Components of TOR and MAP kinase signaling control chemotropism and pathogenicity in the fungal pathogen Verticillium dahliae.

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.

Grapevine Trunk Diseases in Greece: Disease Incidence and Fungi Involved in Discrete Geographical Zones and Varieties.

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.

Fungicide resistance frequencies of Botrytis cinerea greenhouse isolates and molecular detection of a novel SDHI resistance mutation.

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.

The NADPH Oxidase A of Verticillium dahliae Is Essential for Pathogenicity, Normal Development, and Stress Tolerance, and It Interacts with Yap1 to Regulate Redox Homeostasis.

Maintenance of redox homeostasis is vital for aerobic organisms and particularly relevant to plant pathogens. A balance is required between their endogenous ROS production, which is important for their development and pathogenicity, and host-derived oxidative stress. Endogenous ROS in fungi are generated by membrane-bound NADPH oxidase (NOX) complexes and the mitochondrial respiratory chain, while transcription factor Yap1 is a major regulator of the antioxidant response. Here, we investigated the roles of NoxA and Yap1 in fundamental biological processes of the important plant pathogen Verticillium dahliae. Deletion of noxA impaired growth and morphogenesis, compromised formation of hyphopodia, diminished penetration ability and pathogenicity, increased sensitivity against antifungal agents, and dysregulated expression of antioxidant genes. On the other hand, deletion of yap1 resulted in defects in conidial and microsclerotia formation, increased sensitivity against oxidative stress, and down-regulated antioxidant genes. Localized accumulation of ROS was observed before conidial fusion and during the heterokaryon incompatibility reaction upon nonself fusion. The frequency of inviable fusions was not affected by the deletion of Yap1. Analysis of a double knockout mutant revealed an epistatic relationship between noxA and yap1. Our results collectively reveal instrumental roles of NoxA and ROS homeostasis in the biology of V. dahliae.

Endophytic Bacterial Isolates From Halophytes Demonstrate Phytopathogen Biocontrol and Plant Growth Promotion Under High Salinity.

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.

Characterization of Fungi Associated with Olive Fruit Rot and Olive Oil Degradation in Crete, Southern Greece.

In November 2019, a severe outbreak of fruit rot was observed in olive orchards in Crete, southern Greece. Symptoms appeared primarily on fruits and stalks, resembling those caused by anthracnose. Typical symptoms were fruit rot, shrinkage, and mummification, associated commonly with stalk discoloration and fruit drop. Disease incidence was estimated at up to 100% in some cases, and an unprecedented increase in olive oil acidity reaching up to 8% (percentage of oleic acid) in severely affected olive groves was recorded. Thirty-two olive groves were then surveyed, and samples of fruit, stalk, leaf, and shoot were collected. Visual, stereoscopic, and microscopic observations revealed several fungi belonging to the genera Alternaria, Botryosphaeria, Capnodium, Colletotrichum, Fusarium, and Pseudocercospora. Fungal infection in fruits was commonly associated with concomitant infestation by the olive fruit fly Bactrocera oleae along with increased air temperature and relative humidity conditions that prevailed in October and November 2019. Twenty representative fungal strains isolated from symptomatic fruits and stalks were characterized by morphological, physiological, and molecular analyses. By internal transcribed spacer regions of ribosomal DNA region and translation elongation factor 1-α gene sequencing analysis, these isolates were identified as Alternaria spp., A. infectoria, Botryosphaeria dothidea, Colletotrichum boninense sensu lato, Fusarium lateritium, F. solani species complex and Stemphylium amaranthi. Pathogenicity tests on punctured fruits revealed that all isolates were pathogenic; however, F. solani isolates along with B. dothidea were the most virulent, and wounds were necessary for efficient fungal infection. Moreover, as few as 10 spores of F. solani were sufficient to cause significant infection in punctured fruits. F. solani was also capable of infecting olive fruits in the presence of B. oleae, with no additional wounding, in artificial inoculation experiments. Moreover, it was capable of colonizing and affecting olive blossoms. Further analyses of olive oil extracted from fruits artificially inoculated with F. solani indicated a significant increase in oil acidity, K232, K270, and peroxide value, whereas total phenol content was significantly decreased. To the best of our knowledge, this is the first report of F. solani associated with olive fruit rot and olive oil degradation worldwide.

First Report of Stem Canker of Almond Trees Caused by Fusarium solani in Greece.

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.

Paenibacillus alvei K165 and Fusarium oxysporum F2: Potential Biocontrol Agents against Phaeomoniella chlamydospora in Grapevines.

In the last two decades grapevine trunk diseases (GTDs) have emerged as the most significant threat for grapevine sustainability worldwide. The tracheomycotic fungus Phaeomoniella chlamydospora (Pch) is the predominant GTD-associated species and cannot be controlled with available chemicals. In the present study, we evaluated the effectiveness of two microbial strains (Paenibacillus alvei K165 and Fusarium oxysporum F2) against Pch in grapevine. In vitro bioassays, performed in a growth culture medium simulating the xylem environment, indicated that F2 decreased Pch growth and sporulation, whereas K165 did not have any effect on Pch growth. In planta experiments revealed that root-drench and stem-puncture application of K165 and F2 reduced the endophytic relative DNA amount of Pch by 90% and 82%, respectively, compared to controls. However, wood discoloration, the typical symptom of Pch infection, was not reduced in the F2 treated grapevines. Nevertheless, the F2 treated grapevines harbored higher lignin levels compared to mocks, as it was also done by K165. Therefore, F2 and K165 have the potential to be used as biocontrol agents against Pch in grapevines.

Structural Diversity and Highly Specific Host-Pathogen Transcriptional Regulation of Defensin Genes Is Revealed in Tomato.

Defensins are small and rather ubiquitous cysteine-rich anti-microbial peptides. These proteins may act against pathogenic microorganisms either directly (by binding and disrupting membranes) or indirectly (as signaling molecules that participate in the organization of the cellular defense). Even though defensins are widespread across eukaryotes, still, extensive nucleotide and amino acid dissimilarities hamper the elucidation of their response to stimuli and mode of function. In the current study, we screened the Solanum lycopersicum genome for the identification of defensin genes, predicted the relating protein structures, and further studied their transcriptional responses to biotic (Verticillium dahliae, Meloidogyne javanica, Cucumber Mosaic Virus, and Potato Virus Y infections) and abiotic (cold stress) stimuli. Tomato defensin sequences were classified into two groups (C8 and C12). Our data indicate that the transcription of defensin coding genes primarily depends on the specific pathogen recognition patterns of V. dahliae and M. javanica. The immunodetection of plant defensin 1 protein was achieved only in the roots of plants inoculated with V. dahliae. In contrast, the almost null effects of viral infections and cold stress, and the failure to substantially induce the gene transcription suggest that these factors are probably not primarily targeted by the tomato defensin network.

Hex1, the Major Component of Woronin Bodies, Is Required for Normal Development, Pathogenicity, and Stress Response in the Plant Pathogenic Fungus Verticillium dahliae.

Woronin bodies are membrane-bound organelles of filamentous ascomycetes that mediate hyphal compartmentalization by plugging septal pores upon hyphal damage. Their major component is the peroxisomal protein Hex1, which has also been implicated in additional cellular processes in fungi. Here, we analyzed the Hex1 homolog of Verticillium dahliae, an important asexual plant pathogen, and we report its pleiotropic involvement in fungal growth, physiology, stress response, and pathogenicity. Alternative splicing of the Vdhex1 gene can lead to the production of two Hex1 isoforms, which are structurally similar to their Neurospora crassa homolog. We show that VdHex1 is targeted to the septum, consistently with its demonstrated function in sealing hyphal compartments to prevent excessive cytoplasmic bleeding upon injury. Furthermore, our investigation provides direct evidence for significant contributions of Hex1 in growth and morphogenesis, as well as in asexual reproduction capacity. We discovered that Hex1 is required both for normal responses to osmotic stress and factors that affect the cell wall and plasma-membrane integrity, and for normal resistance to oxidative stress and reactive oxygen species (ROS) homeostasis. The Vdhex1 mutant exhibited diminished ability to colonize and cause disease on eggplant. Overall, we show that Hex1 has fundamentally important multifaceted roles in the biology of V. dahliae.

The Role of Proteases in Determining Stomatal Development and Tuning Pore Aperture: A Review.

Plant proteases, the proteolytic enzymes that catalyze protein breakdown and recycling, play an essential role in a variety of biological processes including stomatal development and distribution, as well as, systemic stress responses. In this review, we summarize what is known about the participation of proteases in both stomatal organogenesis and on the stomatal pore aperture tuning, with particular emphasis on their involvement in numerous signaling pathways triggered by abiotic and biotic stressors. There is a compelling body of evidence demonstrating that several proteases are directly or indirectly implicated in the process of stomatal development, affecting stomatal index, density, spacing, as well as, size. In addition, proteases are reported to be involved in a transient adjustment of stomatal aperture, thus orchestrating gas exchange. Consequently, the proteases-mediated regulation of stomatal movements considerably affects plants' ability to cope not only with abiotic stressors, but also to perceive and respond to biotic stimuli. Even though the determining role of proteases on stomatal development and functioning is just beginning to unfold, our understanding of the underlying processes and cellular mechanisms still remains far from being completed.

Characterization of Fungi Associated With Wood Decay of Tree Species and Grapevine in Greece.

A two-year survey was conducted to identify fungi associated with wood decay in a range of tree species and grapevine. Fifty-eight fungal strains isolated from plants of 18 species showing typical wood decay symptoms were characterized by morphological, physiological, and molecular analyses. By 5.8S rRNA gene-ITS sequencing analysis, these isolates were classified into 25 distinct operational taxonomic units, including important phytopathogenic species of the phyla Pezizomycotina and Agaricomycotina, such as Fomitiporia, Inonotus, Phellinus, Inocutis, Fuscoporia, Trametes, Fusarium, Eutypa, Phaeomoniella, Phaeoacremonium, and Pleurostomophora spp. The white rot basidiomycetes Fomitiporia mediterranea (20 isolates, 34.5%) and Inonotus hispidus (6 isolates, 10.3%) were the most prevalent. Pathogenicity tests revealed for the first time that certain fungal species of the genera Fomitiporia, Inonotus, Phellinus, Pleurostomophora, and Fusarium caused wood infection of various tree species in Greece and worldwide. To the best of our knowledge, this is the first report of F. mediterranea as the causal agent of wood decay in pear, pomegranate, kumquat, and silk tree. This is also the first record of Inonotus hispidus, Phellinus pomaceus, Pleurostomophora richardsiae, and Fusarium solani in apple, almond, avocado, and mulberry tree, respectively, whereas P. richardsiae was associated with wood infection of olive tree for the first time in Greece. Cross pathogenicity tests with F. mediterranea strains originated from grapevine applied on other woody hosts and from olive on grapevine demonstrated partial host specificity of the fungus. The potential of F. mediterranea to transinfect hosts other than those originated, along with the host range extension of the fungus, is discussed.

Survival, Persistence, and Infection Efficiency of Verticillium dahliae Passed Through the Digestive System of Sheep.

The present study was carried out to determine the survival, persistence, and infection efficiency of Verticillium dahliae passed through the digestive tract of sheep. Eggplant, turnip, tomato, and pepper plants were artificially inoculated with 32 V. dahliae isolates. At 33 days postinoculation, the disease incidence and severity for eggplant, turnip, tomato, and pepper plants were 99.6, 96.2, 62.9, and 18.0% and 80.1, 49.8, 19.8, and 7.8%, respectively. The infected plant material was used to feed four 1-year-old sheep. Polymerase chain reaction assays revealed the presence of V. dahliae DNA in fecal samples received from animals' rectum on days 1, 2, 3, 4, and 5, whereas the pathogen DNA was not detected on 0, 6, and 7 days after feeding. Pathogenicity tests were conducted by transplanting eggplant plants into soil substrate amended with 20% decomposed manure, collected from the four animals fed with the infested forage. At 52 days after transplanting, manure-treated plants exhibited Verticillium wilt symptoms whereas, 2 months later, disease incidence, disease severity, and percentage of positive V. dahliae isolations from stem tissues were 58.3, 30.7, and 48.3%, respectively. Symptoms or positive isolations were not observed in control plants (transplanted in 100% soil substrate). This is the first report of the active role of V. dahliae passed through the digestive system of sheep as effective inoculum for host plants, in relation to the span persistence and transmission via the sheep carrier.

Phenolic Responses of Resistant and Susceptible Olive Cultivars Induced by Defoliating and Nondefoliating Verticillium dahliae Pathotypes.

Verticillium wilt is the most serious olive disease worldwide. The olive-infecting Verticillium dahliae pathotypes have been classified as defoliating (D) and nondefoliating (ND), and the disease is mainly controlled in olive orchards by using resistant or tolerant cultivars. Limited information is available about the nature of resistance in most of the olive cultivars. In the present study, the phenolic responses of the susceptible to V. dahliae olive cv. Amfissis and the resistant cv. Koroneiki upon D and ND V. dahliae infection were monitored in relation to the fungal DNA levels in the vascular tissues with the purpose to explore the defense mechanisms of olive trees against V. dahliae. Quantitative polymerase chain reaction revealed that the decrease in symptom severity shown in Koroneiki trees was associated with significant reduction in the growth of both V. dahliae pathotypes in the vascular tissues compared with Amfissis. In Koroneiki trees, the levels of o-diphenols and verbascoside were positively associated with the DNA levels of the D and ND pathotypes. In addition, a positive association was observed between the levels of verbascoside and the fungal DNA level in Amfissis trees, whereas a negative association was revealed between the fungal DNA level and the total phenols and oleuropein content in both cultivars. The levels of verbascoside were clearly higher in Koroneiki trees compared with Amfissis trees, indicating for the first time in the literature the involvement of verbascoside in the defense mechanism of olive trees against V. dahliae.