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Vascular wilt diseases caused by Verticillium dahliae Kleb. are difficult to control and lead to increasing losses of many crops worldwide. It can cause disease on not only horticultural crops but also many economically important crops such vegetables, legumes, forest trees, woody and herbaceous plants. Reasons of this situation are various: (i) the specialization of crop production resulted in the accumulation of the pathogen in the soil, especially monoculture production; (ii) the lack of an efficient and safe soil fumigation method; (iii) the production in large amounts of survival structures—microslerotia and melanized hyphae that are resistant to chemical and biological degradation. Due to the lack of effective synthetic agents for eradication of V. dahliae from soil, considerable interest in this paper has been focused on biological control, especially the selection of microorganisms with mycoparasitic activity towards V. dahliae microsclerotia, that can decrease their number in soil. The main attention is paid on the Trichoderma fungi, non pathogenic Fusarium spp., Talaromyces flavus and bacteria Bacillus spp., Pseudomonas spp. and Streptomyces spp. that are discussed in this review. In this work the suppressive effect of organic amendments against this soil-borne pathogen is also mentioned. In addition biofumigation using toxic plant materials, which is an approach to the soil-borne pathogen management could be an effective method to control diseases caused by V. dahliae.

Entomopathogenic fungi (EPF) can be defined as beneficial multifunctional eukaryotic microorganisms that display pivotal ecological services in pest management, with some species possessing the special ability to establish mutualistic relationships with plants. Mass production of these fungi is critical to support affordable widespread commercialization and worldwide field application. Among the mass production methods explored mainly by industry, submerged liquid fermentation is a robust and versatile technology that allows the formation of different types of propagules designated for various applications in pest control. Many hypocrealean EPF are easily culturable on artificial substrates by producing single-celled structures (hyphal bodies, blastospores, and submerged conidia) or multicellular structures (mycelium and microsclerotia). Less frequently, some EPF may form environmentally resistant chlamydospores, but these structures have almost always been overlooked. A continued research pipeline encompassing screening fungal strains, media optimization, and proper formulation techniques aligned with the understanding of molecular cues involved in the formation and storage stability of these propagules is imperative to unlock the full potential and to fine-tune the development of robust and effective biocontrol agents against arthropod pests and vectors of diseases. Finally, we envision a bright future for the submerged liquid fermentation technology to supplement or replace the traditional solid substrate fermentation method for the mass production of many important EPF. Key points • Submerged liquid fermentation (SLF) allows precise control of nutritional and environmental factors • SLF provides a scalable, robust, and cost-effective platform for mycopesticide production • Enhancing formulation, shelf life, and field efficacy of submerged propagules remain crucial • Understanding the molecular mechanisms behind submerged propagule formation is key to advancing SLF technology Graphical abstract

Verticillium wilt of olive (VWO), caused by the widespread soil-borne fungus Verticillium dahliae Kleb., is currently the most serious disease affecting olive trees (Olea europaea L.) in all production areas. An integrated management strategy using eco-friendly approaches such as genetic resistance and biological control is considered the most advisable approach for controlling the disease in commercial olive orchards. This study evaluated a non-pathogenic strain of F. oxysporum (FO12) and the grape marc compost CGR03 for reducing inoculum density of V. dahliae and the disease progress in two olive cultivars with different VWO resistance levels. The experiment was conducted under semi-controlled conditions, using a naturally infested soil with two inoculum densities of V. dahliae. The biocontrol treatments (FO12 and CGR03) were previously selected out of 220 natural products as two of the most effective treatments against the pathogen. FO12 and CGR03 treatments significantly reduced pathogen inoculum density in comparison with that of the control (P = 0.05), with minimum microsclerotium amounts of 0.13 g-1 for FO12 and 0.53 g-1 for CGR03, during the experimental period. CGR03 reduced the progression of the disease compared with that in the control (P = 0.05), and FO12 achieved complete control of VWO, since no plants treated with this biological control strain developed VWO symptoms. This study highlights the effectiveness of these biocontrol treatments, and the potential use of eco-friendly approaches for control of VWO.

Background During the disease cycle, plant pathogenic fungi exhibit a morphological transition between hyphal growth (the phase of active infection) and the production of long-term survival structures that remain dormant during “overwintering.” Verticillium dahliae is a major plant pathogen that produces heavily melanized microsclerotia (MS) that survive in the soil for 14 or more years. These MS are multicellular structures produced during the necrotrophic phase of the disease cycle. Polyketide synthases (PKSs) are responsible for catalyzing production of many secondary metabolites including melanin. While MS contribute to long-term survival, hyphal growth is key for infection and virulence, but the signaling mechanisms by which the pathogen maintains hyphal growth are unclear. Results We analyzed the VdPKSs that contain at least one conserved domain potentially involved in secondary metabolism (SM), and screened the effect of VdPKS deletions in the virulent strain AT13. Among the five VdPKSs whose deletion affected virulence on cotton, we found that VdPKS9 acted epistatically to the VdPKS1 -associated melanin pathway to promote hyphal growth. The decreased hyphal growth in VdPKS9 mutants was accompanied by the up-regulation of melanin biosynthesis and MS formation. Overexpression of VdPKS9 transformed melanized hyphal-type (MH-type) into the albinistic hyaline hyphal-type (AH-type), and VdPKS9 was upregulated in the AH-type population, which also exhibited higher virulence than the MH-type. Conclusions We show that VdPKS9 is a powerful negative regulator of both melanin biosynthesis and MS formation in V. dahliae . These findings provide insight into the mechanism of how plant pathogens promote their virulence by the maintenance of vegetative hyphal growth during infection and colonization of plant hosts, and may provide novel targets for the control of melanin-producing filamentous fungi.

Olive (Olea europaea L.) is one of the first domesticated and cultivated tree species and has historical, social and economical relevance. However, its future as a strategic commodity in Mediterranean agriculture is threatened by diverse biotic (traditional and new/emerging pests and diseases) and abiotic (erosion, climate change) menaces. These problems could also be of relevance for new geographical areas where olive cultivation is not traditional but is increasingly spreading (i.e., South America, Australia, etc). One of the major constraints for olive cultivation is Verticillium wilt, a vascular disease caused by the soil-borne fungus Verticillium dahliae Kleb. In this review we describe how Verticillium wilt of olive (VWO) has become a major problem for olive cultivation during the last two decades. Similar to other vascular diseases, VWO is difficult to manage and single control measure are mostly ineffective. Therefore, an integrated disease management strategy that fits modern sustainable agriculture criteria must be implemented. Multidisciplinary research efforts and advances to understand this pathosystem and to develop appropriate control measures are summarized. The main conclusion is that a holistic approach is the best strategy to effectively control VWO, integrating biological, chemical, physical, and cultural approaches.

Rhipicephalus microplus , commonly known as the cattle tick, is responsible for causing severe economic losses in livestock production in several countries. The utilization of entomopathogens in infested pastures may represent a sustainable and eco-friendly alternative for tick control. This study evaluated the effectiveness of combining entomopathogenic fungi (EPF, Metarhizium spp.) and entomopathogenic nematodes (EPN, Heterorhabditis bacteriophora ) for controlling R. microplus . Laboratory assays tested sub-doses of M. robertsii IP 146 conidia in combination with H . bacteriophora HP88 infective juveniles against engorged females of R. microplus , whereas field trials assessed a granular formulation containing M. robertsii microsclerotia applied with H. bacteriophora infective juveniles in infested field plots to target the tick’s non-parasitic phase during rainy and dry seasons. In laboratory experiments, the co-application of fungal sub-doses and nematodes demonstrated synergistic effects, significantly enhancing tick control . Field applications during the rainy season achieved tick population reductions of 54.09% ( M. robertsii ), 38.11% ( H. bacteriophora ), and 46.72% (combination). During the dry season, only the fungal formulation significantly reduced tick populations, with 26.27% efficacy. These findings underscore the potential of EPF and EPN, either singly or in combination, as complementary tools to traditional chemical methods for sustainable cattle tick management.

Background Melanin plays important roles in morphological development, survival, host–pathogen interactions and in the virulence of phytopathogenic fungi. In Verticillum dahliae , increases in melanin are recognized as markers of maturation of microsclerotia which ensures the long-term survival and stress tolerance, while decreases in melanin are correlated with increased hyphal growth in the host. The conserved upstream components of the VdCmr1-regulated pathway controlling melanin production in V. dahliae have been extensively identified, but the direct activators of this pathway are still unclear. Results We identified two genes encoding conserved C2H2-type zinc finger proteins VdZFP1 and VdZFP2 adjacent to VdPKS9 , a gene encoding a negative regulator of both melanin biosynthesis and microsclerotia formation in V. dahliae. Both VdZFP1 and VdZFP2 were induced during microsclerotia development and were involved in melanin deposition. Their localization changed from cytoplasmic to nuclear in response to osmotic pressure. VdZFP1 and VdZFP2 act as modulators of microsclerotia melanization in V. dahliae , as confirmed by melanin biosynthesis inhibition and supplementation with the melanin pathway intermediate scytalone in albino strains. The results indicate that VdZFP1 and VdZFP2 participate in melanin biosynthesis by positively regulating VdCmr1 . Based on the results obtained with yeast one- and two-hybrid (Y1H and Y2H) and bimolecular fluorescence complementation (BiFC) systems, we determined the melanin biosynthesis relies on the direct interactions among VdZFP1, VdZFP2 and VdCmr1, and these interactions occur on the cell walls of microsclerotia. Additionally, VdZFP1 and/or VdZFP2 mutants displayed increased sensitivity to stress factors rather than alterations in pathogenicity, reflecting the importance of melanin in stress tolerance of V. dahliae . Conclusions Our results revealed that VdZFP1 and VdZFP2 positively regulate VdCmr1 to promote melanin deposition during microsclerotia development, providing novel insight into the regulation of melanin biosynthesis in V. dahliae .

• Verticillium dahliae nuclear transcription factors Som1 and Vta3 can rescue adhesion in a FLO8-deficient Saccharomyces cerevisiae strain. Som1 and Vta3 induce the expression of the yeast FLO1 and FLO11 genes encoding adhesins. Som1 and Vta3 are sequentially required for root penetration and colonisation of the plant host by V. dahliae. • The SOM1 and VTA3 genes were deleted and their functions in fungus-induced plant pathogenesis were studied using genetic, cell biology, proteomic and plant pathogenicity experiments. • Som1 supports fungal adhesion and root penetration and is required earlier than Vta3 in the colonisation of plant root surfaces and tomato plant infection. Som1 controls septa positioning and the size of vacuoles, and subsequently hyphal development including aerial hyphae formation and normal hyphal branching. Som1 and Vta3 control conidiation, microsclerotia formation, and antagonise in oxidative stress responses. The molecular function of Som1 is conserved between the plant pathogen V. dahliae and the opportunistic human pathogen Aspergillus fumigatus. • Som1 controls genes for initial steps of plant root penetration, adhesion, oxidative stress response and VTA3 expression to allow subsequent root colonisation. Both Som1 and Vta3 regulate developmental genetic networks required for conidiation, microsclerotia formation and pathogenicity of V. dahliae.

The Serendipitaceae family was erected in 2016 to accommodate the Sebacinales ‘group B’ clade, which contains peculiar species of cultivable root–associated fungi involved in symbiotic associations with a wide range of plant species. Here we report the isolation of a new Serendipita species which was obtained from protocorms of the terrestrial orchid Epidendrum fulgens cultivated in a greenhouse. This species is described based on phylogenetic analysis and on its microscopic and ultrastructural features in pure culture and in association with the host’s protocorms. Its genome size was estimated using flow cytometry, and its capacity to promote the germination of E. fulgens seeds and to associate with roots of Arabidopsis thaliana was also investigated. Serendipita restingae sp. nov. is closely related to Serendipita sp. MAFF305841, isolated from Microtis rara (Orchidaceae), from which it differs by 14.2% in the ITS region and by 6.5% in the LSU region. It produces microsclerotia formed of non-monilioid hyphae, a feature that was not reported for the Sebacinales hitherto. Serendipita restingae promoted the germination of E. fulgens seeds, forming typical mycorrhizal pelotons within protocorm cells. It was also able to colonize the roots of Arabidopsis thaliana under in vitro conditions. Arabidopsis plants grown in association with S. restingae increased their biomass more than fourfold. Serendipita restingae is the first Serendipitaceae species described for the Americas.

ObjectivesMicrosclerotia (MS), anti-stress structures produced by many filamentous fungi, have been proven to be a great substitute for conidia in the production of insecticides within entomogenous fungi. NADPH oxidase (Nox) is a highly conserved ROS-response protein family that is widespread in eukaryotes and plays distinct roles in environmental fitness among various filamentous fungi. However, it is not clear whether the formation of MS and pathogenicity in entomogenous fungi is regulated by the Nox inside. In this study, we reported the presence of NADPH oxidase homologs in a great potential biocontrol fungus, Metarhizium rileyi, and further showed multiple biological functions.ResultsThree Nox homologous genes in M. rileyi showed high expression throughout the entire process of MS formation. Targeted deletion of MrNoxA, MrNoxB and MrNoxR all led to a decrease in MS yield and impaired morphology. Moreover, the anti-adversity assay showed that they are indispensable for growth, osmotic pressure and oxidative stress regulation in Metarhizium rileyi. Most importantly, △MrNoxR and △MrNoxA but not △MrNoxB showed a dramatic reduction in virulence via inoculation. The normality of appressoria might be unaffected in mutants since there are no striking differences in virulence compared with WT by topical injections.ConclusionOur results revealed that NADPH oxidase plays important roles in growth regulation, MS formation and pathogenicity in M. rileyi, perhaps in the ROS response and hyphal polarity.