Insect Pathogenic Fungus Research Articles

Efficacy of entomopathogenic fungi, nematodes and spinetoram combinations for integrated management of Thrips tabaci.

Two consecutive field trials using a blend of entomopathogens in combination with a new chemistry insecticide were conducted to determine treatment effects on onion thrips (Thrips tabaci Lindeman) populations, crop damage, plant development, crop yield and impact on natural enemies. Products were tested in an onion cropping system and included the insect pathogenic fungus Beauveria bassiana (isolate WG-11), an entomopathogenic nematode Heterorhabditis bacteriophora (strain VS) and the new-chemistry chemical insecticide spinetoram. In all treatments, a significant decrease in thrips per plant population was detected in both trials. Overall, dual application of entomopathogens and insecticide was more effective than singly applied treatments. The lowest number of thrips larvae (1.96 and 3.85) and adults (0.00 and 0.00) were recorded when treated with dual application of B. bassiana and spinetoram at 7 days post application (DPA) after the second spray application in 2017and 2018, respectively. Damage on onion plants was considerably decreased in all treatments relative to the control. The lowest damage was observed on onion plants treated with B. bassiana + spinetoram at 7 DPA after the second spray application during both years. A significant decrease in the number of natural enemies (beetles, spiders, mites, lacewings, ants and bugs) on onion plants was recorded during both years. Insect pathogens when applied alone and in combination with each other considerably protected arthropod natural enemies compared to insecticide application applied alone. Significant increase in plant agronomic traits was observed compared to the control. Among all the treatments, B. bassiana + spinetoram produced maximum leaf length, leaf weight, total leaves, neck diameter, bulb diameter, number of rings per bulb, bulb weight, dry matter and plant yield following the 2017and 2018 applications, respectively. The findings of the study reveal the potential of using insect pathogens and insecticide for control of T. tabaci. However, combinations containing spinetoram are harmful to nontarget organisms, whereas biological control agents help in protecting biodiversity in onion agroecosystems. © 2023 Society of Chemical Industry.

Lipids as a key element of insect defense systems.

The relationship between insect pathogenic fungi and their insect hosts is a classic example of a co-evolutionary arms race between pathogen and target host: parasites evolve towards mechanisms that increase their advantage over the host, and the host increasingly strengthens its defenses. The present review summarizes the literature data describing the direct and indirect role of lipids as an important defense mechanism during fungal infection. Insect defense mechanisms comprise anatomical and physiological barriers, and cellular and humoral response mechanisms. The entomopathogenic fungi have the unique ability to digest the insect cuticle by producing hydrolytic enzymes with chitin-, lipo- and proteolytic activity; besides the oral tract, cuticle pays the way for fungal entry within the host. The key factor in insect resistance to fungal infection is the presence of certain types of lipids (free fatty acids, waxes or hydrocarbons) which can promote or inhibit fungal attachment to cuticle, and might also have antifungal activity. Lipids are considered as an important source of energy, and as triglycerides are stored in the fat body, a structure analogous to the liver and adipose tissue in vertebrates. In addition, the fat body plays a key role in innate humoral immunity by producing a range of bactericidal proteins and polypeptides, one of which is lysozyme. Energy derived from lipid metabolism is used by hemocytes to migrate to the site of fungal infection, and for phagocytosis, nodulation and encapsulation. One polyunsaturated fatty acid, arachidonic acid, is used in the synthesis of eicosanoids, which play several crucial roles in insect physiology and immunology. Apolipoprotein III is important compound with antifungal activity, which can modulate insect cellular response and is considered as important signal molecule.

Synergistic effects of the entomopathogenic fungus Isaria javanica and low doses of dinotefuran on the efficient control of the rice pest Sogatella furcifera

The rice planthopper, Sogatella furcifera, is a piercing-sucking insect pest of rice, Oryza sativa. It is responsible for significant crop yield losses, and has developed moderate to high resistance to several commonly used chemical insecticides. We investigated the effects of the insect fungal pathogen Isaria javanica, alone and in combination with the chemical insecticide dinotefuran, on S. furcifera under both laboratory and field conditions. Our results show that I. javanica displays high infection efficiency and mortality for different stages of S. furcifera, reducing adult survival, female oviposition and ovary development. Laboratory bioassays showed that the combined use of I. javanica with a low dose (4–16 mg L–1) of dinotefuran resulted in higher mortality in S. furcifera than the use of I. javanica or dinotefuran alone. The combined treatment also had more significant effects on several host enzymes, including superoxide dismutase, catalase, peroxidase, and prophenol oxidase activities. In field trials, I. javanica effectively suppressed populations of rice planthoppers to low levels (22–64% of the level in untreated plots). Additional field experiments showed synergistic effects, i.e., enhanced efficiency, for the control of S. furcifera populations using the combination of a low dose of I. javanica (1×104 conidia mL–1) and a low dose of dinotefuran (~4.8–19.2% of normal field use levels), with control effects of >90% and a population level under 50 insects per 100 hills at 3–14 days post-treatment. Our findings indicate that the entomogenous fungus I. javanica offers an attractive biological control addition as part of the integrated pest management (IPM) practices for the control of rice plant pests.

Autophagy-Related Gene 4 Participates in the Asexual Development, Stress Response and Virulence of Filamentous Insect Pathogenic Fungus Beauveria bassiana.

Autophagy is a conserved mechanism for the turnover of intracellular components. Among the 'core' autophagy-related genes (ATGs), the cysteine protease Atg4 plays an important role in the activation of Atg8 by exposing the glycine residue at its extreme carboxyl terminus. In the insect fungal pathogen Beauveria bassiana, a yeast ortholog of Atg4 was identified and functionally analyzed. Ablation of the BbATG4 gene blocks the autophagic process during fungal growth under aerial and submerged conditions. Gene loss did not affect fungal radial growth on various nutrients, but ΔBbatg4 exhibited an impaired ability to accumulate biomass. The mutant displayed increased sensitivity to stress caused by menadione and hydrogen peroxide. ΔBbatg4 generated abnormal conidiophores with reduced production of conidia. Additionally, fungal dimorphism was significantly attenuated in gene disruption mutants. Disruption of BbATG4 resulted in significantly weakened virulence in topical and intrahemocoel injection assays. Our study indicates that BbAtg4 contributes to the lifecycle of B. bassiana via its autophagic roles.

Metode Isolasi Jamur Patogen Serangga (Aschersonia Placenta) Dengan Menggunakan Media Potato Sucrose Agar (PSA)

Cultivating oranges is one of the main fruit commodities in Bali. Citrus cultivation often experiences problems, one of which is insect pests and plant diseases. Pests that often attack are whitefly. Whiteflies cause damage directly or indirectly. It directly results in stunted plant leaves because the whitefly sucks on the young shoots and leaves. Indirectly, the whitefly has been reported as a vector of various diseases caused by viruses. Based on this, it is necessary to control efforts, one of which is by using biological control of insect pathogenic fungi. The pathogenic fungus found in association with the citrus whitefly is Aschersonia placenta. The insect pathogen A. placenta naturally exists in nature and has potential as a biological control agent, however special handling is required in its utilization. One such treatment is the isolation of the fungus from the field for mass cultivation in the laboratory. Isolation poetry using Potato Sucrose Agar (PSA) was the best, of the four isolation methods performed. The method is detailed as follows: the fungal stroma is rinsed with sterile distilled water, with 70% alcohol, then rinsed again with sterile distilled water before being planted in PSA media.

Two Secretory T2 RNases Act as Cytotoxic Factors Contributing to the Virulence of an Insect Fungal Pathogen.

RNase T2 members are secreted by several pathogens or parasites during infection, playing various roles in pathogen-host interaction. However, functions of those members in biocontrol microbes targeting their hosts are still unknown. Here, we report that an insect fungal pathogen, Beauveria bassiana, produces two secretory RNase T2 members that act as cytotoxic factors, which were examined by insect bioassays using the targeted gene(s) disruption and overexpression strains. Overexpression strains displayed dramatically increased virulence, which was concurrent with few fungal cells and hemocytes in hemocoel, suggesting a cytotoxicity of the overexpressed gene products. In vitro assays using yeast-expressed proteins verified the cytotoxicity of the two members against insect cells, to which the cytotoxic effect was dependent on their RNases enzyme activities and glycosylation modification. Moreover, the excessive humoral immune responses triggered by the two ribonucleases were examined. These results suggested prospects of these two T2 ribonucleases for improvement of biocontrol agents.

Tolerance to UV-B radiation of the entomopathogenic fungus Metarhizium rileyi

Soybean, corn, and cotton crops are afflicted by several noctuid pests and the development of bioinsecticides could help control these pests. The fungus Metarhizium rileyi has the greatest potential because its epizootics decimate caterpillar populations in the absence of insecticide applications. However, insect-pathogenic fungi when used for insect control in agriculture have low survival mainly due to the deleterious effects of ultraviolet radiation and heat from solar radiation. In this study, fourteen isolates of M. rileyi were studied and compared with isolates ARSEF 324 and ARSEF 2575 of Metarhizium acridum and Metarhizium robertsii, respectively, whose sensitivity to UV-B radiation had previously been studied. Conidia were exposed at room temperature (ca. 26 °C) to 847.90 mWm−2 of Quaite-weighted UV-B using two fluorescent lamps. The plates containing the conidial suspensions were irradiated for 1, 2, and 3 h, providing doses of 3.05, 6.10, and 9.16 kJ m2, respectively. A wide variability in conidial UV-B tolerance was found among the fourteen isolates of M. rileyi. Isolate CNPSo-Mr 150 was the most tolerant isolate (germination above 80% after 2 h exposure), which was comparable to ARSEF 324 (germination above 90% after 2 h exposure), the most tolerant Metarhizium species. The least tolerant isolates were CNPSo-Mr 141, CNPSo-Mr 142, CNPSo-Mr 156, and CNPSo-Mr 597. Nine M. rileyi isolates exhibited similar tolerance to UV-B radiation as ARSEF 2575 (germination above 50% after 2 h exposure). In conclusion, the majority of M. rileyi isolates studied can endure 1 or 2 h of UV-B radiation exposure. However, after 3 h of exposure, the germination of all studied isolates reduced below 40%, except for CNPSo-Mr 150 and ARSEF 324.

Root colonization of industrial hemp (Cannabis sativa L.) by the endophytic fungi Metarhizium and Pochonia improves growth

Metarhizium is an endophytic, insect pathogenic fungus. Here we investigated the time-course of systemic and rhizospheric colonization by Metarhizium and its influence on the growth of industrial hemp (Cannabis sativa L.). Fungal colonization of hemp root, stem and leaf as well as rhizospheric soil, and agronomic parameters of hemp, were tested in four Metarhizium species with broad- (Metarhizium robertsii, Metarhizium anisopliae, Metarhizium brunneum) and narrow-insect (Metarhizium acridum) host ranges, as well as Pochonia chlamydosporia, a phylogenetically related endophytic fungus that is a nematode pathogen. Root and rhizosphere colonization were found in all Metarhizium species and Pochonia at 10, 20 and 30 days post-inoculation. Pochonia showed greatest root colonization compared to that of all Metarhizium species. Endophytic root colonization by Metarhizium was confirmed by confocal fluorescence microscopy using green fluorescent protein-expressing fungal strains. The genes, cat, mpl and EF1α were tested using a qPCR assay for Metarhizium and hemp. Root colonization by Metarhizium and Pochonia positively influenced shoot length, stem weight and root weight of hemp during the early stages of colonization. Our results suggest the potential for the application of Metarhizium and Pochonia as biocontrol and plant growth promoter agents during hemp colonization.

Ada2 and Ada3 Regulate Hyphal Growth, Asexual Development, and Pathogenicity in Beauveria bassiana by Maintaining Gcn5 Acetyltransferase Activity.

The histone acetyltransferase (HAT) Gcn5 ortholog is essential for a variety of fungi. Here, we characterize the roles of Ada2 and Ada3, which are functionally linked to Gcn5, in the insect-pathogenic fungus Beauveria bassiana. Loss of Ada2 and Ada3 led to severe hyphal growth defects on rich and minimal media and drastic decreases in blastospore yield and conidiation capacity, with abnormal conidia-producing structures. ΔAda2 and ΔAda3 exhibited a delay in conidial germination and increased sensitivity to multiple chemical stresses and heat shock. Nearly all their pathogenicity was lost, and their ability to secrete extracellular enzymes, Pr1 proteases and chitinases for cuticle degradation was reduced. A yeast two-hybrid assay demonstrated that Ada2 binds to Ada3 and directly interacts with Gcn5, confirming the existence of a yeast-like Ada3-Ada2-Gcn5 HAT complex in this fungus. Additionally, deletion of the Ada genes reduced the activity of Gcn5, especially in the ΔAda2 strain, which was consistent with the acetylation level of histone H3 determined by Western blotting. These results illustrate the dependence of Gcn5 enzyme activity on Ada2 and Ada3 in fungal hyphal growth, asexual development, multiple stress responses, and pathogenicity in B. bassiana. IMPORTANCE The histone acetyltransferase Gcn5 ortholog contributes significantly to the growth and development of various fungi. In this study, we found that Ada2 and Ada3 have critical regulatory effects on Gcn5 enzyme activity and influence the acetylation of histone H3. Deletion of Ada2 or Ada3 decreased the fungal growth rate and asexual conidial yield and increased susceptibility to multiple stresses in Beauveria bassiana. Importantly, Ada genes are vital virulence factors, and their deletion caused the most virulence loss, mainly by inhibiting the activity of a series of hydrolytic enzymes and the dimorphic transition ability. These findings provide a new perspective on the function of the Gcn5 acetyltransferase complex in pathogens.

Potential of 3-octanone as a lure and kill agent for control of the Brown garden snail

The brown garden snail (Cornu aspersum) is a major agricultural pest, causing damage to a wide range of economically important crops. Withdrawal or restricted use of pollutant molluscicides like metaldehyde has prompted a search for more benign control products. This study investigated the response of snails to 3-octanone; a volatile organic compound (VOCs) produced by the insect pathogenic fungus Metarhizium brunneum. Concentrations of 1 – 1000 ppm of 3-octanone were first assessed in laboratory choice assays to determine behavioural response. Repellent activity was found at 1000 ppm whereas attractance was found for the lower concentrations of 1, 10 and 100 ppm. These three concentrations of 3-octanone were carried forward in field evaluations to assess potential for use in “lure and kill” strategies. The highest concentration (100 ppm) was the most attractive to the snails but also the most lethal. Even at the lowest concentration this compound proved toxic effects making 3-octanone an excellent candidate for the development as a snail attractant and molluscicide.

Transcriptomic investigation reveals a physiological mechanism for Beauveria bassiana to survive under linoleic acid stress

In integrated pest management program (IPM), the compatibility of mycoinsecticides with bioactive fungicides [e.g., unsaturated fatty acids (UFAs)] has attracted more and more attention; however, the mechanisms underlying fungal resistance to UFAs remain largely unknown. In this study, Beauveria bassiana, an entomopathogenic fungus, was used to explore fungal responses to linoleic acid (LA). Genome-wide expression revealed the transcriptomic responses of fungal cells to LA in a stress-intensity-dependent manner. Enrichment analyses indicated that the up-regulated differentially expressed genes (DEGs) are associated with the metabolism of lipid and fatty acids. Notably, a lipid-droplet protein (BbLar1) maintains the intracellular homeostasis of fatty acids and is crucial to fungal tolerance to LA stress, which significantly contributes to fungal compatibility with UFAs. Additionally, BbLar1 links the lipid droplets to global expression profiles in B.bassiana under LA stress. Our investigations provide an initial framework for improving the efficacy of insect pathogenic fungi in practical application.

Fus3/Kss1-MAP kinase and Ste12-like control distinct biocontrol-traits besides regulation of insect cuticle penetration via phosphorylation cascade in a filamentous fungal pathogen.

Homolog of the yeast Fus3/Kss1 MAP kinase (MAPK) pathway and its target transcription factor, Ste12-like, are involved in penetration of host cuticle /pathogenicity in many ascomycete pathogens. However, details of their interaction during fungal infection, as well as their controlled other virulence-associated traits, are unclear. Ste12-like (BbSte12) and Fus3/Kss1 MAPK homolog (Bbmpk1) interacted in nucleus, and phosphorylation of BbSte12 by Bbmpk1 was essential for penetration of insect cuticle in an insect fungal pathogen, Beauveria bassiana. However, some distinct biocontrol-traits were found to be mediated by Ste12 and Bbmpk1. In contrast to ΔBbmpk1 colony that grew more rapid than wild type strain, inactivation of BbSte12 resulted in the opposite phenotype, which was consistent with their different proliferation rates in insect hemocoel after direct injection of conidia bypass the cuticle. Reduced conidial yield with decreased hydrophobicity was examined in the both mutants, however they displayed distinct conidiogenesis, accompanying with differently altered cell cycle, distinct hyphal branching and septum formation. Moreover, ΔBbmpk1 showed increased tolerance to oxidative agent, whereas the opposite phenotype was seen for ΔBbSte12 strain. RNA sequencing analysis revealed that Bbmpk1 controlled 356 genes depending on BbSte12 during cuticle penetration, but 1077 and 584 genes were independently controlled by Bbmpk1 and BbSte12. BbSte12 and Bbmpk1 separately participate additional pathways for control of conidiation, growth and hyphal differentiation, as well as oxidative stress response besides regulating cuticle penetration via phosphorylation cascade. This article is protected by copyright. All rights reserved.

Defense priming in cabbage (Brassica oleracea) by insect-pathogenic fungi

Defense priming in cabbage (Brassica oleracea) by insect-pathogenic fungi

The infection efficacy of Metarhizium strains (Hypocreales: Clavicipitaceae) against the Queensland fruit fly Bactrocera tryoni (Diptera: Tephritidae).

The Queensland fruit fly (Qfly), Bactrocera tryoni Froggatt, is a devastating pest of Australia's commercial fruit systems. Fruit fly mitigation is heavily centered around the use chemical insecticides, with limited investigation into microbial control alternatives. The wet tropics of northern Queensland is a highly biodiverse ecosystem containing many species of insect pathogenic fungi, but it is unclear whether any of these entomopathogens could contribute to Qfly management programs. In laboratory trials, we investigated the potential for Qfly microbial control by 3 locally sourced strains of entomopathogenic fungi comprising 2 species, Metarhizium guizhouense (Chen and Guo) and Metarhizium lepidiotae (Driver and Milner). Additionally, we evaluated 2 different inoculation methods to derive the most effective way to expose the flies to conidia-either through dry conidia or in a conidial suspension. All 3 strains were successful in causing Qfly mortality. Metarhizium lepidiotae resulted in the highest mean mortality over the trials, while M. guizhouense resulted in the highest mortality in a single replicate. Laboratory experiments revealed exposure through dry conidia to be the most effective method to inoculate the flies. These results suggest that fungal entomopathogens could be a viable pathway to Qfly suppression.

Tandem Multimerization Can Enhance the Structural Homogeneity and Antifungal Activity of the Silkworm Protease Inhibitor BmSPI39

Previous studies have shown that BmSPI39, a serine protease inhibitor of silkworm, can inhibit virulence-related proteases and the conidial germination of insect pathogenic fungi, thereby enhancing the antifungal capacity of Bombyx mori. The recombinant BmSPI39 expressed in Escherichia coli has poor structural homogeneity and is prone to spontaneous multimerization, which greatly limits its development and application. To date, the effect of multimerization on the inhibitory activity and antifungal ability of BmSPI39 remains unknown. It is urgent to explore whether a BmSPI39 tandem multimer with better structural homogeneity, higher activity and a stronger antifungal ability can be obtained by protein engineering. In this study, the expression vectors of BmSPI39 homotype tandem multimers were constructed using the isocaudomer method, and the recombinant proteins of tandem multimers were obtained by prokaryotic expression. The effects of BmSPI39 multimerization on its inhibitory activity and antifungal ability were investigated by protease inhibition and fungal growth inhibition experiments. In-gel activity staining and protease inhibition assays showed that tandem multimerization could not only greatly improve the structural homogeneity of the BmSPI39 protein, but also significantly increase its inhibitory activity against subtilisin and proteinase K. The results of conidial germination assays showed that tandem multimerization could effectively enhance the inhibitory ability of BmSPI39 on the conidial germination of Beauveria bassiana. A fungal growth inhibition assay showed that BmSPI39 tandem multimers had certain inhibitory effects on both Saccharomyces cerevisiae and Candida albicans. The inhibitory ability of BmSPI39 against these the above two fungi could be enhanced by tandem multimerization. In conclusion, this study successfully achieved the soluble expression of tandem multimers of the silkworm protease inhibitor BmSPI39 in E. coli and confirmed that tandem multimerization can improve the structural homogeneity and antifungal ability of BmSPI39. This study will not only help to deepen our understanding of the action mechanism of BmSPI39, but also provide an important theoretical basis and new strategy for cultivating antifungal transgenic silkworms. It will also promote its exogenous production and development and application in the medical field.

Entomopathogenic Fungi-Mediated Solubilization and Induction of Fe Related Genes in Melon and Cucumber Plants

Endophytic insect pathogenic fungi have a multifunctional lifestyle; in addition to its well-known function as biocontrol agents, it may also help plants respond to other biotic and abiotic stresses, such as iron (Fe) deficiency. This study explores M. brunneum EAMa 01/58-Su strain attributes for Fe acquisition. Firstly, direct attributes include siderophore exudation (in vitro assay) and Fe content in shoots and in the substrate (in vivo assay) were evaluated for three strains of Beauveria bassiana and Metarhizium bruneum. The M. brunneum EAMa 01/58-Su strain showed a great ability to exudate iron siderophores (58.4% surface siderophores exudation) and provided higher Fe content in both dry matter and substrate compared to the control and was therefore selected for further research to unravel the possible induction of Fe deficiency responses, Ferric Reductase Activity (FRA), and relative expression of Fe acquisition genes by qRT-PCR in melon and cucumber plants.. In addition, root priming by M. brunneum EAMa 01/58-Su strain elicited Fe deficiency responses at transcriptional level. Our results show an early up-regulation (24, 48 or 72 h post inoculation) of the Fe acquisition genes FRO1, FRO2, IRT1, HA1, and FIT as well as the FRA. These results highlight the mechanisms involved in the Fe acquisition as mediated by IPF M. brunneum EAMa 01/58-Su strain.

Asphyxiation of Metarhizium robertsii during mycelial growth produces conidia with increased stress tolerance via increased expression of stress-related genes

Little is known about the impact of hypoxia and anoxia during mycelial growth on tolerance to different stress conditions of developing fungal conidia. Conidia of the insect-pathogenic fungus Metarhizium robertsii were produced on potato dextrose agar (PDA) medium under normoxia (control = normal oxygen concentrations), continuous hypoxia, and transient anoxia, as well as minimal medium under normoxia. The tolerance of the conidia produced under these different conditions was evaluated in relation to wet heat (heat stress), menadione (oxidative stress), potassium chloride (osmotic stress), UV radiation, and 4-nitroquinoline-1-oxide (=4-NQO genotoxic stress). Growth under hypoxic condition induced higher conidial tolerance of M. robertsii to menadione, KCl, and UV radiation. Transient anoxic condition induced higher conidial tolerance to KCl and UV radiation. Nutritional stress (i.e., minimal medium) induced higher conidial tolerance to heat, menadione, KCl, and UV radiation. However, neither of these treatments induced higher tolerance to 4-NQO. The gene hsp30 and hsp101 encoding a heat shock protein was upregulated under anoxic condition. In conclusion, growth under hypoxia and anoxia produced conidia with higher stress tolerances than conidia produced in normoxic condition. The nutritive stress generated by minimal medium, however, induced much higher stress tolerances. This condition also caused the highest level of gene expression in the hsp30 and hsp101 genes. Thus, the conidia produced under nutritive stress, hypoxia, and anoxia had greater adaptation to stress.

Metarhizium robertsii COH1 functionally complements Schizosaccharomyces pombe Ecl family proteins.

The fission yeast Schizosaccharomyces pombe ecl family genes respond to various starvation signals and induce appropriate intracellular responses, including the extension of chronological lifespan and induction of sexual differentiation. Herein, we propose that the colonization of hemocoel 1 (COH1) protein of Metarhizium robertsii, an insect-pathogenic fungus, is a functional homolog of S. pombe Ecl1 family proteins.

Cytotoxic cytochalasans from cultures of the fungus Metarhizium brunneum TBRC-BCC 79240.

Fourteen new cytochalasans, brunnesins A-N (1-14), along with eleven known compounds, were isolated from the culture extracts of the insect pathogenic fungus Metarhizium brunneum strain TBRC-BCC 79240. The compound structures were established by spectroscopy, X-ray diffraction analysis, and electronic circular dichroism. Compound 4 exhibited antiproliferative activity against all cell lines tested (mammalian), with 50% inhibition concentration (IC50) values ranging from 2.09 to 16.8 μg mL-1. Compounds 6 and 16 were shown to be bioactive only against non-cancerous Vero cells (IC50 4.03 and 0.637 μg mL-1, respectively) whereas compounds 9 and 12 were bioactive only against NCI-H187 small-cell lung cancer cells (IC50 18.59 and 18.54 μg mL-1, respectively). Compounds 7, 13, and 14 showed cytotoxicity against NCI-H187 and Vero cell lines with IC50 values ranging from 3.98-44.81 μg mL-1.

Three Methods Assessing the Association of the Endophytic Entomopathogenic Fungus Metarhizium robertsii with Non-Grafted Grapevine Vitis vinifera.

Characterizing the association of endophytic insect pathogenic fungi (EIPF) with plants is an important step in order to understand their ecology before using them in biological control programs. Since several methods are available, it is challenging to identify the most appropriate for such investigations. Here, we used two strains of Metarhizium robertsii: EF3.5(2) native to the French vineyard environment and ARSEF-2575-GFP a laboratory strain expressing a green fluorescent protein, to compare their potential of association with non-grafted grapevine Vitis vinifera. Three methods were used to evaluate the kinetics of rhizosphere and grapevine endospheric colonization: (i) Droplet Digital (ddPCR), a sensitive molecular method of M. robertsii DNA quantification in different plant parts, (ii) culture-based method to detect the live fungal propagules from plant tissues that grew on the medium, (iii) confocal imaging to observe roots segments. Both strains showed evidence of establishment in the rhizosphere of grapevines according to the culture-based and ddPCR methods, with a significantly higher establishment of strain EF3.5(2) (40% positive plants and quantified median of exp(4.61) c/μL) compared to strain ARSEF-2575-GFP (13% positive plants and quantified median of exp(2.25) c/μL) at 96-98 days post-inoculation. A low incidence of association of both strains in the grapevine root endosphere was found with no significant differences between strains and evaluation methods (15% positive plants inoculated with strain EF3.5(2) and 5% with strain ARSEF-2575-GFP according to culture-based method). ddPCR should be used more extensively to investigate the association between plants and EIPF but always accompanied with at least one method such as culture-based method or confocal microscopy.