Halophytic microbiomes represent an untapped reservoir of stress-adapted microbial functions, yet whether these functions can be transferred across ecological boundaries to enhance crop immune resilience remains unknown. Here we show that Kushneria, a halophyte associated genus with broad environmental resilience, confers robust disease protection in tomato and cucumber plants against fungal and oomycete pathogens despite lacking direct antagonistic activity. Comparative genomics across 26 genomes revealed extensive biosynthetic novelty and conserved catabolic clusters associated with rhizosphere competence, providing a genomic framework for persistence of Kushneria in saline environments. Multiomics profiling associated protection with a distinct host immune regulatory response, characterized by strong induction of core NLR receptors, including a ZAR1 paralogue. This response was further linked to a previously uncharacterized long noncoding RNA, Solyc10r048010.1, connected to the induced NLR network. Our findings establish halophytic endophytes as field-validated and transferable modulators of crop immunity and identify a candidate regulatory RNA/NLR axis associated with Kushneria-induced disease resistance.

Endophytic microbiomes of crop wild relatives (CWRs) adapted to extreme environments, such as halophytes, are promising sources of plant-beneficial bacteria and secondary metabolites for sustainable food production. Here, we analyzed 25 Bacilli isolates obtained from CWRs, halophytes, and other plant species in Crete, Greece. Using a hybrid Illumina-PacBio sequencing approach, we generated high-quality genomes and performed comparative genomics, phylogenetic, and pangenome analyses, complemented by in vitro assays. We identified 312 biosynthetic gene clusters (BGCs), nearly 60% of which showed no similarity to known clusters, revealing extensive unexplored biosynthetic potential. These unique BGCs may constitute an adaptive feature enabling endophytic Bacilli to colonize and interact with host plants. The isolates spanned diverse genera (Bacillus, Paenibacillus, Peribacillus, Neobacillus, Cytobacillus, Rossellomorea), including three novel species. Phenotypic assays of our isolates demonstrated high salinity tolerance (up to 17.5% w/v NaCl) and strong antagonism against major bacterial and fungal phytopathogens. Genome mining further revealed a broad array of putatively plant-beneficial traits related to growth promotion, stress adaptation, host interaction and inhibition of pathogens. Together, these findings show that Bacilli endophytes from wild and halophytic plants possess exceptional phylogenetic novelty, functional diversity, and biosynthetic capacity, providing new genomic and ecological insights into Bacilli associated with plants inhabiting extreme environments. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Introduction Caves represent unique, nutrient-limited windows into the deep biosphere, yet the microbiology of the deep terrestrial subsurface remains remarkably under-explored. In this work, we conducted a rare expedition into Gourgouthakas Cave (Crete, Greece), one of the world’s deepest vertical systems, which had remained untouched by humans for 19 years. Methods We performed a high-resolution vertical profiling of the cavès microbes by sampling rock surfaces across nine different depths down to 1,100 meters. Through extensive cultivation on various media and at different temperatures, we established a biobank of 820 bacterial isolates. Results Taxonomic identification of a 374-isolate subset revealed a diverse community spanning 35 genera and 4 phyla, dominated by Pseudomonas , Aquipseudomonas , Bacillus , and Stenotrophomonas . Beyond characterizing this taxonomic diversity, we explored the biotechnological potential of these subterranean microbes against major agricultural threats. Screening 70 representative isolates against six key pathogens, including Ralstonia solanacearum , Verticillium dahliae , and Phytophthora nicotianae , uncovered a notable group of strains with potent antagonistic activity, particularly within the Pseudomonas and Brevibacillus groups. Genomic sequencing of cave-derived Actinobacteria ( Streptomyces and Nocardiopsis isolates) further highlighted this potential, revealing 142 biosynthetic gene clusters (BGCs), over half of which showed little to no similarity to known clusters, suggesting a hidden reservoir of novel secondary metabolites. Pangenomic analysis of Streptomyces revealed 1,497 unique gene clusters. Finally, ex vivo trials showed that the Aquipseudomonas paracarnis (formerly Pseudomonas sp. ) isolate SRL917 significantly reduced Botrytis cinerea infections on tomato leaves, even surpassing the performance of a commercial biocontrol agent. Discussion Collectively, our results demonstrate that deep karstic systems are not merely geological wonders but vital hotspots for microbial innovation with tangible applications for sustainable agriculture.

Verticillium wilt caused by the soil-borne fungus Verticillium dahliae causes severe losses to a broad range of economically important crops worldwide. Chemical disease management is ineffective; thus, alternative control strategies are needed. Olive-producing countries face the challenge of managing olive mill wastewater (OMW) in an environmentally friendly and agronomically beneficial manner. The proper use of OMW supported by scientific research has been proposed as a valuable means for successful disease management. In this respect, we tested whether soil application of raw OMW can protect vegetable crops against V. dahliae and investigated the potential disease-suppressive mechanisms. OMW inhibited significantly fungal growth, sporulation, hyphae width, and conidial and microsclerotial germination in vitro, and these effects were dose-dependent. Moreover, the addition of OMW in the soil provided sufficient protection of eggplant and tomato against V. dahliae in planta. The high OMW-conferred protection of eggplant was gradually decreased, possibly due to the decreased phenolic content in OMW over time. Bioassays with sterilized soil substrate and OMW, along with isolated microbial strains, revealed that soil- and OMW-originated microbes had no role in disease suppression. Moreover, split-root set-ups suggested a non-systemic OMW-induced resistance mechanism. Root-drench application of OMW in eggplant and tomato plants did not cause significant alterations in the structure of the plant microbiome that could be associated with disease suppressiveness.

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

Field trials were conducted at traditional Mediterranean olive agro-ecosystems grown at two locations (Italy –IT, Greece –GR). Groves were managed for many years using sustainable (S, cover crops, compost application, mulching of pruning biomass) or conventional (C) practices (e.g., soil tillage, burning of pruning residuals). The IT grove was rainfed (rain) while the GR was irrigated (IRR). This study examined the seasonal variation of soil CO2 emission (Rs) to explore the effect of the management options (C, S) on Rs at both sites. The second aim was to test the hypothesis that the seasonal Rs is differentially modulated by soil temperature and moisture, namely that (i) soil moisture limits Rs when it is below the lower limit of the readily available water (RAWLLim) and (ii) soil temperature above a threshold (max_T) reduces Rs even if soil moisture is non limiting. On the whole-season basis, the mean Rs rate at the rainfed site was 2.17 ± 0.06 (SE) at Crain and 2.32 ± 0.06 μmol CO2 m−2 s–1 at Srain plot, while at the irrigated site Rs was about 3.64 ± 0.11 (CIRR) and 4.05 ± 0.15 μmol CO2 m−2 s–1 (SIRR). The seasonal oscillation of Rs was consistent across locations and partitionable in three periods according to DOY (Day of Year) interval: Phase I (DOY 20–103 –GR; 20–118 -IT), Phase II (DOY 141÷257, GR; 142–257, IT) and Phase III (DOY 291–357, GR; 286–350, -IT). Pooling all the Rs data across sites and managements, max_T was ∼ 20 °C discriminating a differential response of Rs when soil moisture was < or > RAWLLim. These differential modulations exerted by temperature and moisture were integrated into a conditional model developed with a repeated random subsampling cross-validation procedure to effectively (R2 = 0.84) predict Rs. This paper mechanistically describes the interaction of the environment (soil moisture and temperature) and the management options (S, C) under various moisture conditions on Rs and would support carbon flux accounting procedures (e.g., regulating ecosystem services) tailored to the estimation of sink/source capability of traditional olive agro-ecosystem within environmental-friendly agricultural domains.

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.

Production from crops of pome, stone fruit, nut, berry fruit, citrus, grapevine, and olive is increasingly threatened by fungal trunk diseases (FTD). These diseases and the consequent production losses are major problems. Many fungi (including Botryosphaeriaceae, Calosphaeriaceae, Diaporthaceae, Diatrypaceae, Nectriaceae, Phaeomoniellaceae, Pleosporaceae, Togniniaceae, Valsaceae) infect host wood, mainly through wounds and subsequent colonization of woody tissues, causing symptoms such as cankers, gummosis, wood rotting, blight and dieback. Propagative plant material, seedlings and fruit play a significant role in pathogen spread. Several abiotic factors (e.g. shifts in cultural practices and climate change) are involved in the disease development. This paper reviews recent literature on FTD of fruit crops, particularly focusing on the European status of pathogen occurrence. Case studies are described related to diseases of apple, citrus, grapevine, berry, nut and stone fruit, and olive trees. Aspects related to epidemiology and the increase in disease incidence along with the future perspectives on the FTD research are also discussed.

Filamentous fungi can sense useful resources and hazards in their environment and direct growth of their hyphae accordingly. Chemotropism ensures access to nutrients, contact with other individuals (e.g., for mating), and interaction with hosts in the case of pathogens. Previous studies have revealed a complex chemotropic sensing landscape during host-pathogen interactions, but the underlying molecular machinery remains poorly characterized. Here we studied mechanisms controlling directed hyphal growth of the important plant-pathogenic fungus Verticillium dahliae towards different chemoattractants. We found that the homologs of the Rag GTPase Gtr1 and the GTPase-activating protein Tsc2, an activator and a repressor of the TOR kinase respectively, play important roles in hyphal chemotropism towards nutrients, plant-derived signals, and heterologous α-pheromone of Fusarium oxysporum. Furthermore, important roles of these regulators were identified in fungal development and pathogenicity. We also found that the mitogen-activated protein kinase (MAPK) Fus3 is required for chemotropism towards nutrients, while the G protein-coupled receptor (GPCR) Ste2 and the MAPK Slt2 control chemosensing of plant-derived signals and α-pheromone. Our study establishes V. dahliae as a suitable model system for the analysis of fungal chemotropism and discovers new components of chemotropic signaling during growth and host-pathogen interactions of V. dahliae.

Common chicory (Cichorium intybus L.) as well as the spiny chicory (Cichorium spinosum L.) or "stamnagathi" in Greek, a bitter-sweet wild green, are two vegetables belonging to the Asteraceae family. Both species are commercially cultivated in outdoor fields and in greenhouses in various regions of Greece and particularly in Crete. During the years, 2014 and 2015 severe damage occurred in crops of C. intybus and C. spinosum in Crete. Typical symptoms in affected plants were marginal spots and lesions on leaves, and leaf blight. Initially, lesions appeared as water-soaked that later became brown, dry and papery, usually surrounded by a thin yellow halo or a brown to black border. Spots were gradually expanded and coalesced each other causing the blight of the leaves and finally the necrosis of the whole plant. In some cases, the diseased incidence reached 20 to 30% causing a significant reduction in yield and marketability of the product. The study of morphological, physiological and biochemical traits of the isolates as well as the pathogenicity tests indicated that the isolated bacteria belonged to group II of LOPAT discriminative tests, which contain the strains of Pseudomonas viridiflava. The phylogenetic analysis based on the B subunit of the gyrase gene (gyrB), and the amplification of a species-specific fragment confirmed P. viridiflava as the causal agent of the disease. This is the first record of the bacterium P. viridiflava as a pathogen of the common chicory in Greece as well as the spiny chicory worldwide. The disease can result in severe crop losses; hence, effective management practices should be investigated and applied.

HomePlant DiseaseVol. 106, No. 8First Report of Pseudophaeomoniella oleae Causing Wood Streaking and Decay on Olive Trees in Greece PreviousNext DISEASE NOTE OPENOpen Access licenseFirst Report of Pseudophaeomoniella oleae Causing Wood Streaking and Decay on Olive Trees in GreeceEmmanouil A. Markakis, Stefanos K. Soultatos, Georgios C. Koubouris, Georgios Psarras, Loukas Kanetis, Anastasia A. Papadaki, and Dimitrios E. GoumasEmmanouil A. Markakis†Corresponding author: E. A. Markakis; E-mail Address: [email protected]https://orcid.org/0000-0002-8791-0669Laboratory of Mycology, Department of Viticulture, Vegetable Crops, Floriculture and Plant Protection, Hellenic Agricultural Organization DIMITRA, Mesa Katsabas 71307, Heraklion, Crete, Greece, Stefanos K. SoultatosLaboratory of Mycology, Department of Viticulture, Vegetable Crops, Floriculture and Plant Protection, Hellenic Agricultural Organization DIMITRA, Mesa Katsabas 71307, Heraklion, Crete, Greece, Georgios C. KoubourisLaboratory of Olive Cultivation, Institute of Olive Tree, Subtropical Crops and Viticulture, Hellenic Agricultural Organization DIMITRA, Mesa Katsabas 71307, Heraklion, Crete, Greece, Georgios PsarrasLaboratory of Plant Physiology and Mineral Nutrition, Institute of Olive Tree, Subtropical Crops and Viticulture, Hellenic Agricultural Organization DIMITRA, Mesa Katsabas 71307, Heraklion, Crete, Greece, Loukas Kanetishttps://orcid.org/0000-0002-1869-558XLaboratory of Plant Pathology, Department of Agricultural Sciences, Biotechnology, and Food Science, Cyprus University of Technology, Limassol, 3036, Cyprus, Anastasia A. PapadakiLaboratory of Mycology, Department of Viticulture, Vegetable Crops, Floriculture and Plant Protection, Hellenic Agricultural Organization DIMITRA, Mesa Katsabas 71307, Heraklion, Crete, Greece, and Dimitrios E. GoumasLaboratory of Plant Pathology, Department of Agriculture, School of Agricultural Sciences, Hellenic Mediterranean University, Stavromenos 71004, Heraklion, Crete, GreeceAffiliationsAuthors and Affiliations Emmanouil A. Markakis1 † Stefanos K. Soultatos1 Georgios C. Koubouris2 Georgios Psarras3 Loukas Kanetis4 Anastasia A. Papadaki1 Dimitrios E. Goumas5 1Laboratory of Mycology, Department of Viticulture, Vegetable Crops, Floriculture and Plant Protection, Hellenic Agricultural Organization DIMITRA, Mesa Katsabas 71307, Heraklion, Crete, Greece 2Laboratory of Olive Cultivation, Institute of Olive Tree, Subtropical Crops and Viticulture, Hellenic Agricultural Organization DIMITRA, Mesa Katsabas 71307, Heraklion, Crete, Greece 3Laboratory of Plant Physiology and Mineral Nutrition, Institute of Olive Tree, Subtropical Crops and Viticulture, Hellenic Agricultural Organization DIMITRA, Mesa Katsabas 71307, Heraklion, Crete, Greece 4Laboratory of Plant Pathology, Department of Agricultural Sciences, Biotechnology, and Food Science, Cyprus University of Technology, Limassol, 3036, Cyprus 5Laboratory of Plant Pathology, Department of Agriculture, School of Agricultural Sciences, Hellenic Mediterranean University, Stavromenos 71004, Heraklion, Crete, Greece Published Online:30 Jun 2022https://doi.org/10.1094/PDIS-10-21-2353-PDNAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleA survey was conducted in olive orchards (Olea europaea L., cv. Koroneiki) showing severe decline in Milatos and Ierapetra (Lasithi, Crete, Greece) in November 2017 and January 2019, respectively. Diseased trees exhibited wilting, yellowing of leaves, twig and branch dieback, and internal discoloration of vascular tissue. Insect infestations were commonly associated with these symptoms. A yeast-like fungus was consistently isolated from discolored vessels previously surface-disinfested with 95% ethanol on acidified potato dextrose agar (APDA). The fungus yielded several dark brown to black and globose to oval pycnidia with dimensions 120 to 330 × 90 to 300 μm (average 179.6 × 143.9 μm) after 25 days of growth on APDA. Emerging colonies were transferred to new PDA and their growth rate was 2.21 mm/day at 24°C in the dark. The sparse aerial mycelium was initially white and turned beige-pinkish in the center after 21 days of growth on PDA. Microscopic observations revealed hyaline, smooth, ampulliform conidiophores, bearing solitary conidia or in slimy heads. Conidia were one-celled, hyaline, smooth, subcylindrical with obtuse ends, and 1.25 to 5.75 × 0.75 to 2.00 μm (average 3.12 × 1.16 μm). Light to dark brown pycnidia, semi-immersed in PDA, with dimensions 150 to 490 × 90 to 320 μm (average 297.7 × 231.0 μm) were evident in 3-week-old cultures. Colony morphology and microscopic features were similar to Pseudophaeomoniella oleae (Crous et al. 2015). DNA from two representative isolates (EML1 and DRAGVR1) was extracted, and their internal transcribed spacer region (ITS) of ribosomal DNA and actin (ACT) and translation elongation factor 1-alpha (TEF1-α) genes were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), ACT-512F/ACT-783R, and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. PCR products were sequenced and deposited in GenBank (accession nos. MZ854242 and MZ854243, OK143463 and OK143464, and OK143465 and OK143466). BLAST search revealed high similarity to GenBank sequences of the ex-type strain of P. oleae for ITS (≥99.50%, NR_137966.1 and KP635972.2), ACT (100%, KP635974.1), and TEF-1a (98.32%, KP635968.1). Based on morphology and phylogenetic analysis of the ITS region, the fungus was identified as P. oleae. Ten 3-year-old olive trees of each of the cultivars Amfissis and Koroneiki were artificially inoculated by drilling a 3-mm-diameter hole into the trunk and injecting 50 μl of a conidia suspension (1 × 107 ml−1) of the isolate DRAGVR1 (Markakis et al. 2017). Another 10 trees of each cultivar treated similarly with sterilized distilled water served as controls. Potted trees were kept under ambient conditions. Fourteen months postinoculation, longitudinal and transverse sections of inoculated trunks revealed wood discoloration extending above and below the inoculation point in both cultivars, whereas no leaf symptoms were observed. P. oleae was consistently reisolated from symptomatic wood tissue and identified by colony morphology. Neither symptoms nor positive isolations were observed in controls. To our knowledge, this is the first report of wood streaking and decay caused by P. oleae in olive trees in Greece. Although the fungus has been isolated previously from decayed olive trees showing vascular wilt in Italy (Crous et al. 2015), this is the first experimental evidence of the pathogenic potential of the species on olive trees worldwide. This disease could potentially be an increasing problem in olive tree growing areas and could result in severe crop losses, so effective management practices should be investigated and applied.The author(s) declare no conflict of interest.References:Carbone, I., and Kohn, L. M. 1999. Mycologia 91:553. https://doi.org/10.2307/3761358Crossref, ISI, Google ScholarCrous, P. W., et al. 2015. Persoonia 34:167. https://doi.org/10.3767/003158515X688433Crossref, ISI, Google ScholarMarkakis, E. A., et al. 2017. Eur. J. Plant Pathol. 149:277. https://doi.org/10.1007/s10658-017-1186-9Crossref, ISI, Google ScholarWhite, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego.Google ScholarThe author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 106, No. 8 August 2022SubscribeISSN:0191-2917e-ISSN:1943-7692 Download Cover Image Metrics Article History Issue Date: 29 Jul 2022Published: 30 Jun 2022First Look: 25 Jan 2022Accepted: 21 Jan 2022 Page: 2263 Information© 2022 The American Phytopathological SocietyKeywordsactindeclineITSOlea europaeaPseudophaeomoniella oleaeTEF1-alphaThe author(s) declare no conflict of interest.PDF download

Verticillium wilt resulting from infection by Verticillium dahliae is one of the most devastating soilborne fungi of the olive tree (Olea europaea L.) worldwide. The pathogen infects a wide variety of plants and can survive in the soil for many years, and chemicals cannot control it. Therefore, sustainable disease management strategies are suggested, with the exploitation of host resistance as the most predominant control measure in practice. In addition, disease risk assessment in commonly used plant genotypes is a prominent issue. In this respect, nine commercially grown Greek olive varieties ('Amfissis', 'Atsiholou', 'Chalkidikis', 'Koroneiki', 'Kothreiki', 'Koutsourelia', 'Mastoidis', 'Megaritiki', and 'Tragolia') and one variety of international interest ('Picual') were comparatively evaluated for their resistance to V. dahliae. The roots of young plants were immersed in a concentrated conidial suspension in order to perform an artificial inoculation. We evaluated disease reactions in a 140-day assessment period based on external symptoms (disease severity, disease incidence, and mortality) and calculated the relative areas under disease progress curves (relative AUDPC). The process of qPCR was used to evaluate V. dahliae DNA in vascular tissues and plant growth parameters (height and fresh weight). A cumulative stress response was calculated to consider the overall effect of V. dahliae on olive cultivars. The olive varieties resistance to V. dahliae varied significantly, with 'Koroneiki', 'Tragolia', and 'Atsiholou' being the most resistant. Interestingly, most tested varieties showed a significantly low resistance level, suggesting increased risk for the Greek olive industry due to V. dahliae.

Summary Over the last few years, novel synthetic nematicides, such as Salibro™ nematicide (a.s. fluazaindolizine - Reklemel™ active), Velum Prime® (a.s. fluopyram) or Nimitz® (a.s. fluensulfone), have been commercialised in various regions around the world. Whilst considerable scientific information exists on their field efficacy against plant-parasitic nematodes, very little has been published on their bio-compatibility with beneficial soil fungi. In this paper in vitro studies are presented with various nematophagous (Athrobotrys, Monacrosporium, Harposporium, Purpureocillium), entomoparasitic (Beauveria, Isaria) and disease-suppressive (Trichoderma) fungi that were exposed to these nematicides under laboratory conditions. Assessments were made on their impact on radial growth and sporulation of those fungi. Clear differences in sensitivity to the nematicides were seen between the different fungi. Intrinsically, fluopyram showed the strongest adverse effects on the tested fungi that often became already visible at a concentration of 5 ppm (a.s.). Negative effects were significant at higher concentrations of 50 ppm. Fluensulfone showed limited adverse impacts on the tested fungi at 5 ppm (a.s.) but clearly inhibited most of the fungi at 50 ppm (a.s.). Fluazaindolizine had the least impact of the novel nematicides, with no adverse effects recorded on any species at 5 ppm (a.s.), and very minor growth reductions at 50 ppm (a.s.). Even when tested at 250 ppm (a.s.) fluazaindolizine still showed no impact on Purpureocillium lilacinum, as well as only a weak impact on some Trichoderma species. Vydate (a.s. oxamyl), which was often included as a traditional carbamate nematicide in the test, also showed excellent bio-compatibility with the tested fungi at concentrations of from 5 to 50 ppm (a.s.). Overall, the studies showed that beneficial soil fungi differ in their intrinsic sensitivity to these modern nematicides. These interactions may be considered when designing integrated nematode management programmes that leverage endemic or introduced biocontrol agents. However, it should be noted that additional studies under field conditions with recommended label rates of the products are needed to confirm the trends seen in these laboratory data.

Sensitivity of B. cinerea to commonly used fungicides against Gray mold with emphasis to the newer quinone outside inhibitor (QoIs), and succinate dehydrogenase inhibitors (SDHIs) was assessed during a monitoring survey from vegetable greenhouses in four representative regions of Crete. 42% from a total of 168 isolates were simultaneously resistant to boscalid, fluopyram, pyraclostrobin and fenhexamid but not to fludioxonil making this phenylpyrrole fungicide an excellent anti-resistance antifungal agent. Isolates with double resistance to SDHIs and QoIs were found in very high frequencies indicating a selection towards double resistance due to the use of pyraclostrobin-boscalid mixtures. A number of sdhB resistance mutations (H272R, N230I and P225F/H) were found in isolates also carrying the G143A cytb resistance mutation in the above isolates. A novel sdhB point mutation (I274V) was identified for the first time in B. cinerea isolates collected from greenhouses with a fluopyram spray history with specific resistance to SDHIs. A PCR-RFLP diagnostic assay was developed for the detection of this mutation in the sdhB gene. Mutations P225F/H and I274V were found to be associated with fitness penalties in terms of mycelial growth, sporulation or pathogenicity. Results suggest that, in order to retain effective control of gray mold in Crete, appropriate anti-resistance strategies should be implemented taking into account the high double SDHI and QoI resistance frequencies. Additional studies for monitoring the already known and the new SDHI-resistance mutations, are necessary in order to hinder the further spread and establishment of single or double resistant isolates of B. cinerea detected in greenhouses in Crete.

In the present study, combined interactions of the root-knot nematode Meloidoyne javanica (M.j.) with the soil-borne fungi Verticillium dahliae (V.d.), Fusarium oxysporum f.sp. radicis-cucumerinum (F.o.r.c.) or Monosporascus cannonballus (M.c.) against susceptible plant hosts were evaluated. Direct and indirect interactions were tested by applying each pathogen (nematode or fungus) alone or together on the whole plant root system or on each of the two sides of a split-root set-up in all possible combinations. Plant-fungi-nematode interactions were estimated by measuring various disease and growth parameters on host plants. A significant increase of verticillium wilt symptoms was observed in eggplant when V.d. and M.j. were applied separately in split-root plants compared with symptoms in whole root plants inoculated with both pathogens. Root and stem rot and root-knot symptoms in cucumber were more severe when F.o.r.c. was combined with M.j. in a split-root set-up than when plants were only inoculated with a single pathogen on one part of the split-root set-up. No significant associations were observed in the case of melon-M.c.-M.j. interaction. Gene expression bioassays for cucumber-F.o.r.c.-M.j. interaction revealed increased transcriptomic activity for PAL1 gene in plants treated with F.o.r.c at 3 days postinoculation (d.p.i.), whereas high transcriptomic level for DEFENSIN gene was observed primarily in M.j.-treated plants at 7 d.p.i. The possible interactions between the abovementioned pathosystems are presented and discussed for the first time in literature.

Almond (Prunus dulcis) is an important crop for Greece grown on 15.130 ha in 2019. In September 2019, a severe stem canker disease was observed in 6-year-old trees of cv Marta grafted on the rootstock 'F675C14', in a new almond grove of cvs Marta, Soleta, Antonela, Belona and Laurete, in Vlachiana, Heraklion, Crete, Greece. Only cv Marta trees were affected. Diseased trees exhibited cankers on trunks and branches with pale yellow to red-colored gum excreting from cankers, yellowing, leaf fall, twig and branch dieback, bark and wood tissue discoloration. Severely affected trees were killed. A Fusarium-like fungus was consistently isolated from symptomatic wood tissue previously surface-disinfested with 95% ethanol, on acidified potato dextrose agar (APDA). Emerging colonies were transferred to new PDA and the growth rate of the fungus was 7.86 mm/day at 24 °C in the dark. The abundant aerial mycelium was initially white, turning into pale orange in the centre after 7 days of growth on PDA. Microscopic observations revealed hyaline conidiophores measuring 26.74 ± 20.44 μm in length, developing microconidia 5.00 to 9.50 × 2.50 to 4.75 μm (average 6.64 × 3.50 μm) and macroconidia 10.00 to 23.25 × 3.75 to 5.50 μm (average 16.42 × 4.50 μm) in size. DNA from one representative single-spore isolate (code KOUB.AM.VR1) was extracted and the internal transcribed spacer region (ITS) of ribosomal DNA and translation elongation factor 1-alpha (EF 1-a) genes were amplified using the primer pairs ITS1/ITS4 (White et al. 1990) and EF1-F/EF2-R (O'Donnell et al. 1998), respectively. The PCR products were sequenced and deposited in GenBank (accession Nos. MW547397 and MW554492). Based on morphological characteristics (Leslie and Summerell 2006) and a BLAST search with 100.00% and 99.38% identity to published F. solani ITS and EF 1-a sequences in GenBank (KX034335.1, DQ247636.1) the fungus was identified as F. solani. Eight 3-year-old almond trees of cv. Marta were artificially inoculated in March 2020 by making a 6.0-mm-diameter hole into the trunk, inserting a 6-mm-diameter mycelial disc taken from a 10-day-old PDA culture, sealing the hole with cellophane membrane and covering with adhesive paper tape. Another eight trees of the same cultivar were mock-inoculated with sterilized PDA discs and served as controls. Potted trees were kept under ambient conditions. One month post inoculation, yellow gum was evident excreting around the inoculation point in F. solani-treated trees but not in the controls. Seven months post inoculation, longitudinal and transverse sections of inoculated trunks revealed internal and external symptoms similar to those observed under natural infection conditions and F. solani was steadily re-isolated from symptomatic wood tissue and identified by colony morphology. Neither symptoms nor positive isolations were observed in control trunks. Pathogenicity tests were repeated twice. Fusarium solani has been reported as the causal agent of stem canker or wood decay diseases in several woody hosts including bitternut hickory, black walnut, mulberry and pistachio trees (Crespo et al. 2019; Markakis et al. 2017; Park and Juzwik 2012; Tisserat 1987). To the best of our knowledge, this is the first worldwide report of stem canker caused by F. solani on almond tree. This disease could potentially be an increasing problem in almond growing areas and result in severe crop losses. Hence, effective management practices should be investigated and applied.

Halophytic endophytes potentially contribute to the host’s adaptation to adverse environments, improving its tolerance against various biotic and abiotic stresses. Here, we identified the culturable endophytic bacteria of three crop wild relative (CWR) halophytes: Cakile maritima , Matthiola tricuspidata , and Crithmum maritimum . In the present study, the potential of these isolates to improve crop adaptations to various stresses was investigated, using both in vitro and in-planta approaches. Endophytic isolates were identified by their 16S rRNA gene sequence and evaluated for their ability to: grow in vitro in high levels of NaCl; inhibit the growth of the economically important phytopathogens Verticillium dahliae , Ralstonia solanacearum , and Clavibacter michiganensis and the human pathogen Aspergillus fumigatus; provide salt tolerance in-planta ; and provide growth promoting effect in-planta . Genomes of selected isolates were sequenced. In total, 115 endophytic isolates were identified. At least 16 isolates demonstrated growth under increased salinity, plant growth promotion and phytopathogen antagonistic activity. Three showed in-planta suppression of Verticillium growth. Furthermore, representatives of three novel species were identified: two Pseudomonas species and one Arthrobacter . This study provides proof-of-concept that the endophytes from CWR halophytes can be used as “bio-inoculants,” for the enhancement of growth and stress tolerance in crops, including the high-salinity stress.