Increasing the survival and efficacy of entomopathogenic nematodes on exposed surfaces by Pickering emulsion formulations offers new venue for foliar pest management.

Formulation technology has been the primordial focus to improve the low viability and erratic infectivity of entomopathogenic nematodes (EPNs) for foliar application. Adaptability to the fluctuating environment is a key trait in ensuring the survival and efficacy of EPNs. Hence, tailoring formulations towards EPNs foliar applications would effectively deliver consistent and reliable results for above-ground applications. EPNs survival and activity were characterized in novel Pickering emulsion post-application in planta cotton foliage. Two different types of novel formulations, Titanium Pickering emulsion (TPE) and Silica Pickering emulsion Gel (SPEG), were tailored for EPNs foliar applications. We report an extension of survival and infectivity to 96 hrs under controlled conditions by SPEG formulations for survival of IJ's on cotton foliage. In addition, survival of IJs (LT50) was extended from 14hrs in water to > 80 hrs and > 40 hrs by SPEG and TPE respectively. SPEG accounted for the slowest decrease of live IJs per surface area in comparison to TPE and control samples over time, exhibiting a 6-fold increase at 48 hrs. Under extreme conditions, survival and efficacy were extended for 8hrs in SPEG compared to merely 2hrs in control. Potential implications and possible mechanisms of protection are discussed.

Tolerance of Steinernema carpocapsae infective juveniles in novel nanoparticle formulations to ultraviolet radiation.

Entomopathogenic nematodes (EPNs) are susceptible to abiotic environmental factors including ultraviolet (UV) radiation, which affects the survival and efficacy. This study evaluated nanoparticle (NP) formulations for protecting Steinernema carpocapsae infective juveniles (IJs) from UV radiation. First, silica-NH2 NPs at oil-to-water ratios of 2:8, 3:7 and 4:6 were compared with Barricade Fire Gel (1 % and 2 %) and a water control (aqueous IJs) by exposing IJs to UV light (254 nm) for 0, 10 and 20 min. Barricade gel (especially 2 % Barricade) significantly improved IJs viability after UV treatment, while all three NPs had adverse effects on IJ viability after UV radiation. Subsequently, two silica (SiO2 basic and advanced) and one titania (TiO2) based formulations were tested with Barricade (1 % and 2 %) and a water control. The titania-NH2 NPs provided the highest UV protection, and IJ viability and virulence were not reduced even after 20-min UV. Except TiO2, only 2 % Barricade at 10-min UV and SiO2 basic at 20-min UV had lower IJ mortality than the water control. Only TiO2 formulated IJs caused higher insect mortality and infection levels than aqueous IJs after UV treatment. The UV tolerance of TiO2 was further examined by assessing the number of nematodes invading the hosts. Consistent with virulence tests, the number of invading nematodes in titania-NH2 NPs did not decrease after UV radiation for 10 or 20 min compared with the no-UV control. The anti-UV capability of titania-NH2 NPs has promise as a tool to enhance biocontrol efficacy of EPNs under field conditions.

The Entomopathogenic Fungi Metarhizium brunneum and Beauveria bassiana Promote Systemic Immunity and Confer Resistance to a Broad Range of Pests and Pathogens in Tomato.

Biocontrol agents can control pathogens by reenforcing systemic plant resistance through systemic acquired resistance (SAR) or induced systemic resistance (ISR). Trichoderma spp. can activate the plant immune system through ISR, priming molecular mechanisms of defense against pathogens. Entomopathogenic fungi (EPF) can infect a wide range of arthropod pests and play an important role in reducing pests' population. Here, we investigated the mechanisms by which EPF control plant diseases. We tested two well studied EPF, Metarhizium brunneum isolate Mb7 and Beauveria bassiana as the commercial product Velifer, for their ability to induce systemic immunity and disease resistance against several fungal and bacterial phytopathogens, and their ability to promote plant growth. We compared the activity of these EPF to an established biocontrol agent, Trichoderma harzianum T39, a known inducer of systemic plant immunity and broad disease resistance. The three fungal agents were effective against several fungal and bacterial plant pathogens and arthropod pests. Our results indicate that EPF induce systemic plant immunity and disease resistance by activating the plant host defense machinery, as evidenced by increases in reactive oxygen species production and defense gene expression, and that EPF promote plant growth. EPF should be considered as control means for Tuta absoluta. We demonstrate that, with some exceptions, biocontrol in tomato can be equally potent by the tested EPF and T. harzianum T39, against both insect pests and plant pathogens. Taken together, our findings suggest that EPF may find use in broad-spectrum pest and disease management and as plant growth promoting agents.

Comparative response of Metarhizium brunneum to the cuticles of susceptible and resistant hosts.

Earlier studies demonstrated that Metarhizium brunneum, usually a broad-host pathogen of arthropods, is unable to complete its pathogenic life cycle when inoculated on the fungus-resistant tick, Hyalomma excavatum engorged females. While the fungus penetrates the cuticle of fungus-susceptible tick, Rhipicephalus annulatus females, it is unable to penetrate the cuticle of fungus-resistant tick, and even perishes on its surface. This is probably due to high concentration of antifungal fatty acids and probably also due to a hypersensitive-like response of the tick. To understand the metabolic pathways occurring in the fungal hyphae upon encountering the cuticles, we compared the response of the fungus to cuticle from susceptible and resistant tick cuticles by 2D-gels. The intracellular proteomes of M. brunneum Mb7 exposed to cuticle of the fungus-susceptible tick, R. annulatus, and to the fungus-resistant tick, H. excavatum engorged females were compared after exposure to either cuticles. By means of liquid chromatography-mass spectrometry/mass spectrometry we identified in both proteomes common proteins involved in biological processes as well as unique proteins identified only in the proteome of fungus exposed to fungus-resistant tick cuticle. These proteins were identified in high probability as heat shock proteins, four key enzymes of the glyoxylate cycle, and proteins associated with hypoxia, and exposure to antifungal drugs. These findings are discussed within the M. brunneum-tick pathosystem in relation to tick resistance and host resistance in general.

Activity of Metarhizium brunneum and Beauveria bassiana against early developmental stages of the false codling moth Thaumatotibia leucotreta.

This study evaluated the efficacy of two entomopathogenic Hypocrealean fungi, Metarhizium brunneum (laboratory isolate) and Beauveria bassiana (the commercial product Botanigard), for preventative control of the false codling moth, Thaumatotibia leucotreta. The mortality of eggs and first instar larvae was studied in three different assays. First, fungal virulence was examined under optimal laboratory conditions (25 °C, 85% RH) by placing T. leucotreta eggs on conidia-impregnated filter paper. One-day-old eggs and first instar larvae were susceptible to both fungi. In contrast, 5-day-old eggs (advanced embryo development) were susceptible to M. brunneum, but not to B. bassiana. The activity of both fungi against eggs was assessed under two humidity regimes: 85% RH-optimal for fungal germination, and 60% RH-the average humidity in the laboratory. Pieces of parchment paper serving as oviposition surfaces were treated with each of the fungi and introduced to gravid females at different time points after inoculation (0, 2, 7 and 14 days). Although the tested fungal species differed in their virulence to T. leucotreta eggs, both reduced hatching rate under both humidity regimes to 8.3-58.3%, compared to 71.7-83.3% in the control treatments. To evaluate reduction of T. leucotreta infestation of fruit, 'Ori' citrus fruit (easy peeler Citrus sinensis) were treated with each of the fungi. Eggs were placed on the fruit peels and the fruit were maintained under room conditions (25 °C, 60% RH). Between 41.7% and 54.1% of fruit in control groups were infested by the T. leucotreta larvae. Treatments with either of the fungi resulted in about 16% infestation of the fruit with larvae, a marked (3.3-fold) reduction.

Differential gene expression in tomato fruit and Colletotrichum gloeosporioides during colonization of the RNAi-SlPH tomato line with reduced fruit acidity and higher pH.

BACKGROUND: The destructive phytopathogen Colletotrichum gloeosporioides causes anthracnose disease in fruit. During host colonization, it secretes ammonia, which modulates environmental pH and regulates gene expression, contributing to pathogenicity. However, the effect of host pH environment on pathogen colonization has never been evaluated. Development of an isogenic tomato line with reduced expression of the gene for acidity, SlPH (Solyc10g074790.1.1), enabled this analysis. Total RNA from C. gloeosporioides colonizing wild-type (WT) and RNAi-SlPH tomato lines was sequenced and gene-expression patterns were compared. RESULTS: C. gloeosporioides inoculation of the RNAi-SlPH line with pH 5.96 compared to the WT line with pH 4.2 showed 30% higher colonization and reduced ammonia accumulation. Large-scale comparative transcriptome analysis of the colonized RNAi-SlPH and WT lines revealed their different mechanisms of colonization-pattern activation: whereas the WT tomato upregulated 13-LOX (lipoxygenase), jasmonic acid and glutamate biosynthesis pathways, it downregulated processes related to chlorogenic acid biosynthesis II, phenylpropanoid biosynthesis and hydroxycinnamic acid tyramine amide biosynthesis; the RNAi-SlPH line upregulated UDP-D-galacturonate biosynthesis I and free phenylpropanoid acid biosynthesis, but mainly downregulated pathways related to sugar metabolism, such as the glyoxylate cycle and L-arabinose degradation II. Comparison of C. gloeosporioides gene expression during colonization of the WT and RNAi-SlPH lines showed that the fungus upregulates ammonia and nitrogen transport and the gamma-aminobutyric acid metabolic process during colonization of the WT, while on the RNAi-SlPH tomato, it mainly upregulates the nitrate metabolic process. CONCLUSIONS: Modulation of tomato acidity and pH had significant phenotypic effects on C. gloeosporioides development. The fungus showed increased colonization on the neutral RNAi-SlPH fruit, and limited colonization on the WT acidic fruit. The change in environmental pH resulted in different defense responses for the two tomato lines. Interestingly, the WT line showed upregulation of jasmonate pathways and glutamate accumulation, supporting the reduced symptom development and increased ammonia accumulation, as the fungus might utilize glutamate to accumulate ammonia and increase environmental pH for better expression of pathogenicity factors. This was not found in the RNAi-SlPH line which downregulated sugar metabolism and upregulated the phenylpropanoid pathway, leading to host susceptibility.

Mutation of AREA affects growth, sporulation, nitrogen regulation, and pathogenicity in Colletotrichum gloeosporioides.

The GATA transcription factor AreA is a global nitrogen regulator that restricts the utilization of complex and poor nitrogen sources in the presence of good nitrogen sources in microorganisms. In this study, we report the biological function of an AreA homolog (the CgareA gene) in the fruit postharvest pathogen Colletotrichum gloeosporioides. Targeted gene deletion mutants of areA exhibited significant reductions in vegetative growth, increases in conidia production, and slight decreases in conidial germination rates. Quantitative RT-PCR (qRT-PCR) analysis revealed that the expression of AreA was highly induced under nitrogen-limiting conditions. Moreover, compared to wild-type and complemented strains, nitrogen metabolism-related genes were misregulated in ΔareA mutant strains. Pathogenicity assays indicated that the virulence of ΔareA mutant strains were affected by the nitrogen content, but not the carbon content, of fruit hosts. Taken together, our results indicate that CgareA plays a critical role in fungal development, conidia production, regulation of nitrogen metabolism and virulence in Colletotrichum gloeosporioides.

A Role of AREB in the Regulation of PACC-Dependent Acid-Expressed-Genes and Pathogenicity of Colletotrichum gloeosporioides.

Gene expression regulation by pH in filamentous fungi and yeasts is controlled by the PACC/RIM101 transcription factor. In Colletotrichum gloeosporioides, PACC is known to act as positive regulator of alkaline-expressed genes, and this regulation was shown to contribute to fungal pathogenicity. PACC is also a negative regulator of acid-expressed genes, however; the mechanism of downregulation of acid-expressed genes by PACC and their contribution to C. gloeosporioides pathogenicity is not well understood. RNA sequencing data analysis was employed to demonstrate that PACC transcription factor binding sites (TFBS) are significantly overrepresented in the promoter of PACC-upregulated, alkaline-expressed genes. In contrast, they are not overrepresented in the PACC-downregulated, acid-expressed genes. Instead, acid-expressed genes showed overrepresentation of AREB GATA TFBS in C. gloeosporioides and in homologs of five other ascomycetes genomes. The areB promoter contains PACC TFBS; its transcript was upregulated at pH 7 and repressed in ΔpacC. Furthermore, acid-expressed genes were found to be constitutively upregulated in ΔareB during alkalizing conditions. The areB mutants showed significantly reduced ammonia secretion and pathogenicity on tomato fruit. Present results indicate that PACC activates areB expression, thereby conditionally repressing acid-expressed genes and contributing critically to C. gloeosporioides pathogenicity.

Simultaneous transcriptome analysis of Colletotrichum gloeosporioides and tomato fruit pathosystem reveals novel fungal pathogenicity and fruit defense strategies.

The fungus Colletotrichum gloeosporioides breaches the fruit cuticle but remains quiescent until fruit ripening signals a switch to necrotrophy, culminating in devastating anthracnose disease. There is a need to understand the distinct fungal arms strategy and the simultaneous fruit response. Transcriptome analysis of fungal-fruit interactions was carried out concurrently in the appressoria, quiescent and necrotrophic stages. Conidia germinating on unripe fruit cuticle showed stage-specific transcription that was accompanied by massive fruit defense responses. The subsequent quiescent stage showed the development of dendritic-like structures and swollen hyphae within the fruit epidermis. The quiescent fungal transcriptome was characterized by activation of chromatin remodeling genes and unsuspected environmental alkalization. Fruit response was portrayed by continued highly integrated massive up-regulation of defense genes. During cuticle infection of green or ripe fruit, fungi recapitulate the same developmental stages but with differing quiescent time spans. The necrotrophic stage showed a dramatic shift in fungal metabolism and up-regulation of pathogenicity factors. Fruit response to necrotrophy showed activation of the salicylic acid pathway, climaxing in cell death. Transcriptome analysis of C. gloeosporioides infection of fruit reveals its distinct stage-specific lifestyle and the concurrent changing fruit response, deepening our perception of the unfolding fungal-fruit arms and defenses race.

Seismic sensor-based management of the red palm weevil Rhynchophorus ferrugineus in date palm plantations.

BACKGROUND: The red palm weevil (RPW), Rhynchophorus ferrugineus, is one of the gravest threats to palm trees. The challenge in monitoring RPW primarily arises from the inconspicuous presence of larvae within the stem, which is often devoid of noticeable symptoms. This study looks at the use of seismic sensors in RPW management in commercial date palm plantations. It explores whether the data garnered from the sensor domain, and its translation into the health status of date palms, can reliably inform precise decision-making. RESULTS: Sensor and damage index values, as gauged by the Agrint IoTree seismic sensor, vividly mirrored RPW colonization activity. They also accurately portrayed the impact of three distinct insecticides: imidacloprid, phosphine, and entomopathogenic nematodes. The seismic values and damage index of healthy untreated palms strongly supported the decision to pursue tree recovery. Furthermore, this facilitated the computation of recovery pace discrepancies across the tested treatments, measured as the number of days required for tree restoration. CONCLUSIONS: Our findings underscore the practicality of employing seismic sensors, as exemplified by the IoTree system and its network services, to both monitor and assess palm tree health. Furthermore, it validates their efficacy in evaluating the efficiency of management strategies adopted against RPW, all grounded in a wealth of sensor-derived data. © 2023 Society of Chemical Industry.

Efficacy of Entomopathogenic Fungi as Prevention against Early Life Stages of the Red Palm Weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae) in Laboratory and Greenhouse Trials.

The red palm weevil (RPW) Rhynchophorus ferrugineus is a highly destructive invasive pest for palms whose management is mainly by application of synthetic pesticides. As a key pest of date palm plantations, it is necessary to integrate environmentally safe measures for its management. Entomopathogenic fungi (EPF) have been primarily studied as a preventative control measure due to the horizontal transfer of conidia within the RPW population. We previously demonstrated the horizontal transmission of fungal conidia from an egg-laying surface to the female weevil and then to the eggs and larvae. Based on that strategy, this study aimed to evaluate the virulence of commercial EPF products and laboratory EPF preparations to RPW females and their progeny, and their ability to protect palms against infestation. As such, it serves as a screening platform for field experiments. Mortality rates of females and eggs depended on the applied treatment formulation and fungal strain. Velifer®, a Beauveria bassiana product, and Metarhizium brunneum (Mb7) resulted in 60-88% female mortality. Mb7-as a conidial suspension or powder-resulted in 18-21% egg-hatching rates, approximately 3 times less than in the non-treated control. Treating palms with Mb7 suspension or dry formulation significantly inhibits infestation signs and results in protection. These results lay the foundation for investigating the protective rate of EPF products against RPW in date plantations.

Type VII secretion system and its effect on group B Streptococcus virulence in isolates obtained from newborns with early onset disease and colonized pregnant women.

Introduction: GBS may cause a devastating disease in newborns. In early onset disease of the newborn the bacteria are acquired from the colonized mother during delivery. We characterized type VII secretion system (T7SS), exporting small proteins of the WXG100 superfamily, in group B Streptococci (GBS) isolates from pregnant colonized women and newborns with early onset disease (EOD) to better understand T7SS contribution to virulence in these different clinical scenarios. Methods: GBS genomes [N=33, 17 EOD isolates (serotype III/ST17) and 16 colonizing isolates (12 serotype VI/ST1, one serotype VI/ST19, one serotype VI/ST6, and two serotype 3/ST19)] were analyzed for presence of T7SS genes and genes encoding WXG100 proteins. We also perform bioinformatic analysis. Galleria mellonella larvae were used to compare virulence between colonizing, EOD, and mutant EOD isolates. The EOD isolate number 118659 (III/ST17) was used for knocking out the essC gene encoding a membrane-bound ATPase, considered the driver of T7SS. Results: Most GBS T7SS loci encoded core component genes: essC, membrane-embedded proteins (essA; essB), modulators of T7SS activity (esaA; esaB; esaC) and effectors: [esxA (SAG1039); esxB (SAG1030)].Bioinformatic analysis indicated that based on sequence type (ST) the clinicalGBS isolates encode at least three distinct subtypes of T7SS machinery. In all ST1isolates we identified two copies of esxA gene (encoding putative WXG100proteins), when only 23.5% of the ST17 isolates harbored the esxA gene. Five ST17isolates encoded two copies of the essC gene. Orphaned WXG100 molecule(SAG0230), distinct from T7SS locus, were found in all tested strains, except inST17 strains where the locus was found in only 23.5% of the isolates. In ST6 andST19 isolates most of the structure T7SS genes were missing. EOD isolates demonstrated enhanced virulence in G. mellonella modelcompared to colonizing isolates. The 118659DessC strain was attenuated in itskilling ability, and the larvae were more effective in eradicating 118659DessC. Conclusions: We demonstrated that T7SS plays a role during infection. Knocking out the essC gene, considered the driver of T7SS, decreased the virulence of ST17 responsible for EOD, causing them to be less virulent comparable to the virulence observed in colonizing isolates.

The Dissemination of Metarhizium brunneum Conidia by Females of the Red Palm Weevil, Rhynchophorus ferrugineus, Suggests a New Mechanism for Prevention Practices.

Direct contact between the conidia of entomopathogenic fungi (EPF) and their host is a prerequisite to successful infection; the host can, therefore, be infected by both direct treatment and by transmission of fungal inoculum from infested surfaces. This unique characteristic makes EPF especially relevant for the control of cryptic insects. In the case of the red palm weevil (RPW) Rhynchophorus ferrugineus, the eggs and larvae are almost inaccessible to direct-contact treatment. The objective of the present study was to investigate the mechanism of conidia transmission from a treated surface to host eggs and larvae. Foam pieces infested with Metarhizium brunneum conidial powder, conidial suspension, or distilled water were used as a laying surface for RPW females. The number of eggs laid was not affected by the EPF treatments and ranged from 2 to 14 eggs per female. However, hatching rate and larval survival were significantly reduced in the conidial powder treatment, resulted in 1.5% hatching and no live larvae. In the conidial suspension treatment, 21% of laid eggs hatched, compared to 72% in the control treatment. In both M. brunneum treatments, females' proboscis, front legs and ovipositor were covered with conidia. The females transferred conidia in both treatments to the laying holes, reaching up to 15 mm in depth. This resulted in reduced egg-hatching rate and significant larval mortality due to fungal infection. The stronger effect on egg and larval survival using dry conidia seemed to result from better conidial adhesion to the female weevil in this formulation. In future studies, this dissemination mechanism will be examined as a prevention strategy in date plantations.

Pathogen Eradication in Garlic in the Phytobiome Context: Should We Aim for Complete Cleaning?

Global food production is challenged by plant pathogens that cause significant crop losses. Fungi, bacteria, and viruses have long threatened sustainable and profitable agriculture. The danger is even higher in vegetatively propagated horticultural crops, such as garlic. Currently, quarantine, rouging infected plants, and control of natural vectors are used as the main means of disease and pest control in garlic crops. Agricultural biotechnology, meristem-tip culture, and cryotherapy offer solutions for virus eradication and for the multiplication of 'clean stocks', but at the same time, impact the symbiotic and beneficial components of the garlic microbiome. Our research involves the first metatranscriptomic analysis of the microbiome of garlic bulb tissue, PCR analyses, and a biological assay of endophytes and pathogens. We have demonstrated that in vitro sanitation methods, such as shoot tip culture or cryotherapy can alter the garlic microbiome. Shoot tip culture proved ineffective in virus elimination, but reduced bacterial load and eliminated fungal infections. Conversely, cryotherapy was efficient in virus eradication but demolished other components of the garlic microbiome. Garlic plants sanitized by cryotherapy exhibited a lower survival rate, and a longer in vitro regeneration period. The question arises whether total eradication of viruses, at the expense of other microflora, is necessary, or if a partial reduction in the pathogenic load would suffice for sanitized garlic production. We explore this question from both scientific and commercial perspectives.

Resistant ticks inhibit Metarhizium infection prior to haemocoel invasion by reducing fungal viability on the cuticle surface.

We studied disease progression of, and host responses to, four species in the Metarhizium anisopliae complex expressing green fluorescent protein (GFP). We compared development and determined their relative levels of virulence against two susceptible arthropods, the cattle tick Rhipicephalus annulatus and the lepidopteran Galleria mellonella, and two resistant ticks, Hyalomma excavatum and Rhipicephalus sanguineus. Metarhizium brunneum Ma7 caused the greatest mortality of R. annulatus, Metarhizium robertsii ARSEF 2575 and Metarhizium pingshaense PPRC51 exhibited intermediate levels of virulence, and Metarhizium majus PPRC27 caused low mortality of cattle ticks. Conidia of all four species germinated on all hosts examined, but on resistant hosts, sustained hyphal growth was inhibited and GFP emission steadily and significantly decreased over time, suggesting a loss of fungal viability. Cuticle penetration was observed only for the three most virulent species infecting susceptible hosts. Cuticles of resistant and susceptible engorged female ticks showed significant increases in red autofluorescence at sites immediately under fungal hyphae. This is the first report (i) of tick mortality occurring after cuticle penetration but prior to haemocoel colonization and (ii) that resistant ticks do not support development of Metarhizium germlings on the outer surface of the cuticle. Whether reduced Metarhizium viability on resistant tick cuticles is due to antibiosis or limited nutrient availability is unknown.

Survival and efficacy of entomopathogenic nematodes on exposed surfaces.

Entomopathogenic nematodes (EPN) species differ in their capability to withstand rapid desiccation (RD). Infective juveniles of Steinernema carpocapsae are a better adaptable and tolerant than Steinernema feltiae or Heterorhabditis bacteriophora as, an optimal RH of > 90% is required by S. feltiae and H. bacteriophora while maintaining RH equivalent to 74% could sustain survival of S. carpocapsae under RD. Our findings from infectivity suggest that following application, shrunk IJs are acquired passively by the larvae, probably rehydrate and resume infection within the insect gut. Water loss rate is a key factor affecting survival of S. carpocapsae on exposed surfaces. The present study provides the foundation for characterizing mechanism of rapid rate of water loss in EPN. ATR-FTIR is a rapid and reliable method for analysis of water loss. Changes in peak intensity was observed at 3100-3600 cm-1 (OH bonds of water), 2854 cm-1 (CH stretching of symmetric CH2, acyl chains), 2924 cm-1 (CH stretching of anti-symmetric CH2, lipid packing heterogeneity), 1634 cm-1 (amide I bonds) indicate major regions for hydration dependent changes in all EPN species. FTIR data also indicates that, S. carpocapsae contains strong water interacting regions in their biochemical profile, which could be an influencing factor in their water holding capacity under RD. ATR-FTIR were correlated to water content determined gravimetrically by using Partial Least square -Regression and FTIR multivariate method, which could be used to screen a formulation's potential to maintain or delay the rate of water loss in a rapid and efficient manner.

The SWC4 subunit of the SWR1 chromatin remodeling complex is involved in varying virulence of Metarhizium brunneum isolates offering role of epigenetic regulation of pathogenicity.

The host - pathogen interaction is a multifactorial process subject to a co-evolutionary arms race consisting of rapid changes in both host and pathogen, controlled at the genetic and epigenetic levels. Previously, we showed intra-species variation in disease progression and pathogenicity in aphids for Metarhizium brunneum isolates MbK and Mb7. Herein, we compared genomic, epigenetic, and metabolomic variations between these isolates and their effects on pathogenicity. Genomic variation could not completely explain the observed differences between the isolates. However, differential N6-adenine methylation (6 mA) and its correlation to reduced expression of the essential SWC4 subunit of SWR1 chromatin-remodelling complex (SWR1-C) led us to hypothesize a role for swc4 in the varying pathogenicity. Mutagenesis of the essential swc4 gene in MbKisolate resulted in reduction of secondary-metabolite (SM) secretion and impaired virulence in Galleria mellonella. Our results suggest the role of SWC4 in the regulation of SMs and the role of both SWC4 and SWR1-C in virulence of M. brunneum isolates. A better understanding of epigenetic regulation of SM production and secretion in entomopathogenic fungi may enable theirmanipulation for better biocontrol performance, and expand possibilities for environmentally friendly pest control.

Encapsulation of Bacillus thuringiensis in an inverse Pickering emulsion for pest control applications.

Here, we present an inverse Pickering emulsion-based formulation for Bacillus thuringiensis serovar aizawai (BtA) encapsulations utilized towards pest control applications. The emulsification was carried out by high shear homogenization process via ULTRA-TURRAX®. The water-in-mineral oil emulsions were stabilized by commercial hydrophobic silica. Different silica contents and water/oil ratios were studied. Stable emulsions were obtained at 2 and 3 wt% silica at 30% and 20% water volumes, respectively. The structure of the Pickering emulsions were characterized by laser scanning confocal microscopy and cryogenic scanning electron microscopy. The BtA cells, spores and crystals were encapsulated in the water droplets of the inverse Pickering emulsions. An emulsion composed of 3 wt% silica and 30% water was found to be the most suitable for encapsulation. The pest control efficiency of the encapsulated BtA against Spodoptera littoralis first instar larvae was tested. The studied BtA/emulsion system exhibited a mortality rate of 92%. However, the non-formulated BtA has shown 71% mortality, and the emulsion alone resulted in only 9% mortality. These findings confirm that an emulsion with encapsulated BtA can function as an efficient formulation for biopesticides.

Individual Coating of Entomopathogenic Nematodes with Titania (TiO2) Nanoparticles Based on Oil-in-Water Pickering Emulsion: A New Formulation for Biopesticides.

This study presents a new eco-friendly formulation of entomopathogenic nematodes (EPNs) based on individual coating of EPNs with titanium dioxide (TiO2) nanoparticles (NPs) and mineral oil via oil-in-water Pickering emulsions. Mineral oil-in-water emulsions stabilized by amine-functionalized titanium dioxide (TiO2-NH2) particles were prepared. 40:60 and 50:50 oil-water volume ratios using 2 wt % TiO2-NH2 particles were found to be the most stable emulsions with a droplet size suitable for the formulation and were further studied for their toxicity against the incorporated EPNs. Carboxyfluorescein was covalently bonded to TiO2-NH2 NPs, and the resulting composite was observed via fluorescence confocal microscopy. The dry coating was evaluated using SEM and confocal microscopy, which showed significant nematode coverage by the particles and oil. The final formulation was biocompatible with the studied EPNs, where the viability of the EPNs in the formulation was equivalent to control aqueous suspension after 120 days. Finally, yields of nematodes from infected Galleria mellonella cadavers collected for 150 days showed no significant differences (P > 0.05) using the tested emulsions compared to the control containing nematodes in water.

Intraspecies variation of Metarhizium brunneum against the green peach aphid, Myzus persicae, provides insight into the complexity of disease progression.

BACKGROUND: Intensive application of chemical insecticides is required for aphid pest control. Among the biorational alternatives, entomopathogenic fungi are the most sustainable biocontrol agents; those of the order Hypocreales attack and cause fungal disease in arthropod hosts, with variations in host susceptibility attributed to both fungal and host characteristics. We evaluated inter- and intraspecies variations in Metarhizium spp. virulence and differences in fungal disease progression on adult and nymph stages of the green peach aphid, Myzus persicae (Sulzer), a parthenogenetically reproducing insect species. RESULTS: Minor interspecies diversity was detected between the generalist Metarhizium species examined. Interestingly, significant intraspecies diversity was observed between Metarhizium brunneum isolates Mb7 and MbK. Infected adult aphids demonstrated similar disease progression for both isolates, mortality rates of more than 80% and fivefold reduction in fecundity. However, nymph mortality was detected only following MbK infection, with 50% mortality and significant reduction in molting rates. Confocal laser scanning microscopy demonstrated the variation in the disease stages of conidial adhesion and hemocoel colonization on each examined day post inoculation for each isolate. Significantly faster disease progression was observed in MbK-infected versus Mb7-infected nymphs, the latter demonstrating a higher percentage of uninfected nymphs accompanied with aphid molting. CONCLUSIONS: The observed intraspecies variation suggests that altered conidial adhesion to the nymph cuticle is a major factor affecting virulence. We prove the role of nymph ecdysis as a defense mechanism disrupting fungal infection. Because significant differences were observed between closely related isolates, this study emphasizes the importance of appropriate isolate selection for biological control. © 2021 Society of Chemical Industry.