Production and characterization of Trichoderma asperellum chitinases and their use in synergy with Bacillus thuringiensis for lepidopteran control.

BACKGROUND: Despite their known negative effects on ecosystems and human health, synthetic pesticides are still largely used to control crop insect pests. Currently, the biopesticide market for insect biocontrol mainly relies on the entomopathogenic bacterium Bacillus thuringiensis (Bt). New biocontrol tools for crop protection might derive from fungi, in particular from Trichoderma spp., which are known producers of chitinases and other bioactive compounds able to negatively affect insect survival. RESULTS: In this study, we first developed an environmentally sustainable production process for obtaining chitinases from Trichoderma asperellum ICC012. Then, we investigated the biological effects of this chitinase preparation - alone or in combination with a Bt-based product - when orally administered to two lepidopteran species. Our results demonstrate that T. asperellum efficiently produces a multi-enzymatic cocktail able to alter the chitin microfibril network of the insect peritrophic matrix, resulting in delayed development and larval death. The co-administration of T. asperellum chitinases and sublethal concentrations of Bt toxins increased larval mortality. This synergistic effect was likely due to the higher amount of Bt toxins that passed the damaged peritrophic matrix and reached the target receptors on the midgut cells of chitinase-treated insects. CONCLUSION: Our findings may contribute to the development of an integrated pest management technology based on fungal chitinases that increase the efficacy of Bt-based products, mitigating the risk of Bt-resistance development. © 2024 Society of Chemical Industry.

Analysis of apocarotenoid volatiles from lettuce (Lactuca sativa) induced by insect herbivores and characterization of carotenoid cleavage dioxygenase gene.

Plant apocarotenoids have been shown to have a diverse biological role in herbivore-plant interactions. Despite their importance, little is known about herbivores' effect on apocarotenoid emissions in Lactuca sativa. In this study, we examined changes in apocarotenoid emissions in lettuce leaves after infestation by two insects, viz., Spodoptera littoralis larvae and Myzus persicae aphids. We found that ß-ionone and ß-cyclocitral showed higher concentrations than the other apocarotenoids, with a significant increase as per the intensity of infestation of both herbivore species. Furthermore, we performed functional characterization of Lactuca sativa carotenoid cleavage dioxygenase 1 (LsCCD1) genes. Three LsCCD1 genes were overexpressed in E. coli strains, and recombinant proteins were assayed for cleavage activity on an array of carotenoid substrates. The LsCCD1 protein cleaved ß-carotene at the 9,10 (9',10') positions producing ß-ionone. The transcript analysis of LsCCD1 genes revealed differential expression patterns under varying levels of herbivores' infestation, but the results were inconsistent with the pattern of ß-ionone concentrations. Our results suggest that LsCCD1 is involved in the production of ß-ionone, but other regulatory factors might be involved in its induction in response to herbivory. These results provide new insights into apocarotenoid production in response to insect herbivory in lettuce. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03511-4.

Identification and characterization of three nearly identical linalool/nerolidol synthase from Acorus calamus.

Acorus calamus is a perennial aromatic medicinal plant from the Acorusaceae family, known for its pharmaceutical and medicinal value. A combined chemical, biochemical, and molecular study was conducted to evaluate the differential accumulation of volatile organic compounds (VOCs) in rhizomes and leaves of A. calamus essential oil. Here, we performed VOC profiling and transcriptome-based identification and functional characterization of terpene synthase (TPS) genes. A total of 110 VOCs were detected from the rhizomes and leaves of A. calamus, and some VOCs showed significant differences between them. The further transcriptome-based analysis led to the identification of six putative TPSs genes. In phylogenetic analysis, three TPSs belonged to the TPS-g clade, one to each of the TPS-a, TPS-c, and TPS-e clades. The heterologous E. coli-based expression of recombinant TPSs identified three genes (AcTPS3, AcTPS4, and AcTPS5) as bifunctional linalool/nerolidol synthase. The correlation of TPS gene expression and VOC metabolite profiles supported the function of these genes in A. calamus. Our findings provide a roadmap for future efforts to enhance the molecular mechanisms of terpene biosynthesis and our understanding of Acorus-insect interactions.

A Review of the Botany, Volatile Composition, Biochemical and Molecular Aspects, and Traditional Uses of Laurus nobilis.

Laurus nobilis L. is an aromatic medicinal plant widely cultivated in many world regions. L. nobilis has been increasingly acknowledged over the years as it provides an essential contribution to the food and pharmaceutical industries and cultural integrity. The commercial value of this species derives from its essential oil, whose application might be extended to various industries. The chemical composition of the essential oil depends on environmental conditions, location, and season during which the plants are collected, drying methods, extraction, and analytical conditions. The characterization and chemotyping of L. nobilis essential oil are extremely important because the changes in composition can affect biological activities. Several aspects of the plant's secondary metabolism, particularly volatile production in L. nobilis, are still unknown. However, understanding the molecular basis of flavor and aroma production is not an easy task to accomplish. Nevertheless, the time-limited efforts for conservation and the unavailability of knowledge about genetic diversity are probably the major reasons for the lack of breeding programs in L. nobilis. The present review gathers the scientific evidence on the research carried out on Laurus nobilis L., considering its cultivation, volatile composition, biochemical and molecular aspects, and antioxidant and antimicrobial activities.

Identification of a wild carrot as carrot psylla (Bactericera trigonica) attractant and host plant chemistry.

Carrot psylla is one of the devastating pests of carrot throughout northern Europe and the Mediterranean basin. Here we characterized the behavioral response of psylla females towards different carrot germplasm and identified the chemical cues involved in the host selection of psylla females by oviposition choice experiments and metabolic profiling of leaf volatiles. In choice assays, carrot psylla displayed differential responses to tested 14 germplasm. Among germplasm, wild accessions 21793 and 20465 were highly preferred by carrot psylla, while wild accessions 20465 and the orange cultivar Nairobi were less. In non-choice experiments conducted only with this four-germplasm revealed that the carrot psylla females gave higher preference to the Nairobi and wild accession 20465, indicating the vicinity to other host plants in the same area might affect female preference. Moreover, the nymph development and survival experiments showed the lowest nymphs survival rate on the wild accessions 21793 and 20497. Furthermore, the volatile emissions among different carrot cultivars infested with psylla showed qualitative and quantitative differences versus intact plants. Among these volatiles, apiol, ß-asarone, myristicin, and sabinene showed a relationship with psyllas growth and survival. We also showed that myristicin and sabinene exogenous applications caused a dramatic reduction in the number of eggs laid by psylla and subsequent nymph survival. This is an initial study of the volatiles that mediate attraction and oviposition preference of carrot psylla in response to its host plant. The results from this study provide baseline information for the development of new control strategies against carrot psylla.

Characterization of terpene synthase genes potentially involved in black fig fly (Silba adipata) interactions with Ficus carica.

The black fig fly (Silba adipata) is one of the major pests of figs worldwide. This study investigated the effect of pollination on black fig fly infestation and volatile emission during fruit development of facultative parthenocarpic Ficus carica. The results from in-field oviposition preference of black fig fly, olfactory analysis, and fruit volatile profiles indicate that the black fig fly gave a strong preference to unpollinated figs that showed higher emissions of volatile organic compounds. Terpenes are known to be important compounds determining many insect-plant interactions, so we report a transcriptome-based identification and functional characterization of a terpene synthase (TPS) gene family in F. carica. The protein expression in Escherichia coli of eight terpene synthases (TPSs) revealed that three were monoterpene synthases belonging to the TPS-b clade, with FcTPS6 catalyzing the formation of 1,8-cineole while the other two converted GPP into linalool. Four sesquiterpene synthases from the TPS-a clade catalyze the formation of germacrene D (FcTPS1), E-ß-caryophyllene (FcTPS2), cadinene (FcTPS3) and δ-elemene (FcTPS5) while one sesquiterpene synthase FcTPS4 from the TPS-b clade showed nerolidol synthase activity. Most of the enzymatic products closely matched the volatile terpenes emitted from fig fruits and all the genes were expressed during fruit development. This study provides new insights into fig-insect interactions and understanding the molecular mechanisms of terpene biosynthesis and could provide the foundations for sustainable pest management strategies.

Effects of tillage practices on soil microbiome and agricultural parameters.

No-tillage (NT) is a common soil-conservation management practice with known agricultural advantages and drawbacks. However, its short- and long-term effects on the soil microbiome have not been well established. Here, we compared conventional (CT), minimal (MT) and NT practices in two agricultural fields in the north of Israel over a period of 3 years. Edaphic properties, plant-associated pests, weed species abundance and soil microbial community structure were assessed to examine the effects of tillage. Tillage significantly altered physical and chemical soil properties, and a significant increase in hydrolytic and redox microbial activities was observed in NT soils from both sites. Consistent with this, the microbial community structure of NT samples diverged significantly over time from those of CT samples. Repetitive tillage and even a single tillage event caused significant changes in the relative abundance of microorganisms at taxonomic levels ranging from phylum to OTU. However, no significant difference between treatments was found in microbial community alpha-diversity or crop yield. Conversely, higher levels of weed diversity and some pests number were found in NT samples. Overall, we demonstrate that tillage plays a major role in shaping microbial community structure, and in influencing additional environmental, ecological and agricultural soil parameters.

Analysis of apocarotenoid volatiles during the development of Ficus carica fruits and characterization of carotenoid cleavage dioxygenase genes.

In plants the oxidative cleavage of carotenoid substrates produces volatile apocarotenoids, including ß-ionone, 6-methyl-5-hepten-2-ol, and α-ionone; these compounds are important in herbivore-plant communication. Combined chemical, biochemical, and molecular studies were conducted to evaluate the differential accumulation of carotenoids and volatile apocarotenoids during the development of pollinated and parthenocarpic fig fruits. Pollinated fig fruits showed less emission of apocarotenoid volatiles than the parthenocarpic figs, while in the case of carotenoid pigments, pollinated figs manifested higher accumulation. The apocarotenoids, 6-methyl-5-hepten-2-ol and ß-cyclogeraniol, showed a marked increase after the two weeks of hand-pollination in pollinated and parthenocarpic figs; but afterwards these volatile levels decreased during further fruit development. In addition, we report a transcriptome-based identification and functional characterization of the carotenoid cleavage dioxygenase (FcCCD) genes. These genes were overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. The recombinant FcCCD1A enzyme showed specificity for the 9,10 (9',10') double bond position of cyclic carotenoids to generate α-ionone and ß-ionone, while FcCCD1B cleaved lycopene and an acyclic moiety of δ-carotene, producing 6-methyl-5-hepten-2-one. The qRT-PCR analysis of FcCCD genes revealed differential gene expression during fig fruit development. Our results suggest a role for the FcCCD1genes in apocarotenoid biosynthesis in fig fruits.

Profiling of volatile terpenes from almond (Prunus dulcis) young fruits and characterization of seven terpene synthase genes.

Almond (Prunus dulcis) is an agricultural and economically important fruit tree from the Rosaceae family used in the food industry. The monoterpenes and sesquiterpenes perform important ecological functions such as insecticidal and antifeedant activities against various insects. The young fruits of the different almond varieties were found to produce considerable amounts of terpene volatiles, including linalool and geraniol. To identify terpene synthases (TPSs) involved in the production of these volatile terpenes, existing genome databases of the Rosaceae were screened for almond genes with significant sequence similarity to other plants TPSs. Bioinformatics analysis led to the identification of seven putative TPSs genes with complete open reading frames. We characterized the enzymes encoded by these seven complementary DNAs: the monoterpene synthases PdTPS1, PdTPS3, PdTPS5, and PdTPS6 belong to the TPS-b clade, which catalyzes the formation of ß-phellandrene, geraniol, linalool, and farnesene, respectively. The sesquiterpene synthases PdTPS2 and PdTPS4, which belong to the TPS-a clade mainly catalyze the formation of bergamotene, while another sesquiterpene synthase, PdTPS7, from the TPS-g clade showed nerolidol synthase activity. The qRT-PCR analysis revealed that the various tissues of almond varieties showed differential transcription for all these PdTPSs genes.

A Pyrus communis gene for p-hydroxystyrene biosynthesis, has a role in defense against the pear psylla Cacopsylla biden.

Styrene analogs are known to be naturally synthesized in the leaves of pears and in other plant species, including several trees in the Styracaceae family. Styrene analogs are potential contributors to the aroma of wine, perfumes, pharmaceuticals, and other fermented foods and beverages. In addition, styrene analogs perform important ecological functions such as insecticidal and antifeedant activities against insects. We showed here that exogenous applications of styrene and p-hydroxystyrene caused a dramatic reduction the number of eggs laid by psylla and of subsequent nymph survival. Despite their importance specific reactions that lead to the biosynthesis of the styrene analogs in pear are unknown. To identify genes involved in the synthesis of these metabolites, existing genome databases of the Rosaceae were screened for pear genes with significant sequence similarity to bacterial phenolic acid decarboxylase. Herein described are the isolation and characterization of a pear phenolic acid decarboxylase, designated PyPAD1, which catalyzed the decarboxylation of p-coumaric acid and ferulic acid to p-hydroxystyrene and 3-methoxy-4-hydroxystyrene respectively. Its apparent Km values for p-coumaric acid and ferulic acid were 34.42 and 84.64 µM, respectively. The PyPAD1 preferred p-coumaric acid to ferulic acid as a substrate by a factor of 2.4 when comparing catalytic efficiencies in vitro. Expression analysis of PyPAD1 showed that the gene was transcribed in all five pear genotypes examined. However, transcript abundance was increased in correlation with the presence of p-hydroxystyrene in resistant cultivars Py-701 and Py-760 and in the sensitive cultivar Spadona when grafted on these resistant cultivars. Thus, PyPAD1 appears to be responsible for the decarboxylation of the p-coumaric acid, and for the production of metabolites that are active against pear psylla.

Grafting on resistant interstocks reduces scion susceptibility to pear psylla, Cacopsylla bidens.

BACKGROUND: Pear psylla is a major obstacle to efficient integrated pest management in pear orchards in Israel and around the world. We used two accessions with natural resistance to pear psylla Cacopsylla bidens (Sulc) - Py.760-261 (760) and Py.701-202 (701), both apparently of Pyrus communis L. origin - as interstock grafts to confer psylla resistance to the commercially important 'Spadona Estiva' (Pyrus communis) scion (Spadona) cultivar. The interaction of the interstocks with quince (Cydonia oblong Mill.) and Pyrus betulifolia Bunge rootstocks was also tested. RESULTS: Usage of Py.760-261 (760) and Py.701-202 (701) as interstocks for the psylla-sensitive Spadona resulted in a five-fold decrease in the C. bidens population, apparently as a consequence of antibiosis affecting nymph survival. Additionally, psylla survival was negatively correlated with the interstock length and amount of foliage. The yield and fruit quality of Spadona grafted on the '701' interstock equaled or even exceeded those of the control in fruit quantity, fruit size and soluble solids content, especially on P. betulifolia rootstock. CONCLUSION: Susceptibility to pear psylla decreased significantly following grafting of commercial Spadona on resistant interstock. This is the first demonstration of increased resistance to pear psylla conferred by the use of resistant interstock in pear trees and among the few examples demonstrating transfer of resistance to insects from the interstock in fruit trees. © 2017 Society of Chemical Industry.

Control of the Tomato Leafminer, Tuta absoluta (Lepidoptera: Gelechiidae), in Open-Field Tomatoes by Indigenous Natural Enemies Occurring in Israel.

The tomato leafminer, Tuta absoluta (Meyrick), had established in Israel by 2010, attacking both open-field tomatoes and greenhouse crops.We searched for its natural enemies in open-field tomatoes, and tried to determine their potential for controlling this pest. We surveyed the local natural enemies in open tomato fields and measured their impact on pest populations in an unsprayed field. We assessed the suppressive ability of the dominant hemipteran predator, Nesidiocoris tenuis Reuter, against T. absoluta under controlled laboratory conditions and evaluated the impact of its augmentation on T. absoluta control in open-field tomatoes. We found five natural enemy species:the predator, N. tenuis, two braconids, and two eulophids. Predation accounted for 64.5±9.2% (mean ± SE) of T. absoluta larval mortality, whereas parasitism accounted for 20.96±7.5%. Together, they eliminated the pest population at tomato harvest time. Under controlled conditions, predation by N. tenuis rose from 58 to 72% with increased density of T. absoluta, suggesting positive density dependence. The reduction of T. absoluta (83%) by N. tenuis was higher than that of Bemisia tabaci (32%), suggesting a preference of N. tenuis for T. absoluta. Augmentation of N.tenuis was as effective as conventional treatment insecticide treatment, and plant damage was low and did not seem to affect yield. Results indicate that reduced pesticide use enables indigenous natural enemies, particularly N.tenuis, to successfully control T. absoluta and prevent crop damage in open-field tomatoes.

Two pear accessions evaluated for susceptibility to pear psylla Cacopsylla bidens (Sulc) in Israel.

BACKGROUND: The pear psylla, Cacopsylla bidens (Sulc), is one of the most damaging pests of commercial pear orchards in Israel. Psylla control is a major obstacle to efficient integrated pest management, necessitating research on cultivars with natural resistance to pear psylla. Recently, two pear accessions (Py.760-261 and Py.701-202) from the local Newe Ya'ar fruit tree live collection were identified as having apparent resistance to pear psylla. Our goal was to evaluate the resistance of these two accessions relative to the commercial cultivar Spadona Estiva, and to identify whether the resistance mechanisms in the former interfere with insect colonisation of the plant (antixenosis) or inhibit insect growth, development, reproduction and survival (antibiosis). RESULTS: Settlement and development of C. bidens was evaluated under natural conditions (pear orchard), semi-natural conditions (potted plants), and on detached branches and leaves (laboratory). Our results indicate that the selection Py.760-261 is 10 times more resistant than Spadona while Py.701-202 is five times more resistant. CONCLUSIONS: The resistance mechanism in both accessions appears to be antibiosis affecting nymph survival. These resistant accessions may be used as rootstock or as a source of resistant genes in breeding programmes.