstock Richter 110. The initial molecular identification of the isolated mycobiome at the genus level was carried out by sequencing the universal internal transcribed spacer (ITS) locus, while subsequent species-level identification of the Diaporthe isolates was performed through phylogenetic approaches coupled with morphological characterization. Based on the combined analysis, five phylogenetically distinct Diaporthe spp. were identified in this study, taxonomically assigned to D. ampelina, D. eres, D. foeniculina, D. serafiniae, and D. novem. Pathogenicity trials demonstrated that the most aggressive species were D. ampelina followed by D. eres, while the remaining species were classified as opportunistic or weak pathogens of grapevine. Overall, accurate identification and monitoring of Diaporthe species involved in propagation material infections are important in order to develop species-specific effective management strategies in grapevine nurseries.

Background 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. Results 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% as indicated by zero cross-reactivity to common pathogens found in olive trees. Moreover, detection of V. dahliae in purified DNA gave a sensitivity of 100% (Ct < 30) and 80% (Ct > 30) while the detection of the fungus in unpurified crude wood extracts showed a sensitivity of 80% when multisampling was implemented. The superiority of the LAMP methodology regarding robustness and sensitivity was demonstrated when only LAMP was able to detect V. dahliae in crude samples from naturally infected trees with very low infection levels, while nested PCR and SYBR qPCR failed to detect the pathogen in an unpurified form. Conclusions This study describes the development of a new real time LAMP assay, targeting the ITS region of the rRNA gene of V. dahliae in olive trees combined with a 3D-printed portable device for field testing using a tablet. The assay is characterized by high sensitivity and specificity as well as ability to operate using directly crude samples such as woody tissue or petioles. The reported methodology is setting the basis for the development of an on-site detection methodology for V. dahliae in olive trees, but also for other plant pathogens.

Pythium root rot has been reported in several countries, but in Greece this disease was first detected in 2021, causing severe yield losses in a hydroponic lettuce crop. Isolations, morphological and molecular characterization, as well as pathogenicity assays identified a Pythium Cluster B2a species causing the disease in hydroponically grown lettuce. This is the first report of Pythium Cluster B2a sp. causing lettuce root rot in Greece.

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.

Ceratocystis ficicola causes vascular wilt of fig trees in Japan, invading root systems and the main stems eventually leading to tree death. In surveys from 2018 to 2020 in fig orchards in Greece, this fungus was detected in two separated regions. The fungus was consistently isolated from infected wood and from rhizosphere soil. The isolates were identified based on multi-locus phylogenetic analyses of rpb2, bt1 and tef1 gene regions and detailed morphological characteristics, including comparisons with an ex-type isolate of C. ficicola from Japan. The pathogenicity of Greek isolates was proven on Ficus carica and F. benjamina plants. Ceratocystis ficicola is a soilborne pathogen, and the occurrence of vascular wilt outbreaks suggest that the pathogen spreads within and between orchards with infested soil and wood debris during ploughing. The pathogen is also spreading in Greece with infected propagation material. This is the first detailed report of C. ficicola outside Japan, and there is concern over potential spread of the pathogen to other Mediterranean countries, where approx. 70% of the world fig production occurs.

Pear brown rot and blossom blight caused by Monilinia laxa seriously affect pear production worldwide. Here, we compared the transcriptomic profiles of petals after inoculation with M. laxa using two pear cultivars with different levels of sensitivity to disease (Sissy, a relatively tolerant cultivar, and Kristalli, a highly susceptible cultivar). Physiological indexes were also monitored in the petals of both cultivars at 2 h and 48 h after infection (2 HAI and 48 HAI). RNA-seq data and weighted gene co-expression network analysis (WGCNA) allowed the identification of key genes and pathways involved in immune- and defense-related responses that were specific for each cultivar in a time-dependent manner. In particular, in the Kristalli cultivar, a significant transcriptome reprogramming occurred early at 2 HAI and was accompanied either by suppression of key differentially expressed genes (DEGs) involved in the modulation of any defense responses or by activation of DEGs acting as sensitivity factors promoting susceptibility. In contrast to the considerably high number of DEGs induced early in the Kristalli cultivar, upregulation of specific DEGs involved in pathogen perception and signal transduction, biosynthesis of secondary and primary metabolism, and other defense-related responses was delayed in the Sissy cultivar, occurring at 48 HAI. The WGCNA highlighted one module that was significantly and highly correlated to the relatively tolerant cultivar. Six hub genes were identified within this module, including three WRKY transcription factor-encoding genes: WRKY 65 (pycom05g27470), WRKY 71 (pycom10g22220), and WRKY28 (pycom17g13130), which may play a crucial role in enhancing the tolerance of pear petals to M. laxa . Our results will provide insights into the interplay of the molecular mechanisms underlying immune responses of petals at the pear– M. laxa pathosystem.

The biocontrol bacterium Paenibacillus alvei K165 has the ability to protect Arabidopsis thaliana against Verticillium dahliae. A direct antagonistic action of strain K165 against V. dahliae was ruled out, making it likely that K165-mediated protection results from induced systemic resistance (ISR) in the host. K165-mediated protection was tested in various Arabidopsis mutants and transgenic plants impaired in defense signaling pathways, including NahG (transgenic line degrading salicylic acid [SA]), etr1-1 (insensitive to ethylene), jar1-1 (insensitive to jasmonate), npr1-1 (nonexpressing NPR1 protein), pad3-1 (phytoalexin deficient), pad4-1 (phytoalexin deficient), eds5/sid1 (enhanced disease susceptibility), and sid2 (SA-induction deficient). ISR was blocked in Arabidopsis mutants npr1-1, eds5/sid1, and sid2, indicating that components of the pathway from isochorismate and a functional NPR1 play a crucial role in the K165-mediated ISR. Furthermore, the concomitant activation and increased transient accumulation of the PR-1, PR-2, and PR-5 genes were observed in the treatment in which both the inducing bacterial strain and the challenging pathogen were present in the rhizosphere of the A. thaliana plants.

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.

Olive anthracnose outbreaks caused by the Colletotrichum species complex in the Mediterranean region decrease both fruit yield and olive oil production while also drastically degrading olive oil quality. The presence of various Colletotrichum species able to produce disease symptoms in olive fruits significantly deteriorates the efforts for an efficient crop protection strategy. In this report, the major olive productive area of Peloponnese was screened for Colletotrichum species capable of generating anthracnose symptoms. Olive fruits of 12 different olive cultivars were collected from 60 groves distributed analogously in the Peloponnese. Thirty-two fungal strains isolated from asymptomatic olive drupes were identified morphologically as Colletotrichum spp. and were multilocus genetically analyzed. The 32 isolates were grouped into two primary lineages resembling the previously characterized Colletotrichum acutatum and Colletotrichum nymphaeae based on the conducted genetic analysis for five genetic loci. The virulence of 16 Colletotrichum spp. strains were evaluated in a detached fruit assay of 10 Greek olive cultivars. The results clearly suggested that fungal isolates belonging to both C. acutatum and C. nymphaeae exhibited different levels of pathogenicity in a cultivar-dependent manner. Thus, cultivars examined in terms of the % Disease Index (%DI) were divided into highly tolerant, tolerant, and susceptible, and those analyzed regarding the % Disease Severity Index (%DSI) were divided into tolerant and susceptible. Our results suggest that the Greek cultivars of Athinolia and Megaritiki are highly tolerant to the vast majority of Colletotrichum strains isolated from Peloponnesian groves and consist of a significant genetic material for the future design of crop protection programs against anthracnose breakouts.

The wilt-inducing strains of Fusarium oxysporum are responsible for severe damage to many economically important plant species. The most cost-effective and environmentally safe method for the management of Fusarium wilt is the use of resistant cultivars when they are available. In the present study, the Arabidopsis genotype with disruptions in the β-amylase 3 (BAM3) gene, which encodes the major hydrolytic enzyme that degrades starch to maltose, had significantly lower susceptibility to Fusarium oxysporum f. sp. raphani (For) compared to wild-type (wt) plants. It showed the lowest disease severity and contained reduced quantities of fungal DNA in the plant vascular tissues when analyzed with real-time PCR. Through metabolomic analysis using gas chromatography (GC)–mass spectrometry (MS) and gene-expression analysis by reverse-transcription quantitative PCR (RT-qPCR), we observed that defense responses of Arabidopsis bam3 mutants are associated with starch-degradation enzymes, the corresponding modification of the carbohydrate balance, and alterations in sugar (glucose, sucrose, trehalose, and myo-inositol) and auxin metabolism.