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

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 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

Evaluation of Olive Varieties Resistance for Sustainable Management of Verticillium Wilt

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.</i

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

<i>Paenibacillus alvei</i> K165 and <i>Fusarium oxysporum</i> F2: Potential Biocontrol Agents against <i>Phaeomoniella chlamydospora</i> 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

First report of Pseudophaeomoniella oleae causing wood streaking and decay on olive trees in Greece

A 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, globose to oval pycnidia with dimensions 120–330 × 90–300 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, turning into beige-pinkish in the centre after 21 days of growth on PDA. Microscopic observations revealed hyaline, smooth, ampulliform conidiophores, bearing conidia solitary or in slimy heads. Conidia were 1-celled, hyaline, smooth, subcylindrical with obtuse ends, 1.25–5.75 × 0.75–2.00 μm (average 3.12 × 1.16 μm) in size. Light to dark brown pycnidia, semi-immersed in PDA, with dimensions 150–490 × 90–320 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, 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-MZ854243, OK143463-OK143464 and OK143465-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 cvs Amfissis and Koroneiki were artificially inoculated by drilling a 3-mm-diameter hole into the trunk and injecting 50 μl of a 1 × 107 ml-1 conidia suspension of the isolate DRAGVR1 (Markakis et al. 2017). Another ten trees of each cv treated similarly with sterilized distilled water served as controls. Potted trees were kept under ambient conditions. Fourteen months post inoculation, 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 steadily re-isolated 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 result in severe crop losses. Hence, effective management practices should be investigated and applied