The effect of natural products used as pesticides on the soil microbiota: OECD 216 nitrogen transformation test fails to identify effects that were detected via q-PCR microbial abundance measurement.

BACKGROUND: Natural products present an environmentally attractive alternative to synthetic pesticides which have been implicated in the off-target effect. Currently, the assessment of pesticide toxicity on soil microorganisms relies on the OECD 216 N transformation assay (OECD stands for the Organisation Economic Co-operation and Development, which is a key international standard-setting organisation). We tested the hypotheses that (i) the OECD 216 assay fails to identify unacceptable effects of pesticides on soil microbiota compared to more advanced molecular and standardized tests, and (ii) the natural products tested (dihydrochalcone, isoflavone, aliphatic phenol, and spinosad) are less toxic to soil microbiota compared to a synthetic pesticide compound (3,5-dichloraniline). We determined the following in three different soils: (i) ammonium (NH4 +) and nitrate (NO3 -) soil concentrations, as dictated by the OECD 216 test, and (ii) the abundance of phylogenetically (bacteria and fungi) and functionally distinct microbial groups [ammonia-oxidizing archaea (AOA) and bacteria (AOB)] using quantitative polymerase chain reaction (q-PCR). RESULTS: All pesticides tested exhibited limited persistence, with spinosad demonstrating the highest persistence. None of the pesticides tested showed clear dose-dependent effects on NH4 + and NO3 - levels and the observed effects were <25% of the control, suggesting no unacceptable impacts on soil microorganisms. In contrast, q-PCR measurements revealed (i) distinct negative effects on the abundance of total bacteria and fungi, which were though limited to one of the studied soils, and (ii) a significant reduction in the abundance of both AOA and AOB across soils. This reduction was attributed to both natural products and 3,5-dichloraniline. CONCLUSION: Our findings strongly advocate for a revision of the current regulatory framework regarding the toxicity of pesticides to soil microbiota, which should integrate advanced and well-standardized tools. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

2-oxoglutarate-dependent dioxygenases drive expansion of steroidal alkaloid structural diversity in the genus Solanum.

Solanum steroidal glycoalkaloids (SGAs) are renowned defence metabolites exhibiting spectacular structural diversity. Genes and enzymes generating the SGA precursor pathway, SGA scaffold and glycosylated forms have been largely identified. Yet, the majority of downstream metabolic steps creating the vast repertoire of SGAs remain untapped. Here, we discovered that members of the 2-OXOGLUTARATE-DEPENDENT DIOXYGENASE (2-ODD) family play a prominent role in SGA metabolism, carrying out three distinct backbone-modifying oxidative steps in addition to the three formerly reported pathway reactions. The GLYCOALKALOID METABOLISM34 (GAME34) enzyme catalyses the conversion of core SGAs to habrochaitosides in wild tomato S. habrochaites. Cultivated tomato plants overexpressing GAME34 ectopically accumulate habrochaitosides. These habrochaitoside enriched plants extracts potently inhibit Puccinia spp. spore germination, a significant Solanaceae crops fungal pathogen. Another 2-ODD enzyme, GAME33, acts as a desaturase (via hydroxylation and E/F ring rearrangement) forming unique, yet unreported SGAs. Conversion of bitter α-tomatine to ripe fruit, nonbitter SGAs (e.g. esculeoside A) requires two hydroxylations; while the known GAME31 2-ODD enzyme catalyses hydroxytomatine formation, we find that GAME40 catalyses the penultimate step in the pathway and generates acetoxy-hydroxytomatine towards esculeosides accumulation. Our results highlight the significant contribution of 2-ODD enzymes to the remarkable structural diversity found in plant steroidal specialized metabolism.

Regulation of two homodimer hexosaminidases in the mycoparasitic fungus Trichoderma asperellum by glucosamine.

Trichoderma asperellum is a mycoparasitic fungus which is used as a biocontrol agent against plant pathogens. Its hydrolytic enzymes take part in its parasitic interaction, degrading the pathogen cell wall and thereby helping to control disease. One of those enzymes, beta- N-acetyl- d-glucosaminidase (GlcNAcase), degrades chitin, which is a major component of the cell wall of many plant-pathogenic fungi. Two GlcNAcases of T. asperellum T203, designated EXC1Y and EXC2Y, were purified, their genes and their promoters were sequenced, and their regulation was studied. The enzymes share homology (59% identity) but are easily distinguished by PAGE assay. Biochemical characterization, Edman degradation, and mass spectrometry demonstrated that EXC1Y and EXC2Y are both active as homodimers. Both genes are up-regulated by glucosamine (GlcN), in contrast to two endochitinases of this fungus. GlcN induces the secretion of several proteins (including a beta-glucosidase), among which EXC1Y is the most abundant. An exc2y knockout was constructed, to study the regulation of EXC1Y expression and secretion. The fungus has the ability to store a high amount of this enzyme in an active form and secrete it into the medium later.

Expression regulation of the endochitinase chit36 from Trichoderma asperellum (T. harzianum T-203).

The presence of the endochitinase CHIT36 from Trichoderma harzianum TM was assessed in several antagonistic Trichoderma strains belonging to different molecular taxonomic groups. CHIT37 from T. harzianum CECT 2413 was sequenced and found to display 89% homology with CHIT36 at the amino acid level. Northern analysis showed that chit36Y from T. asperellum is regulated both by glucose and nitrogen levels. Stress conditions, colloidal chitin and N-acetyl-glucosamine are effective inducers of this gene. The promoter of chit36Y was cloned and was used to direct expression of a gfp reporter gene in Trichoderma transformants. Confrontation experiments with the plant pathogen Rhizoctonia solani revealed that direct contact between the fungi is not necessary for gfp expression. The R. solani-inducing factor appears to be a soluble molecule capable of diffusing through a dialysis membrane (<12 kDa). CHIT36 recombinant protein from the yeast Pichia pastoris was active against different phytopathogens, confirming the importance of this endochitinase in the mycoparasitic activity of Trichoderma antagonistic strains.

Significance of lytic enzymes from Trichoderma spp. in the biocontrol of fungal plant pathogens.

The use of specific mycolytic soil microorganisms to control plant pathogens is an ecological approach to overcome the problems caused by standard chemical methods of plant protection. The ability to produce lytic enzymes is a widely distributed property of rhizosphere-competent fungi and bacteria. Due to the higher activity of Trichoderma spp. lytic enzymes as compared to the same class of enzymes from other microorganisms and plants, effort is being aimed at improving biocontrol agents and plants by introducing Trichoderma genes via genetic manipulations. An overview is presented of the data currently available on lytic enzymes from the mycoparasitic fungus Trichoderma.