Plant-Entomopathogenic Fungi Interaction: Recent Progress and Future Prospects on Endophytism-Mediated Growth Promotion and Biocontrol.

Entomopathogenic fungi, often acknowledged primarily for their insecticidal properties, fulfill diverse roles within ecosystems. These roles encompass endophytism, antagonism against plant diseases, promotion of the growth of plants, and inhabitation of the rhizosphere, occurring both naturally and upon artificial inoculation, as substantiated by a growing body of contemporary research. Numerous studies have highlighted the beneficial aspects of endophytic colonization. This review aims to systematically organize information concerning the direct (nutrient acquisition and production of phytohormones) and indirect (resistance induction, antibiotic and secondary metabolite production, siderophore production, and mitigation of abiotic and biotic stresses) implications of endophytic colonization. Furthermore, a thorough discussion of these mechanisms is provided. Several challenges, including isolation complexities, classification of novel strains, and the impact of terrestrial location, vegetation type, and anthropogenic reluctance to use fungal entomopathogens, have been recognized as hurdles. However, recent advancements in biotechnology within microbial research hold promising solutions to many of these challenges. Ultimately, the current constraints delineate potential future avenues for leveraging endophytic fungal entomopathogens as dual microbial control agents.

Ligands of HMG-like dorsal switch protein 1 of Spodoptera exigua leads to mortality in diamondback moth, Plutellaxylostella.

HMG-like dorsal switch protein 1 (DSP1) is the insect homolog of the high mobility group box 1 (HMGB1) protein of the vertebrates. Previous studies confirmed DSP1 in Spodoptera exigua, Tenebrio molitor, and Aedes albopictus, and were analyzed for their immune roles, survivability, and binding affinity with entomopathogenic bacterial metabolites. The present study aimed to predict, and confirm DSP1 in diamondback moth, Plutella xylostella along with the effect of Spodoptera exigua DSP1 ligands in the survivability of this insect. DSP1 of Plutella xylostella (Px-DSP1) consists of 465 amino acids (AA). Phylogeny analysis showed that Px-DSP1 clustered with other Lepidopteran insects where each insect order clustered separately. Domain analysis showed that like other insects, Px-DSP1 contains two HMG boxes (Box A and Box B), one coiled-coil (CC), five Q-rich low complexity (LC), and an acidic tail (AT). Px-DSP1 was expressed in each developmental stage and tissue. The highest expression was in L4 larvae and fat body tissues. Thermal shift assay (TSA) showed the binding affinity of 3-Ethoxy-4-Methoxyphenol (EMP), Phthalimide (PM), and o-Cyanobenzoic acid (CBA) to rDSP1 of Spodoptera exigua. Mortality bioassay showed that all these metabolites were toxic against P. xylostella larvae. Among these, EMP was more toxic providing more than 65% mortality at 500 ppm concentration. However, PM and CBA also showed more than 60 and 50% mortality, respectively at 500 ppm concentration. We assume that like Se-DSP1, these compounds also bind with Px-DSP1 which leads to the inhibition of DSP1-mediated immunity and impose the mortality of Plutella xylostella larvae.

Heat shock increases the anti-inflammatory and anti-obesity activity of soybean by increasing polyphenol, antioxidant and aglycon form isoflavones.

We know that heat shock can activate the functional components in soybeans, but we don't know the type, level, and duration of heat shock for maximum activation. To address this, the present study investigated the changes in functional components like polyphenols, antioxidants, and isoflavones in soybeans at various temperature levels and durations with their respective functionality or health benefits. For this, treated seed samples were extracted with 70 % ethanol. Heat shock at 60 °C for 2 h increased polyphenol content (60.67 % of control) and antioxidant activity for both ABTS (41.14 % of control) and DPPH (217.72 % of control). This also increased the beneficial aglycone form of isoflavones that includes daidzein (8.36-fold of control), glycitein (3.85-fold of control) and genistein (20.50-fold of control) but decreased the harmful ß-glucoside form (3.65-fold) including daiazin (1.84-fold of control); glycitin (1.45-fold of control) and genistin (23.88-fold of control) over untreated dry seed. This may happen because of the conversion of conjugated ß-glucoside isoflavones to their aglycone forms that have various health benefits. Maximum inhibition of NO production in RAW 264.7 cells was achieved by samples elicited for 2 h with 300 µg/mL concentration. This sample also confirmed the maximum anti-obesity activity treated against 3-T-3L1 cells. This study summarized that heat shock at 60 °C for 2 h increased polyphenols, antioxidants, and aglycon isoflavone in soybeans resulting in increased anti-inflammatory and anti-obesity activity.

Spätzle processing enzyme is required to activate dorsal switch protein 1 induced Toll immune signalling pathway in Tenebrio molitor.

Dorsal switch protein 1 (DSP1) acts as a damage-associated molecular pattern (DAMP) molecule to activate immune responses in Tenebrio molitor. From a previous study in Spodoptera exigua, we found that DSP1 activates Toll immune signalling pathway to induce immune responses by melanisation, PLA2 activity and AMP synthesis. However, the target site of DSP1 in this pathway remains unknown. The objective of this study was to determine the role of spätzle processing enzyme in the DSP1 induced toll immune signalling pathway. To address this, we analyzed spätzle processing enzyme (Tm-SPE) of the three-step serine protease cascade of T. molitor Toll pathway. Tm-SPE expressed in all developmental stages and larval tissues. Upon immune challenge, its expression levels were upregulated but significantly reduced after RNA interference (RNAi). In addition, the induction of immune responses upon immune challenge or recombinant DSP1 injection was significantly increased. Loss of function using RNA interference revealed that the Tm-SPE is involved in connecting DSP1 induced immune responses like hemocyte nodule formation, phenoloxidase (PO) activity, phospholipase A2 (PLA2) activity and antimicrobial peptide (AMP) synthesis. These suggest that Tm-SPE controls the DSP1 induced activation of Toll immune signalling pathway required for both cellular and humoral immune responses. However, to confirm the target molecule of DSP1 in three-step proteolytic cascade, we have to check other upstream serine proteases like Spatzle activating enzyme (SAE) or modular serine protease (MSP).

Salicylic Acid, a Plant Hormone, Suppresses Phytophagous Insect Immune Response by Interrupting HMG-Like DSP1.

Salicylic acid is a plant hormone that can mediate various plant physiological processes. Salicylic acid can bind to human high mobility group box 1 (HMGB1) and interrupt its role in mediating immune responses. Dorsal switch protein 1 (DSP1) is an insect homolog of HMGB1. In this study, a DSP1 (Se-DSP1) encoded in Spodoptera exigua, a phytophagous insect, was characterized, and its potential role in immune response was explored. Upon bacterial challenge, Se-DSP1 was localized in the nucleus and released into the hemolymph. The released Se-DSP1 could mediate both cellular and humoral immune responses by activating eicosanoid biosynthesis. Salicylic acid could bind to Se-DSP1 with a high affinity. The immune responses of S. exigua were significantly interrupted by SA feeding. Larvae reared on tomatoes with high endogenous SA levels became more susceptible to entomopathogens. Taken together, these results suggest a tritrophic defensive role of plant SA against phytophagous insects.

HMGB1-like dorsal switch protein 1 of the mealworm, Tenebrio molitor, acts as a damage-associated molecular pattern.

High-mobility group box 1 (HMGB1) is a nuclear protein highly conserved in eukaryotes and ubiquitously expressed to regulate transcription and chromatin remodeling. Dorsal switch protein 1 (DSP1) is its insect homolog. A lepidopteran DSP1 acts as a damage-associated molecular pattern (DAMP) in response to immune challenge. The objective of this study was to determine the role of DAMP in the mealworm beetle, Tenebrio molitor, a coleopteran insect. DSP1 of T. molitor (Tm-DSP1) encodes 536 amino acids and shares sequence similarities with Homo sapiens HMGB1 (56.3%) and Spodoptera exigua DSP1 (59.2%). An antisera raised against S. exigua DSP1 was cross-reactive to Tm-DSP1. Like other insect DSPs, Tm-DSP1 has a relatively long N-terminal extension in addition to two conserved HMG box domains. It was expressed in all developmental stages of T. molitor and different larval tissues. Upon immune challenge, its expression level was upregulated. Its RNA interference (RNAi) treatment resulted in a significant reduction in immune responses measured by hemocyte nodule formation against bacterial infection. In addition, the induction of some antimicrobial peptide genes to the immune challenge was suppressed by its RNAi treatment. Interestingly, phospholipase A2 associated with eicosanoid biosynthesis was significantly suppressed in its catalytic activity by the RNAi treatment specific to Tm-DSP1 expression. Without any pathogen infection, injection of a lepidopteran DSP1 induced both cellular and humoral immune responses. These results suggest that Tm-DSP1 in T. molitor can act as a DAMP molecule and mediate immune responses upon immune challenge.

Immune mediation of HMG-like DSP1 via Toll-Spätzle pathway and its specific inhibition by salicylic acid analogs.

Xenorhabdus hominickii, an entomopathogenic bacterium, inhibits eicosanoid biosynthesis of target insects to suppress their immune responses by inhibiting phospholipase A2 (PLA2) through binding to a damage-associated molecular pattern (DAMP) molecule called dorsal switch protein 1 (DSP1) from Spodoptera exigua, a lepidopteran insect. However, the signalling pathway between DSP1 and PLA2 remains unknown. The objective of this study was to determine whether DSP1 could activate Toll immune signalling pathway to activate PLA2 activation and whether X. hominickii metabolites could inhibit DSP1 to shutdown eicosanoid biosynthesis. Toll-Spätzle (Spz) signalling pathway includes two Spz (SeSpz1 and SeSpz2) and 10 Toll receptors (SeToll1-10) in S. exigua. Loss-of-function approach using RNA interference showed that SeSpz1 and SeToll9 played crucial roles in connecting DSP1 mediation to activate PLA2. Furthermore, a deletion mutant against SeToll9 using CRISPR/Cas9 abolished DSP1 mediation and induced significant immunosuppression. Organic extracts of X. hominickii culture broth could bind to DSP1 at a low micromolar range. Subsequent sequential fractionations along with binding assays led to the identification of seven potent compounds including 3-ethoxy-4-methoxyphenol (EMP). EMP could bind to DSP1 and prevent its translocation to plasma in response to bacterial challenge and suppress the up-regulation of PLA2 activity. These results suggest that X. hominickii inhibits DSP1 and prevents its DAMP role in activating Toll immune signalling pathway including PLA2 activation, leading to significant immunosuppression of target insects.

Antiviral Treatment Reveals a Cooperative Pathogenicity of Baculovirus and Iflavirus in Spodoptera exigua, a Lepidopteran Insect.

NPVThe beet armyworm, Spodoptera exigua, is a serious insect pest infesting various vegetable crops. Two infectious insect viruses, baculovirus and iflavirus, are known to induce epizootics in S. exigua populations. Indeed, some laboratory colonies have appeared to be covertly infected by these viruses. Diagnostic PCR tests detected two different viruses: Spodoptera exigua multiple nucleopolyhedrosis virus (SeMNPV) and iflaviruses (SeIfV1 and SeIfV2). Viral extract from dead larvae of S. exigua could infect Sf9 cells and produce occlusion bodies (OBs). Feeding OBs to asymptomatic larvae of S. exigua caused significant viral disease. Interestingly, both SeIfV1 and SeIfV2 increased their titers at late larval stages. Sterilization of laid eggs with 1% sodium hypochloride significantly reduced SeMNPV titers and increased larval survival rate. Doublestranded RNA (dsRNA) specific to SeIfV1 or SeIfV2 significantly reduced viral titers and increased larval survival rate. To continuously feed dsRNA, a recombinant Escherichia coli HT115 expressing SeIfV1-dsRNA was constructed with an L4440 expression vector. Adding this recombinant E. coli to the artificial diet significantly reduced the SeIfV1 titer and increased larval survival. These results indicate that laboratory colony collapse of S. exigua is induced by multiple viral infections. In addition, either suppression of SeMNPV or SeIfV infection significantly increased larval survival, suggesting a cooperative pathogenicity between baculovirus and iflavirus against S. exigua.

Variations of Indole Metabolites and NRPS-PKS Loci in Two Different Virulent Strains of Xenorhabdus hominickii.

Xenorhabdus hominickii ANU1 is known to be an entomopathogenic bacterium symbiotic to nematode Steinernema monticolum. Another bacterial strain X. hominickii DY1 was isolated from a local population of S. monticolum. This bacterial strain X. hominickii DY1 was found to exhibit high insecticidal activities against lepidopteran and coleopteran species after hemocoelic injection. However, these two X. hominickii strains exhibited significant variations in insecticidal activities, with ANU1 strain being more potent than DY1 strain. To clarify their virulence difference, bacterial culture broths of these two strains were compared for secondary metabolite compositions. GC-MS analysis revealed that these two strains had different compositions, including pyrrolopyrazines, piperazines, cyclopeptides, and indoles. Some of these compounds exhibited inhibitory activities against phospholipase A2 to block eicosanoid biosynthesis and induce significant immunosuppression. They also exhibited significant insecticidal activities after oral feeding, with indole derivatives being the most potent. More kinds of indole derivatives were detected in the culture broth of ANU1 strain. To investigate variations in regulation of secondary metabolite production, expression level of leucine-responsive regulatory protein (Lrp), a global transcription factor, was compared. ANU1 strain exhibited significantly lower Lrp expression level than DY1 strain. To assess genetic variations associated with secondary metabolite synthesis, bacterial loci encoding non-ribosomal protein synthase and polyketide synthase (NRPS-PKS) were compared. Three NRPS and four PKS loci were predicted from the genome of X. hominickii. The two bacterial strains exhibited genetic variations (0.12∼0.67%) in amino acid sequences of these NRPS-PKS. Most NRPS-PKS genes exhibited high expression peaks at stationary phase of bacterial growth. However, their expression levels were significantly different between the two strains. These results suggest that differential virulence of the two bacterial strains is caused by the difference in Lrp expression level, leading to difference in the production of indole compounds and other NRPS-PKS-associated secondary metabolites.

Virulent secondary metabolites of entomopathogenic bacteria genera, Xenorhabdus and Photorhabdus, inhibit phospholipase A2 to suppress host insect immunity.

BACKGROUND: Xenorhabdus and Photorhabdus are entomopathogenic bacteria that cause septicemia and toxemia in insects. They produce secondary metabolites to induce host immunosuppression. Their metabolite compositions vary among bacterial species. Little is known about the relationship between metabolite compositions and the bacterial pathogenicity. The objective of this study was to compare pathogenicity and production of secondary metabolites of 14 bacterial isolates (species or strains) of Xenorhabdus and Photorhabdus. RESULTS: All bacterial isolates exhibited insecticidal activities after hemocoelic injection to Spodoptera exigua (a lepidopteran insect) larvae, with median lethal doses ranging from 168.8 to 641.3 CFU per larva. Bacterial infection also led to immunosuppression by inhibiting eicosanoid biosynthesis. Bacterial culture broth was fractionated into four different organic extracts. All four organic extracts of each bacterial species exhibited insecticidal activities and resulted in immunosuppression. These organic extracts were subjected to GC-MS analysis which predicted 182 compounds, showing differential compositions for 14 bacteria isolates. There were positive correlations between total number of secondary metabolites produced by each bacterial culture broth and its bacterial pathogenicity based on immunosuppression and insecticidal activity. From these correlation results, 70 virulent compounds were selected from secondary metabolites of high virulent bacterial isolates by deducting those of low virulent bacterial isolates. These selected virulent compounds exhibited significant immunosuppressive activities by inhibiting eicosanoid biosynthesis. They also exhibited relatively high insecticidal activities. CONCLUSION: Virulence variation between Xenorhabdus and Photorhabdus is determined by their different compositions of secondary metabolites, of which PLA2 inhibitors play a crucial role.

Immunosuppressive Activities of Novel PLA2 Inhibitors from Xenorhabdus hominickii, an Entomopathogenic Bacterium.

Eicosanoids mediate both cellular and humoral immune responses in insects. Phospholipase A2 (PLA2) catalyzes the first committed step in eicosanoid biosynthesis. It is a common pathogenic target of two entomopathogenic bacteria, Xenorhabdus and Photorhabdus. The objective of this study was to identify novel PLA2 inhibitors from X. hominickii and determine their immunosuppressive activities. To identify novel PLA2 inhibitors, stepwise fractionation of X. hominickii culture broth and subsequent enzyme assays were performed. Eight purified fractions of bacterial metabolites were obtained. Gas chromatography and mass spectrometry (GC-MS) analysis predicted that the main components in these eight fractions were 2-cyanobenzoic acid, dibutylamine, 2-ethyl 1-hexanol, phthalimide (PM), dioctyl terephthalate, docosane, bis (2-ethylhexyl) phthalate, and 3-ethoxy-4-methoxyphenol (EMP). Their synthetic compounds inhibited the activity of PLA2 in hemocytes of a lepidopteran insect, Spodoptera exigua, in a dose-dependent manner. They also showed significant inhibitory activities against immune responses such as prophenoloxidase activation and hemocytic nodulation of S. exigua larvae, with PM and EMP exhibiting the most potent inhibitory activities. These immunosuppressive activities were specific through PLA2 inhibition because an addition of arachidonic acid, a catalytic product of PLA2, significantly rescued such suppressed immune responses. The two most potent compounds (PM and EMP) showed significant insecticidal activities after oral administration. When the compounds were mixed with Bacillus thuringiensis (Bt), they markedly increased Bt pathogenicity. This study identified eight PLA2 inhibitors from bacterial metabolites of X. hominickii and demonstrated their potential as novel insecticides.

Variation in pathogenicity of different strains of Xenorhabdus nematophila; Differential immunosuppressive activities and secondary metabolite production.

Xenorhabdus nematophila, an entomopathogenic bacterium, is mutualistic with the nematode Steinernema carpocapsae. The bacterium produces secondary metabolites to inhibit target insect phospholipase A2 (PLA2) and induce immunosuppression, which is required for the pathogenicity of this bacterium-nematode complex. However, it was unclear if immunosuppressive intensity of the bacteria was correlated with their insecticidal potency. We compared six different X. nematophila strains inhibiting the immune responses of the beet armyworm (Spodoptera exigua) to explain their virulence variations. In addition to four known strains obtained from the Korean Agricultural Culture Collection, we identified two new strains (SK1 and SK2) of X. nematophila from two different isolates of S. carpocapsae. Although all six strains were virulent, they showed significant variation in median lethal bacterial dosage (LD50). The LD50 of most strains was 15-30 CFU/larva, however, the LD50 of the SK1 strain was more than two-fold higher against S. exigua larvae. Immunosuppressive activities of the six strains were measured by comparing hemocyte-spreading behavior and nodule formation; the SK1 strain was significantly less potent than other bacterial strains. These suppressed hemocyte behaviors were recovered by adding arachidonic acid (a catalytic product of PLA2) into all six strains. Bacterial culture broth was fractionated with different organic solvents and the ability to inhibit immune response and PLA2 activity were assessed. All organic extracts had immunosuppressive activities and PLA2-inhibitory activities. GC-MS analysis showed that these organic extracts possessed a total of 87 different compounds. There were variations in chemical components among the six bacterial strains. Organic extracts of SK1 strain, which exhibited the lowest virulence, contained the least number of secondary metabolites.