Efficacy of Entomopathogenic Staphylococcus Bacteria as a Biocontrol Agent against Rhipicephalus microplus Ticks: Assessing Reproductive Inhibition and Mortality Rates.

Rhipicephalus microplus is a persistent ectoparasite of cattle that causes bovine anaplasmosis and babesiosis, causing economic losses worldwide. Chemical treatment is the primary method for tick control, but the emergence of pesticide-resistant ticks is a major challenge. Alternative biocontrol strategies utilizing entomopathogenic microorganisms are being explored. This study aimed to validate the species identification and assess the efficacy of four strains of Staphylococcus bacteria (S. shinii S1 and S-2, S. succinus, and S. xylosus) previously reported as being entomopathogenic to R. microplus ticks. According to the bioassays, S. shinii S-1 exhibited the greatest degree of reproductive inhibition (47%), followed by S. succinus (44.3%) at a concentration of 1 × 108 cfu/mL. S. xylosus displayed decreased reproductive inhibition (6.3%). In an additional bioassay, S. shinii S-1 exhibited a significant larval mortality of 67.63%, followed by S. succinus with 66.75%, S. shinni S-2 with 64.61%, and S. xylosus with 28.18% mortality. The common signs of infection observed on these ticks included swelling, yellowish exudate on the hypostome, and reduced limb mobility and color change, except for S. succinus, which did not cause color changes. These bacteria were naturally found on bovine skin. However, further studies are needed to confirm their potential as promising alternatives or complementary agents to existing acaricidal compounds.

The effect of Xenorhabdus bacteria metabolites on Colorado potato beetle (Leptinotarsa decemlineata) adult feeding and larval survival.

Colorado Potato Beetle (CPB) is one of the most destructive potato pests that can quickly develop resistance to insecticides. Therefore, new safe and effective control strategies that are less susceptible to the development of resistance by CPB are urgently needed. Due to their complex mode of action, the likelihood of resistance development by target pests is generally low with antifeedants. In the present study, we assessed the effect of secondary metabolites of various Xenorhabdus bacteria species and strains on CPB adult feeding and on larval development. The metabolites were applied in the form of cell free supernatants (CFSs) from Xenorhabdus cultures. In bioassay 1, leaves treated with ten Xenorhabdus cultures were fed to CPB adults, and their feeding was assessed daily for one week. In bioassay 2, CPB egg masses were placed on the leaves treated with five bacterial cultures, and larval development to pupae was monitored. Out of the ten Xenorhabdus cultures tested, two strains exhibited a significant reduction in the feeding behavior of Colorado Potato Beetle adults, with reductions of up to 70% compared to the control. The effect of CFSs on larval development was variable, and when treated with X. khoisanae SGI 197, over 90% of larvae died in the first few days before reaching the 2nd instar, and complete mortality was achieved on the 8th day of the experiment. Our study is the first study to demonstrate the antifeedant effect of Xenorhabdus cultures towards herbivorous beetles, and the metabolites of these bacteria may have potential for CPB control. Clearly, the metabolites produced by X. khoisanae SGI-197 may be a promising tool for CPB larvae control with the potential to significantly decrease damage to potato plants.

Microencapsulation of Bacillus thuringiensis strains for the control of Aedes aegypti.

In this study, we investigated the microencapsulation of two strains of the entomopathogenic bacteria Bacillus thuringiensis (B. thuringiensis) (BtMA-750 and BtMA-1114), which are biopesticides of high toxicity for the mosquito vector Aedes aegypti. The encapsulation of different concentrations of microorganisms in starch microparticles was evaluated, and the inverse suspension polymerization technique was explored. It was possible to observe that the higher amounts of the biopesticide caused a slight decrease in the diameter of the particles; however, even when encapsulated, the biopesticide still presents an average diameter that is able to be consumed by the larvae of Aedes aegypti. Furthermore, it was noticed that the presence of both of the B. thuringiensis strains did not affect the thermal stability of the particles. The microencapsulated bacterial strains presented a high number of viable spores and preserved the expression of proteins with molecular masses corresponding to the insecticidal toxins Cry and Cyt, indicating that the encapsulation process was conducted satisfactorily. Finally, the encapsulated strains were tested against Ae. aegypti larvae and maintained 100% larval mortality even after 35 days. Therefore, microencapsulation of B. thuringiensis not only guarantees the bacterial activity, but also prolongs the action of the biopesticide. Collectively, such findings highlight the great potential of the new biopesticides, which may help to reduce the population indices of the mosquito vector Ae. aegypti via a sustainable and environment-friendly route.

Production and Characterization of Photorin, a Novel Proteinaceous Protease Inhibitor from the Entomopathogenic Bacteria Photorhabdus laumondii.

Entomopathogenic bacteria of the genus Photorhabdus secrete protease S (PrtS), which is considered a virulence factor. We found that in the Photorhabdus genomes, immediately after the prtS genes, there are genes that encode small hypothetical proteins homologous to emfourin, a recently discovered protein inhibitor of metalloproteases. The gene of emfourin-like inhibitor from Photorhabdus laumondii subsp. laumondii TT01 was cloned and expressed in Escherichia coli cells. The recombinant protein, named photorin (Phin), was purified by metal-chelate affinity and gel permeation chromatography and characterized. It has been established that Phin is a monomer and inhibits activity of protealysin and thermolysin, which, similar to PrtS, belong to the M4 peptidase family. Inhibition constants were 1.0 ± 0.3 and 10 ± 2 µM, respectively. It was also demonstrated that Phin is able to suppress proteolytic activity of P. laumondii culture fluid (half-maximal inhibition concentration 3.9 ± 0.3 nM). Polyclonal antibodies to Phin were obtained, and it was shown by immunoblotting that P. laumondii cells produce Phin. Thus, the prtS genes in entomopathogenic bacteria of the genus Photorhabdus are colocalized with the genes of emfourin-like inhibitors, which probably regulate activity of the enzyme during infection. Strict regulation of the activity of proteolytic enzymes is essential for functioning of all living systems. At the same time, the principles of regulation of protease activity by protein inhibitors remain poorly understood. Bacterial protease-inhibitor pairs, such as the PrtS and Phin pair, are promising models for in vivo studies of these principles. Bacteria of the genus Photorhabdus have a complex life cycle with multiple hosts, being both nematode symbionts and powerful insect pathogens. This provides a unique opportunity to use the PrtS and Phin pair as a model for studying the principles of protease activity regulation by proteinaceous inhibitors in the context of bacterial interactions with different types of hosts.

Photorhabdus aballayi sp. nov. and Photorhabdus luminescens subsp. venezuelensis subsp. nov., isolated from Heterorhabditis amazonensis entomopathogenic nematodes.

Six Gram-negative, rod-shaped bacterial strains isolated from Heterorhabditis amazonensis entomopathogenic nematodes were characterized to determine their taxonomic position. 16S rRNA and gyrB gene sequences indicate that they belong to the class Gammaproteobacteria, family Morganellaceae and genus Photorhabdus, and that some of them are conspecifics. Two of them, APURET and JART, were selected for further molecular characterization using whole genome- and whole-proteome-based phylogenetic reconstructions and sequence comparisons. Phylogenetic reconstructions using whole genome and whole proteome sequences show that strains APURET and JART are closely related to Photorhabdus luminescens subsp. luminescens ATCC 29999T and to P. luminescens subsp. mexicana MEX47-22T. Moreover, digital DNA-DNA hybridization (dDDH) values between APURET and P. luminescens subsp. luminescens ATCC 29999T, APURET and P. luminescens subsp. mexicana MEX47-22T, and APURET and JART are 61.6, 61.2 and 64.1 %, respectively. These values are below the 70 % divergence threshold that delimits prokaryotic species. dDDH scores between JART and P. luminescens subsp. luminescens ATCC 29999T and between JART and P. luminescens subsp. mexicana MEX47-22T are 71.9 and 74.8 %, respectively. These values are within the 70 and 79 % divergence thresholds that delimit prokaryotic subspecies. Based on these genomic divergence values, APURET and JART represent two different taxa, for which we propose the names: Photorhabdus aballayi sp. nov. with APURET (=CCM 9236T =CCOS 2019T) as type strain and Photorhabdus luminescens subsp. venezuelensis subsp. nov. with JART (=CCM 9235T =CCOS 2021T) as type strain. Our study contributes to a better understanding of the biodiversity of an important bacterial group with enormous biotechnological and agricultural potential.

Interkingdom Signaling of the Insect Pathogen Photorhabdus luminescens with Plants Via the LuxR solo SdiA.

In bacteria, group-coordinated behavior such as biofilm formation or virulence are often mediated via cell-cell communication, a process referred to as quorum sensing (QS). The canonical QS system of Gram-negative bacteria uses N-acyl homoserine lactones (AHLs) as communication molecules, which are produced by LuxI-type synthases and sensed by cognate LuxR-type receptors. These receptors act as transcriptional regulators controlling the expression of specific genes. Some bacteria harbor LuxR-type receptors lacking a cognate LuxI-type synthases, designated as LuxR solos. Among many other LuxR solos, the entomopathogenic enteric bacterium Photorhabdus luminescens harbors a SdiA-like LuxR solo containing an AHL signal-binding domain, for which a respective signal molecule and target genes have not been identified yet. Here we performed SPR analysis to demonstrate that SdiA acts as a bidirectional regulator of transcription, tightly controlling its own expression and the adjacent PluDJC_01670 (aidA) gene in P. luminescens, a gene supposed to be involved in the colonization of eukaryotes. Via qPCR we could further determine that in sdiA deletion mutant strains, aidA is upregulated, indicating that SdiA negatively affects expression of aidA. Furthermore, the ΔsdiA deletion mutant exhibited differences in biofilm formation and motility compared with the wild-type. Finally, using nanoDSF analysis we could identify putative binding ability of SdiA towards diverse AHLs, but also to plant-derived signals, modulating the DNA-binding capacity of SdiA, suggesting that this LuxR solo acts as an important player in interkingdom signaling between P. luminescens and plants.

HETERORHABDITIS BACTERIOPHORA NEMATODES ARE SENSITIVE TO THE BACTERIAL PATHOGEN PHOTORHABDUS ASYMBIOTICA.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora infects a wide range of insect hosts with the aid of its mutualistic bacteria Photorhabdus luminescens. While the mutualistic relationship between H. bacteriophora and P. luminescens and the infectivity of the nematode-bacteria complex have been characterized, how nematode fitness is affected by entomopathogenic bacteria existing in association with other EPN species remains poorly understood. In this study, the survival of H. bacteriophora infective juveniles containing or lacking P. luminescens was tested against the entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus asymbiotica as well as the non-pathogenic Escherichia coli. While X. nematophila and E. coli did not significantly affect the survival of H. bacteriophora, P. asymbiotica exerted a significant effect on nematode survival, particularly on those lacking P. luminescens. These results imply that P. asymbiotica encodes factors that are pathogenic to EPNs. Future efforts will focus on the identification of the bacterial molecular components that induce these effects. This study makes an important contribution to a growing body of research aimed at exploiting the full potential of nematode-bacterial complexes for eliminating noxious insect pests and treating infectious diseases caused by parasitic nematodes.

Cradle for the newborn Monochamus saltuarius: Microbial associates to ward off entomopathogens and disarm plant defense.

The Japanese pine sawyer, Monochamus saltuarius, as a beetle vector of Bursaphelenchus xylophilus (pine wood nematode), is an economically important forest pest in Eurasia. To feed on the phloem and xylem of conifers, M. saltuarius needs to overcome various stress factors, including coping with entomopathogenic bacteria and also various plant secondary compounds (PSCs). As an important adaptation strategy to colonize host trees, M. saltuarius deposit eggs in oviposition pits to shield their progeny. These pits harbor bacterial communities that are involved in the host adaptation of M. saltuarius to the conifers. However, the composition, origin, and functions of these oviposition pit bacteria are rarely understood. In this study, we investigated the bacterial community associated with M. saltuarius oviposition pits and their ability to degrade PSCs. Results showed that the bacterial community structure of M. saltuarius oviposition pits significantly differed from that of uninfected phloem. Also, the oviposition pit bacteria were predicted to be enriched in PSC degradation pathways. The microbial community also harbored a lethal strain of Serratia, which was significantly inhibited. Meanwhile, metatranscriptome analysis indicated that genes involved in PSCs degradation were expressed complementarily among the microbial communities of oviposition pits and secretions. In vitro degradation showed that bacteria cultured from oviposition pits degraded more monoterpenes and flavonoids than bacteria cultured from uninfected phloem isolates. Disinfection of oviposition pits increased the mortality of newly hatched larvae and resulted in a significant decrease in body weight in the early stages. Overall, our results reveal that M. saltuarius construct oviposition pits that harbor a diverse microbial community, with stronger PSCs degradation abilities and a low abundance of entomopathogenic bacteria, resulting in the increased fitness of newly hatched larvae.

Identification and Biocontrol Potential of Entomopathogenic Nematodes and Their Endosymbiotic Bacteria in Apple Orchards against the Codling Moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae).

The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is one of the major pests in pome fruit production worldwide. Heavy treatment of the larvae of C. pomonella with insecticides triggered the development of resistance to many groups of insecticides. In addition, the increasing concern about the adverse effects of synthetic insecticides on human health and the environment has led to the development of sustainable and eco-friendly control practices for C. pomonella. The entomopathogenic nematodes (EPNs) (Steinernema and Heterorhabditis spp.) and their endosymbionts (Xenorhabdus and Photorhabdus spp.) represent a newly emerging approach to controlling a wide range of insect pests. In the present study, field surveys were conducted in apple orchards to isolate and identify EPNs and their endosymbionts and evaluate their insecticidal efficacy on the larvae of C. pomonella. EPNs were isolated from 12 of 100 soil samples (12%). Seven samples were identified as Steinernema feltiae (Filipjev, 1934) (Rhabditida: Steinernematidae), whereas five samples were assigned to Heterorhabditis bacteriophora (Poinar, 1976) (Rhabditida: Heterorhabditidae). The pathogenicity of the EPN species/isolates was screened on the last instar larvae of G. mellonella. The two most pathogenic isolates from each EPN species were tested against fifth instar larvae of C. pomonella under controlled conditions. The maximum mortality (100%) was achieved by all EPN species/isolates at a concentration of 100 IJs/larva 96 h after treatment. The endosymbionts of selected H. bacteriophora and S. feltiae species were identified as Photorhabdus luminescens subsp. kayaii and Xenorhabdus bovienii, respectively. The mortality rates ranged between 25 and 62% when the fifth larval instar larvae of C. pomonella were exposed to the treatment of cell-free supernatants of symbiotic bacteria. In essence, the present survey indicated that EPNs and their symbiotic bacteria have good potential for biological control of C. pomonella.

Identification and environment-friendly biocontrol potential of five different bacteria against Aphis punicae and Aphis illinoisensis (Hemiptera: Aphididae).

The current work is aimed at isolating and identifying new Entomopathogenic bacterium (EPB) strains associated with Steinernema feltiae and assessing the EPB's biocontrol potential on Aphis punicae and Aphis illinoisensis adults in the laboratory. From S. feltiae, five bacterial isolates were isolated and molecularly characterized. Lysinibacillus xylanilyticus strain TU-2, Lysinibacillus xylanilyticus strain BN-13, Serratia liquefaciens strain TU-6, Stenotrophomonas tumulicola strain T5916-2-1b, and Pseudochrobactrum saccharolyticum strain CCUG are the strains. Pathogenicity tests demonstrated that bacterial cells were more toxic against the two aphid species than bacterial cell-free supernatants. S. tumulicola strain T5916-2-1b cells and filtrate were reported to have the strongest potential to kill A. punicae and A. illinoisensis individuals within 6 h after treatment, with 100% mortality of both insects 24 and 48 h after treatment. Based on the results of the study, it looked like endogenous Steinernema-associated EPB could be used directly as a biocontrol agent for A. punicae and A. illinoisensis.

Photorhabdus antumapuensis sp. nov., a novel symbiotic bacterial species associated with Heterorhabditis atacamensis entomopathogenic nematodes.

One motile, Gram-negative, non-spore-forming and rod-shaped symbiotic bacterium, strain UCH-936T, was isolated from Heterorhabditis atacamensis nematodes. Results of biochemical, physiological, molecular and genomic analyses suggest that it represents a new species, which we propose to name Photorhabdus antumapuensis sp. nov. Digital DNA-DNA hybridization shows that strain UCH-936T is more closely related to Photorhabdus kleinii DSM 23513T, but shares solely 50.5 % similarity, which is below the 70% cut-off value that delimits species boundaries in bacteria. Phylogenetic reconstructions using whole-genome sequences show that strain UCH-936T forms a unique clade, suggesting its novel and distinct taxonomic status again. Similarly, comparative genomic analyses shows that the virulence factor flagella-related gene fleR, the type IV pili-related gene pilL and the vibriobactin-related gene vibE are present in the genome of strain UCH-936T but absent in the genomes of its closest relatives. Biochemically and physiologically, UCH-936T differs also from all closely related Photorhabdus species. Therefore, Photorhabdus antumapuensis sp. nov. is proposed as a new species with the type strain UCH-936T (CCCT 21.06T=CCM 9188T=CCOS 1991T).

Efficiency of Bacillus thuringiensis and Bacillus cereus against Rhynchophorus ferrugineus.

The Red Palm Weevil (Rhynchophorus ferrugineus (Oliv.) (Coleoptera, Dryophthoridae) is a well-known palm tree pest that has caused enormous economic damage all over the globe. Insecticides are still the primary method of controlling this pest at this period. However, field populations of RPW have been shown to be resistant to pesticides. Using Bacillus spp. might be one of the options for controlling R. ferruginous. In this study, 23 species of Bacillus spp. were isolated from the rhizosphere of date palm trees in Al Ahsa Oasis, Saudi Arabia. The isolates were identified using 16S rRNA gene sequencing. R. ferrugineus larvae and adults were tested on sugarcane pieces that were treated with the B. thuringiensis strain PDC-AHSAA1 and B. cereus strains (PDC-AHSAA2, PDC-AHSA3 and PDC-AHSA4). The LC50 values for larvae and adults were quite low when they were compared with those of the other isolated strains. The B. thuringiensis strain PDC-AHSAA1 was more effective against both the larvae and adults. The determined LC50 values for B. thuringiensis ranged from 4.19 × 107-3.78 × 109. After 21 days, the data on larval mortality and body weight were evaluated. The surviving larvae that were treated with the bacterial isolates did not acquire a substantial weight. For the RPW larvae and adults, the mortality and corrected mortality death rates were increased by increasing the concentration of B. thuringiensis. In conclusion, Bacillus-treated diets negatively influenced the growth and development of the RPW. This research reported on the interaction between the RPW and the rhizosphere Bacillus spp. and highlighted the tremendous potential for the development of microbial resource-based control strategies for this pest.

Larvicidal activity of Photorhabdus and Xenorhabdus bacteria isolated from insect parasitic nematodes against Aedes aegypti and Aedes albopictus.

Aedes aegypti and Aedes albopictus are important vectors for several arboviruses such as the dengue virus. The chemical control of Aedes spp., which is usually implemented, affects both humans and the environment. The biological control of Aedes spp. with entomopathogenic bacteria such as Photorhabdus and Xenorhabdus may be an alternative method that can overcome such issues. This study aimed to isolate and identify Photorhabdus and Xenorhabdus bacteria from entomopathogenic nematodes (EPNs) collected in Thailand and evaluate their larvicidal properties in controlling A. aegypti and A. albopictus. Colony morphology and recA sequencing of the 118 symbiotic isolated bacteria indicated that most were P. luminescens subsp. akhurstii and X. stockiae with minor prevalence of P. luminescens subsp. hainanensis, P. asymbiotica subsp. australis, X. indica, X. griffiniae, X. japonica, X. thuongxuanensis, and X. eapokensis. The larvicidal bioassay with the third- and fourth-instar mosquito larvae suggested that a whole-cell suspension of X. griffiniae (bMSN3.3_TH) had the highest efficiency in eradicating A. aegypti and A. albopictus, with 90 ± 3.71% and 81 ± 2.13% mortality, respectively, after 96 h exposure. In contrast, 1% of ethyl acetate extracted from X. indica (bSNK8.5_TH) showed reduced mortality for A. aegypti of only 50 ± 3.66% after 96 h exposure. The results indicate that both X. griffiniae (bMSN3.3_TH) and X. indica (bSNK8.5_TH) could be used as biocontrol agents against Aedes larvae.

The Insect Pathogen Photorhabdus luminescens Protects Plants from Phytopathogenic Fusarium graminearum via Chitin Degradation.

Phytopathogens represent a large agricultural challenge. The use of chemical pesticides is harmful to the environment, animals, and humans. Therefore, new sustainable and biological alternatives are urgently needed. The insect-pathogenic bacterium Photorhabdus luminescens, already used in combination with entomopathogenic nematodes (EPNs) as a biocontrol agent, is characterized by two different phenotypic cell forms, called primary (1°) and secondary (2°). The 1° cells are symbiotic with EPNs and are used for biocontrol, and the 2° cells are unable to undergo symbiosis with EPNs, remain in the soil after insect infection, and specifically interact with plant roots. A previous RNA sequencing (RNAseq) analysis showed that genes encoding the exochitinase Chi2A and chitin binding protein (CBP) are highly upregulated in 2° cells exposed to plant root exudates. Here, we investigate Chi2A and CBP functions and demonstrate that both are necessary for P. luminescens 2° cells to inhibit the growth of the phytopathogenic fungus Fusarium graminearum. We provide evidence that Chi2A digests chitin and thereby inhibits fungal growth. Furthermore, we show that 2° cells specifically colonize fungal hyphae as one of the first mechanisms to protect plants from fungal phytopathogens. Finally, soil pot bioassays proved plant protection from F. graminearum by 2° cells, where Chi2A and CPB were essential for this process. This work gives molecular insights into the new applicability of P. luminescens as a plant-growth-promoting and plant-protecting organism in agriculture. IMPORTANCE The enteric enterobacterium Photorhabdus luminescens is already being used as a bioinsecticide since it is highly pathogenic toward a broad range of insects. However, the bacteria exist in two phenotypically different cell types, called 1° and 2° cells. Whereas only 1° cells are symbiotic with their nematode partner to infect insects, 2° cells were shown to remain in the soil after an insect infection cycle. It was demonstrated that 2° cells specifically interact with plant roots. Here, we show that the bacteria are beneficial for the plants by protecting them from phytopathogenic fungi. Specific colonization of the fungus mycelium as well as chitin-degrading activity mediated by the chitin binding protein (CBP) and the chitinase Chi2A are essential for this process. Our data give evidence for the novel future applicability of P. luminescens as a plant-growth-promoting organism and biopesticide.

Citrobacter freundii, a natural associate of the Colorado potato beetle, increases larval susceptibility to Bacillus thuringiensis.

BACKGROUND: We assume that certain representatives of gut microflora mediate immune changes during dysbiosis, accelerating septicemia caused by Bacillus thuringiensis. RESULTS: Co-introduction of Citrobacter freundii with Bacillus thuringiensis var. tenebrionis (morrisoni) (Bt) led to an increase in Colorado potato beetle (CPB) larval mortality to 69.0% (1.3-5×) and a synergistic effect was observed from day 1 to day 6. Ultrathin sections of the CPB midgut showed autophagosome formation and partial destruction of gut microvilli under the influence of Bt, which was accompanied by pronounced hypersecretion of the endoplasmic reticulum with apocrine vesicle formation and oncotic changes in cells under the action of C. freundii. The destruction of gut tissues was accompanied by suppression of detoxification processes under the action of the bacteria and a decrease (2.8-3.5×) in the concentration of lipid oxidation products during Bt infection. In the first hours post combined treatment, we registered a slight increase in the total hemocyte count (THC) especially a predomination (1.4×) of immune-competent plasmatocytes. Oral administration of symbiotic and entomopathogenic bacteria to the CPB larvae significantly decreased the THC (1.4×) after 24 h and increased (1.1-1.5×) the detoxifying enzymes level in the lymph. These changes are likely to be associated with the destruction of hemocytes and the need to remove the toxic products of reactive oxygen species. CONCLUSION: The obtained results indicate that feeding of C. freundii and B. thuringiensis to the CPB larvae is accompanied by tissue changes that significantly affect the cellular and humoral immunity of the insect, increasing its susceptibility to Bt. © 2022 Society of Chemical Industry.

Beyond Soil-Dwelling Actinobacteria: Fantastic Antibiotics and Where to Find Them.

Bacterial secondary metabolites represent an invaluable source of bioactive molecules for the pharmaceutical and agrochemical industries. Although screening campaigns for the discovery of new compounds have traditionally been strongly biased towards the study of soil-dwelling Actinobacteria, the current antibiotic resistance and discovery crisis has brought a considerable amount of attention to the study of previously neglected bacterial sources of secondary metabolites. The development and application of new screening, sequencing, genetic manipulation, cultivation and bioinformatic techniques have revealed several other groups of bacteria as producers of striking chemical novelty. Biosynthetic machineries evolved from independent taxonomic origins and under completely different ecological requirements and selective pressures are responsible for these structural innovations. In this review, we summarize the most important discoveries related to secondary metabolites from alternative bacterial sources, trying to provide the reader with a broad perspective on how technical novelties have facilitated the access to the bacterial metabolic dark matter.

Isolation, Identification, and Biocontrol Potential of Entomopathogenic Nematodes and Associated Bacteria against Virachola livia (Lepidoptera: Lycaenidae) and Ectomyelois ceratoniae (Lepidoptera: Pyralidae).

Virachola livia (Lepidoptera: Lycaenidae) and Ectomyelois ceratoniae (Lepidoptera: Pyralidae) are the key pests of pomegranates in Saudi Arabia that are managed mainly using broad-spectrum pesticides. Interactions between the entomopathogenic nematodes (EPNs) Steinernematids, and Heterorhabditids, and their entomopathogenic bacterial symbionts (EPBs) have long been considered monoxenic 2-partner associations responsible for killing insects and, therefore, are widely used in insect pest biocontrol. However, there are limited reports identifying such organisms in Taif, Saudi Arabia. The current study aimed to identify the EPNs and their associated bacteria isolated from Taif, Saudi Arabia, and evaluate their biocontrol potential on third instar larvae of V. livia and E. ceratoniae under laboratory conditions. A total of 35 EPN isolates belonging to Steinernema (20) and Heterorhabditis (15) were recovered from 320 soil samples. Twenty-six isolates of symbiotic or associated bacteria were isolated from EPNs and molecularly identified as Xenorhabdus (6 isolates), Photorhabdus (4 isolates), Pseudomonas (7), or Stenotrophomonas (9). A pathogenicity assay revealed that Steinernema spp. were more virulent than Heterorhabditis spp. against the two pomegranate insects, with LC50 values of 18.5 and 13.6 infective juveniles (IJs)/larva of V. livia for Steinernema spp. and 52 and 32.4 IJs/larva of V. livia for Heterorhabditis spp. at 48 and 72 h post-treatment, respectively. Moreover, LC50 values of 9 and 6.6 IJs/larva (Steinernema spp.) and 34.4 and 26.6 IJs/larva (Heterorhabditis spp.) were recorded for E. ceratoniae larvae at 48 and 72 h post-treatment. In addition, the EPB Stenotrophomonas maltophilia CQ1, isolated from Steinernema spp., surpassed Pseudomonas mosselii SJ10, associated with Heterorhabditis spp., in their ability to kill V. livia or E. ceratoniae larvae within 6 h post-application, resulting in 100% mortality in both insects after 24 and 48 h of exposure. We conclude that either application of EPNs' IJs or their associated EPBs could serve as potential biocontrol agents for V. livia and E. ceratoniae.

A review on hydroxy anthraquinones from bacteria: crosstalk's of structures and biological activities.

Anthraquinones (AQ), unveiling large structural diversity, among polyketides demonstrate a wide range of applications. The hydroxy anthraquinones (HAQ), a group of anthraquinone derivatives, are secondary metabolites produced by bacteria and eukaryotes. Plant-based HAQ are well-studied unlike bacterial HAQ and applied as herbal medicine for centuries. Bacteria are known to synthesize a wide variety of structurally diversified HAQ through polyketide pathways using polyketide synthases (I, II & III) principally through polyketide synthase-II. The actinobacteria especially the genus Streptomyces and Micromonospora represent a rich source of HAQ, however novel HAQ are reported from the rare actinobacteria genera (Salinospora, Actinoplanes, Amycoloptosis, Verrucosispora, Xenorhabdus, and Photorhabdus. Though several reviews are available on AQ produced by plants and fungi, however none on bacterial AQ. The current review focused on sources of bacterial HAQ and their structural diversity and biological activities along with toxicity and side effects.

The endophytic bacterial entomopathogen Serratia marcescens promotes plant growth and improves resistance against Nilaparvata lugens in rice.

Entomopathogenic bacteria are commonly used as biological agents to control different insect pests. However, little is known about the role of bacterial entomopathogens as endophytes in regulating both plant growth and resistance against insect pests. Here, we applied the entomopathogenic bacterium Serratia marcescens S-JS1 via rice seed inoculation and evaluated its effects on host plant growth and resistance against the rice pest Nilaparvata lugens. Furthermore, the induction of defense-related secondary metabolites by the bacterium was assessed by GC-MS/MS. We showed that S-JS1 was able to endophytically colonize the roots and shoots of rice seedlings following seed inoculation. Colonized plants showed increased seed germination (9.4-13.3 %), root (8.2-36.4 %) and shoot lengths (4.1-22.3 %), and root (26.7-69.3 %) and shoot fresh weights (19.0-49.0 %) compared to plants without inoculation. We also identified the production of indole-3-acetic acid by S-JS1, which is likely involved in enhancing rice plant growth. In a two-choice test, N. lugens adults preferred to feed on untreated control plants than on plants treated with S-JS1. In the no-choice feeding tests, the survival of N. lugens nymphs that fed on S-JS1-treated plants was significantly lower than that of nymphs that fed on untreated plants. Additionally, seeds treated with 109 cfu/mL S-JS1 resulted in elevated levels of secondary metabolites, which may be associated with N. lugens resistance in rice plants. Therefore, we suggest that the entomopathogenic bacterium S. marcescens be considered a potentially promising endophyte for use in an innovative strategy for the integrated management of insect pests.

Uncovering Nematicidal Natural Products from Xenorhabdus Bacteria.

Parasitic nematodes infect different species of animals and plants. Root-knot nematodes are members of the genus Meloidogyne, which is distributed worldwide and parasitizes numerous plants, including vegetables, fruits, and crops. To reduce the global burden of nematode infections, only a few chemical therapeutic classes are currently available. The majority of nematicides are prohibited due to their harmful effects on the environment and public health. This study was intended to identify new nematicidal natural products (NPs) from the bacterial genus Xenorhabdus, which exists in symbiosis with Steinernema nematodes. Cell-free culture supernatants of Xenorhabdus bacteria were used for nematicidal bioassay, and high mortality rates for Caenorhabditis elegans and Meloidogyne javanica were observed. Promoter exchange mutants of biosynthetic gene clusters encoding nonribosomal peptide synthetases (NRPS) or NRPS-polyketide synthase hybrids in Xenorhabdus bacteria carrying additionally a hfq deletion produce a single NP class, which have been tested for their bioactivity. Among the NPs tested, fabclavines, rhabdopeptides, and xenocoumacins were highly toxic to nematodes and resulted in mortalities of 95.3, 74.6, and 72.6% to C. elegans and 82.0, 90.0, and 85.3% to M. javanica, respectively. The findings of such nematicidal NPs can provide templates for uncovering effective and environmentally safe alternatives to commercially available nematicides.