Carbon regulation of environmental pH by secreted small molecules that modulate pathogenicity in phytopathogenic fungi

[EN]Fruit pathogens can contribute to the acidification or alkalinization of the host environment. This capability has been used to divide fungal pathogens into acidifying and/or alkalinizing classes. Here, we show that diverse classes of fungal pathogens—Colletotrichum gloeosporioides, Penicillium expansum, Aspergillus nidulans and Fusarium oxysporum—secrete small pH-affecting molecules. These molecules modify the environmental pH, which dictates acidic or alkaline colonizing strategies, and induce the expression of PACC-dependent genes. We show that, in many organisms, acidification is induced under carbon excess, i.e. 175 mm sucrose (the most abundant sugar in fruits). In contrast, alkalinization occurs under conditions of carbon deprivation, i.e. less than 15 mm sucrose. The carbon source is metabolized by glucose oxidase (gox2) to gluconic acid, contributing to medium acidification, whereas catalysed deamination of non-preferred carbon sources, such as the amino acid glutamate, by glutamate dehydrogenase 2 (gdh2), results in the secretion of ammonia

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

IntroductionGBS 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.MethodsGBS 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.ResultsMost 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.ConclusionsWe 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

Preventative Approach to Microbial Control of Capnodis tenebrionis by Soil Application of Metarhizium brunneum and Beauveria bassiana

Management of the Mediterranean flat-headed root-borer, Capnodis tenebrionis, is critical due to the larvae’s root localization. Neonate larvae can be exposed to natural enemies before penetrating the roots. Application of Metarhizium brunneum strain Mb7 and Beauveria bassiana strain GHA formulations on rice granules was investigated for their efficacy against C. tenebrionis larvae. Mb7 application, evaluated on apricot twigs, significantly and dose-dependently reduced colonization rates of neonates, with highest mortality at 108 conidia/g soil. Neonate susceptibility to Mb7 and GHA was evaluated on potted rootstocks (GF677 almond × peach, 2729 plum) planted in entomopathogenic fungi (EPF)-premixed soil (1.3–1.6 × 105 conidia/cm3 soil) or in EPF-free soil surface-treated with 5 g Mb7 fungal granules (1.25 × 109 conidia). Larval colonization rates were reduced 7.4-fold in 2729 by both fungi; only Mb7 completely prevented colonization of GF677 by larvae. Larvae inside plant galleries exhibited mycosis with EPF-treated soils and both fungi proliferated on larval frass. Mb7 conidia germinated in the rhizosphere of GF677, and conidia of both fungi remained viable throughout the trial. Galleria baiting technique was used on EPF-treated soil to evaluate EPF infectivity over time; Mb7 and GHA persisted 180 and 90 days post inoculation, respectively. The formulation (fungus-covered rice grains), delivery method (mixing with soil) and persistence (3–6 months) of Mb7 and GHA are feasible for potential field application to control C. tenebrionis

Comprehensive Analysis of Management Strategies for Red Palm Weevil in Date Palm Settings, Emphasizing Sensor-Based Infestation Detection

The red palm weevil (Rhynchophorus ferrugineus) inflicts widespread damage in date palm plantations and urban settings, leading to stand loss and safety concerns, intensified by the economic and ecological burdens of synthetic preventive treatments. A novel approach emphasizes detecting weevil-infested palms through sensors and targeting control efforts at colonized trees. This study analyzes two datasets: field manager-reported results from settings with preventive pest control versus seismic sensor-monitored units. Sensor data translate into damage index values, exploring curative treatments, attack likelihood based on infestation history, and the impact of sensor-based management on infestation incidence decline. Findings reveal lower weevil infestation in urban areas and preventive-treated palm settings. A strong correlation exists between sensor indications and grower decisions in date plantations. Palms previously infested are more prone to damage. Long-term sensor-based management reduces weevil-affected palm numbers significantly. The high infestation in sensor-equipped date palm settings stems from a gap between sensor-identified and preventively treated palms. Controlling weevils based on sensor indications aligns practically with grower curative treatments, preventing significant damage. Weevils target previously infested palms, optimizing pest control through a continuous strategy integrating sensor monitoring with prompt response treatments. This proves highly effective, offering a cost-efficient alternative to preventive treatment reliance

Interactions of Metarhizium brunneum-7 with Phytophagous Mites Following Different Application Strategies

Metarhizium brunneum is a generalist entomopathogenic fungus known to be virulent against Acari. We investigated Metarhizium brunneum-7 (Mb7) interactions in three systems of phytophagous mites and their respective plant hosts: Volkamer lemon (Citrus volkameriana) and the citrus rust mite Phyllocoptruta oleivora; common bean (Phaseolus vulgaris) and the two-spotted spider mite Tetranychus urticae; and spring onion (Allium cepa) and the bulb mite Rhizoglyphus robini. All three mite species were susceptible to directly applied Mb7 conidia. Results obtained using the standard method for studying endophytic colonization vs. live confocal imaging of plant tissues using the green fluorescent protein (GFP)-transformed fungus differed markedly, demonstrating that microscopy validation was more definite than the standard process of recovery from plant tissue. Endophytic colonization was observed in conidium-infiltrated citrus leaves and in roots of onion plants treated with soil-drenched conidia, but not in common bean treated by either spray or drench of conidia. Endophytic colonization of citrus leaves did not affect the citrus mite population. Drench application in common bean reduced two-spotted mite population. Similarly, drench application in onion reduced bulb mite population. This study emphasizes the importance of the host plant effects on Mb7 control efficacy of mite pests, and the merits of live-imaging techniques in studying endophytic interaction

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

Characterization of the phenotypic and genotypic tolerance to abiotic stresses of natural populations of Heterorhabditis bacteriophora

Abstract Entomopathogenic nematodes are effective biocontrol agents against arthropod pests. However, their efficacy is limited due to sensitivity to environmental extremes. The objective of the present study was to establish a foundation of genetic-based selection tools for beneficial traits of heat and desiccation tolerance in entomopathogenic nematodes. Screening of natural populations enabled us to create a diverse genetic and phenotypic pool. Gene expression patterns and genomic variation were studied in natural isolates. Heterorhabditis isolates were phenotyped by heat- and desiccation-stress bioassays to determine their survival rates compared to a commercial line. Transcriptomic study was carried out for the commercial line, a high heat-tolerant strain, and for the natural, low heat-tolerant isolate. The results revealed a higher number of upregulated vs. downregulated transcripts in both isolates vs. their respective controls. Functional annotation of the differentially expressed transcripts revealed several known stress-related genes and pathways uniquely expressed. Genome sequencing of isolates with varied degrees of stress tolerance indicated variation among the isolates regardless of their phenotypic characterization. The obtained data lays the groundwork for future studies aimed at identifying genes and molecular markers as genetic selection tools for enhancement of entomopathogenic nematodes ability to withstand environmental stress conditions

Diversity of Bacterial Biota in Capnodis tenebrionis (Coleoptera: Buprestidae) Larvae

The bacterial biota in larvae of Capnodis tenebrionis, a serious pest of cultivated stone-fruit trees in the West Palearctic, was revealed for the first time using the MiSeq platform. The core bacterial community remained the same in neonates whether upon hatching or grown on peach plants or an artificial diet, suggesting that C. tenebrionis larvae acquire much of their bacterial biome from the parent adult. Reads affiliated with class levels Gammaproteobacteria and Alphaproteobacteria (phylum Proteobacteria ca. 86%), and Actinobacteria (ca. 14%) were highly abundant. Most diverse reads belong to the families Xanthomonadaceae (50%), Methylobacteriaceae (20%), Hyphomicrobiaceae (9%), Micrococcaceae (7%) and Geodermatophilaceae (4.5%). About two-thirds of the reads are affiliated with the genera Lysobacter, Microvirga, Methylobacterium, and Arthrobacter, which encompass species displaying cellulolytic and lipolytic activities. This study provides a foundation for future studies to elucidate the roles of bacterial biota in C. tenebrionis

Novel Technique for Quantifying Adhesion of Metarhizium anisopliae Conidia to the Tick Cuticle▿

The present study describes an accurate quantitative method for quantifying the adherence of conidia to the arthropod cuticle and the dynamics of conidial germination on the host. The method was developed using conidia of Metarhizium anisopliae var. anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae) and engorged Rhipicephalus annulatus (Say) (Arachnida: Ixodidae) females and was also verified for M. anisopliae var. acridum Driver et Milner (Hypocreales: Clavicipitaceae) and Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) larvae. This novel method is based on using an organic solvent (dichloromethane [DCM]) to remove the adhered conidia from the tick cuticle, suspending the conidia in a detergent solution, and then counting them using a hemocytometer. To confirm the efficacy of the method, scanning electron microscopy (SEM) was used to observe the conidial adherence to and removal from the tick cuticle. As the concentration of conidia in the suspension increased, there were correlating increases in both the number of conidia adhering to engorged female R. annulatus and tick mortality. However, no correlation was observed between a tick's susceptibility to fungal infection and the amount of adhered conidia. These findings support the commonly accepted understanding of the nature of the adhesion process. The mechanism enabling the removal of the adhered conidia from the host cuticle is discussed

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