Entomopathogenic Bacterium Bacillus Thuringiensis Research Articles

Protatrans – growth biostimulants for centomopathogenic bacteria <i>Bacillus thuringiensis</i>

The study investigates the use of protatran compounds as growth stimulators for Bacillus thuringiensis bacteria, which are widely used as producers of biopesticides. Cultivation of the Bacillus thuringiensis strain subsp. kurstaki was carried out in a Luria-Bertani (LB) liquid medium. Protatrans (2-Me-C6H4OCH2COO- were added to the NN+(CH2CH2OH)3 (1), 4-Cl-C6H4 -SCH2COO-NN(CHCHOH) (2) and 4-Cl-CSOCHCOO NN+(CH2CH2OH)3 (3) media in concentrations of 1×10-4–1×10-8wt %. The LB medium without the addition of compounds 1–3 was used as a control. Cultures were incubated at a temperature of 30°C for 24 hours. The number of Bacillus thuringiensis cells was determined by serial dilution. The maximum growth was observed in a medium containing 1×10-4wt % of protatran 3. The number of cells was almost 10 times (966.7%) higher than in the control. In media with 1×10-5, 1×10-6, 1×10-7 and 1×10-8 wt % of compound 3, the number of cells was 4–7 times higher than in the control (by 371.7–666.7%). Protatrans 1 and 2 had a positive effect on Bacillus thuringiensis, increasing the number of cells by 83–292% compared to control. Therefore, it was demonstrated for the first time that commercially available non-toxic protatran compounds in microconcentrations are powerful growth stimulators for the entomopathogenic bacteria Bacillus thuringiensis. This indicates the potential for significant improvement and cost reduction of biotechnology for the production of bacterial insecticides based on Bacillus thuringiensis, used in agriculture, forestry and homesteads to control harmful insects.

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

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. 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. 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.

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.

Assessment of bio-formulations of indigenous strains of Bacillus thuringiensis, Metarhizium robertsii and Metarhizium majus for management of the rhinoceros beetle, Oryctes rhinoceros L., in field

BackgroundAmong all the coconut pests, rhinoceros beetle causes acute and serious damage to coconut palm. Management of this pest is very difficult due to its nocturnal activity, and also, it damages the emerging leaf inside the bud. Management of rhinoceros beetle using entomopathogens will be of great importance as it is economical and ecofriendly. Studies were carried out to decipher the biocontrol potential of indigenous entomopathogenic bacterium (Bacillus thuringiensis) and entomopathogenic fungi (Metarhizium robertsii and M. majus) against Oryctes rhinoceros in the field, individually as well as in combination, by soil drench as well as by topical spray method.ResultsThe study showed that B. thuringiensis strain NBAIR-BTAN4 showed 24.8% mortality at 5th week in soil drench method and 24% mortality in topical spray method. M. robertsii showed 24% mortality at 5th week in soil drench method as well as topical spray method. Similarly, M. majus showed 24% mortality in soil drench method and 23.2% in topical spray method at 5th week. In combination, NBAIR-BTAN4 + M. robertsii showed 40.8 and 44% in soil drench and topical spray method at 5th week, respectively. Combination of NBAIR-BTAN4 + M. majus showed 44.8 and 40.8% in soil drench and topical spray method at 5th week, respectively. Combination of NBAIR-BTAN4 + M. robertsii + M. majus showed 52.8 and 57.6% mortality at 5th week in soil drench and topical spray method, respectively.ConclusionThe present study showed application B. thuringiensis in combination with M. robertsii and M. majus is effective in management rhinoceros beetle in coconut orchard. Study also indicated that soil drench method is more promising strategy than topical spray method in managing larval population of the beetles and also confirmed that B. thuringiensis, M. robertsii and M. majus are compatible and they seemed to have a synergistic effect in controlling the pest in coconut orchard.

Evaluation and economic analysis of ecofriendly biological approaches for the management of shoot and fruit borer (Earias vittella F.) of okra

During the years 2020 and 2021, the effects of various biopesticides and the egg parasitoid Trichogramma chilonis on the okra shoot and fruit borer were investigated in open fields. All interventions outperformed over the untreated control. The entomopathogenic bacterium Bacillus thuringiensis was the most promising biopesticide tested, with the lowest shoot (5.49% and 6.87% in 2020 and 2021, respectively) and fruit damage (4.95% and 5.65% in 2020 and 2021, respectively), followed by the entomopathogenic fungi Beauveria bassiana (7.08%, 8.04% shoot damage and 6.78% and 6.73% fruit damage during 2020 and 2021, respectively). Interestingly, all biopesticides evaluated were shown to be safe for the polyphagous predators occurring in the okra habitat, such as the ladybird beetles Menochilus sexmaculatus and Micraspis discolour and the spiders Marpissa spp. and Oxyopes lineatipes. However, emamectin benzoate 5% SG was the most effective treatment in terms of minimizing okra shoot and fruit damage, resulting in a maximum percent reduction over control (PROC) (85.54 and 80.90 against shoot damage and 76.60 and 71.33 against fruit damage during 2020 and 2021, respectively). Each treatment's economics were also analysed. The experimental plots treated with emamectin benzoate had the highest cost:benefit ratio (1:11.16), while B. thuringiensis had the highest (1:7.06) among the biopesticide / parasitoid releasing plots.

A single transcription factor facilitates an insect host combating Bacillus thuringiensis infection while maintaining fitness

Maintaining fitness during pathogen infection is vital for host survival as an excessive response can be as detrimental as the infection itself. Fitness costs are frequently associated with insect hosts countering the toxic effect of the entomopathogenic bacterium Bacillus thuringiensis (Bt), which delay the evolution of resistance to this pathogen. The insect pest Plutella xylostella has evolved a mechanism to resist Bt toxins without incurring significant fitness costs. Here, we reveal that non-phosphorylated and phosphorylated forms of a MAPK-modulated transcription factor fushi tarazu factor 1 (FTZ-F1) can respectively orchestrate down-regulation of Bt Cry1Ac toxin receptors and up-regulation of non-receptor paralogs via two distinct binding sites, thereby presenting Bt toxin resistance without growth penalty. Our findings reveal how host organisms can co-opt a master molecular switch to overcome pathogen invasion with low cost, and contribute to understanding the underlying mechanism of growth-defense tradeoffs during host-pathogen interactions in P. xylostella.

STUDY OF EFFECTIVITY Bacillus thuringiensis BASED BIO-INSECTICIDE AGAINST Oryctes rhinoceros LARVAE AT SHADE HOUSE

Oryctes rhinoceros is an important pest in oil palm plantations. Adult stage of the beetle causes damage, however larval stage is very important to be controlled to break the cycle of life. An entomopathogenic bacterium Bacillus thuringiensis is one of biological agents to control these insects. Its toxic protein content provides specific insect targets as stomach poison. Objectives of the research was to study the impact of B. thuringiensis propagated in bio-urine enriched with 5 per cent molasses towards Oryctes larvae. The research was carried out in the shade house of Plant Protection Study Program, Faculty of Agriculture, Sriwijaya University from August to November 2021. Experiment was designed in a randomized complete block design with 7 treatments and 4 replications. A total of 20 ml of bio-insecticide was dissolved in 280 ml of water, sprayed evenly on the soil mixed with male palm flowers as feed of larvae. The treatments were 6 isolates of B. thuringiensis isolated from soil in oilpalm plantation, namely with codes: C14, C15, A15, OJ, BK, and LK as well. The results showed density of B. thuringiensis spores in bio-urine media was different in each isolate. The highest spore density in isolate code LK was 4.83 x 1010 spores/ml and the lowest (in isolate A15) was at 3.5 x 1010 spores/ml. Mortality rates were significantly different between isolate treatments starting from day 3 to day 12 of observation. C15 isolate lead the highest mortality rate of 100% on day 12 while other isolates showed mortality data below 100% (88-98%). Body weight and length showed significantly differences on days 0, 6 and 12 after application. Symptoms of infection begin with a change in skin color from white to brown, dark brown and black. Death is characterized by a soft body texture and wet rot.

Oral Immune Priming Treatment Alters Microbiome Composition in the Red Flour Beetle Tribolium castaneum.

It is now well-established that the microbiome is relevant for many of an organism’s properties and that its composition reacts dynamically to various conditions. The microbiome interacts with host immunity and can play important roles in the defenses against pathogens. In invertebrates, immune priming, that is, improved survival upon secondary exposure to a previously encountered pathogen, can be dependent upon the presence of the gut microbiome. However, it is currently unknown whether the microbiome changes upon priming treatment. We here addressed this question in a well-established model for immune priming, the red flour beetle Tribolium castaneum exposed to the entomopathogenic bacterium Bacillus thuringiensis (Bt). After priming treatments, the microbiota composition of beetle larvae was assessed by deep sequencing of the V1-V2 region of the bacterial 16S rRNA gene. We compared the effect of two established routes of priming treatments in this system: injection priming with heat-killed Bt and oral priming via ingestion of filtered sterilized bacterial spore culture supernatants. For oral priming, we used several strains of Bt known to vary in their ability to induce priming. Our study revealed changes in microbiome composition following the oral priming treatment with two different strains of Bt, only one of which (Bt tenebrionis, Btt) is known to lead to improved survival. In contrast, injection priming treatment with the same bacterial strain did not result in microbiome changes. Combined with the previous results indicating that oral priming with Btt depends on the larval microbiome, this suggests that certain members of the microbiome could be involved in forming an oral priming response in the red flour beetle.

Main and Ratooned Rice Pest Populations in Lowland Rice Fields, South Sumatra Applied Bioinsecticide from Entomopathogens

The pests that attack each plant phase also varies. The aimed of this study was to compare the main population of rice insect pests and ratooned in lowland rice field in the vegetative, flowering and milk ripening phase. The main bioinsecticide rice sprayed on rice canopy were comes from the entomopathogenic fungal conidia Metarhizium anisopliae, the entomopathogenic bacterium Bacillus thuringiensis, and as a control area without the application of bioinsecticide or synthetic insecticide. The results showed that population of fake white pests (Cnaphalocrosis medinalis) and white leafhoppers pest (Covana spectra) were more prevalent in vegetative phase rice compared to other types of pests such as green leafhoppers (Nephotettix spp.), Brown leafhoppers (Nilaparvata lugens), zig zag leafhoppers. (Recillia dorsalis), white-back leafhoppers (Sogatella furcifera), grasshoppers, mole cricket (Gryllotalpa gryllotalpa), and yellow rice stem borer (Scirpophaga incertulas). The Leptocorisa acuta and green ladybugs (Nezara viridula) began to attack the flowering and milk ripening phase, which was when the rice was aged about 54-68 days after transplanting (dat). Whereas in ratooned rice, the pest population of L. acuta has appeared on the first observation on rice aged 9 days after harvest (dah) until the ratooned rice was aged 58 days after harvesting. The population of pests was higher in land without bioinsecticide application compared to land applied of bioinsecticides. In ratooned rice, the pest population between the land applied with bioinsecticide M. anisopliae and B. thuringiensis was not significantly different from the land applied by B. thuringiensis which was sold in the market.

Propagation Of Entomopathogenic Bacteria Bacillus Thuringiensis In Various Agricultural Waste and Its Effectivity Against Oryctes Rinoceros (Coleoptera:Scarabaeidae)

Oryctes rhinoceros is an important pest on oil palm plantations, from immature plants to mature plants. Due to the long life cycle of Oryctes, control is not only aimed at the imago but also the larvae. Control using entomopathogenic bacterium Bacillus thuringiensis is expected to overcome this problem. The materials used as a medium for propagation are agricultural waste that is easy to obtain, cheap and safe for the environment. The research objective was to study the use of agricultural waste on growth of B. thuringiensis and its effectiveness against Oryctes larvae. The results showed bio-urine media enriched with 5% molasses produced the highest spore density compared to other media. The highest mortality rate of Oryctes larvae was 70% in B. thuringiensis treatment which was produced in bio-urine medium enriched with 5% molasses. Weight of Oryctes larvae reflects number of dead larvae. The more that die, the greater the difference in body weight. B. thuringiensis can be produced in a variety of agricultural waste media and was found to be effective in controlling Oryctes larvae.

Ecological advantages of using microbiomethod in an integrated plant protection system

The article presents an analysis of current domestic and world literature data on the use of microbiomethod in plant protection. It has been proven, that biological preparations based on different biotypes of entomopathogenic bacteria Bacillus thuringiensis, due to their high specificity to various insect species and harmlessness to adult bees, warm-blooded and freshwater organisms, remain at the forefront of integrated plant protection systems worldwide. Their amount is up to 95% of all bioinsecticides. Being natural elements of biocenoses, entomopathogenic microorganisms do not harm plants. Moreover, the introduction of a number of bacteria into the soil and on the surface of plants promotes the growth of crops and increase their yield.Given the economic non-competitiveness of bioinsecticides compared to chemical insecticides, their use in Integrated Pest Management (IPM) Systems is appropriate for the control of phytophages against which they are highly effective. The negative result of chemical insecticides is not only the pollution of the environment and products, but also the formation and spread of resistant races and pest populations. The higher cost of microbiomethod is justified by the lack of negative long-term effects of massive use of chemical insecticides.

The regulation landscape of MAPK signaling cascade for thwarting Bacillus thuringiensis infection in an insect host.

Host-pathogen interactions are central components of ecological networks where the MAPK signaling pathways act as central hubs of these complex interactions. We have previously shown that an insect hormone modulated MAPK signaling cascade participates as a general switch to trans-regulate differential expression of diverse midgut genes in the diamondback moth, Plutella xylostella (L.) to cope with the insecticidal action of Cry1Ac toxin, produced by the entomopathogenic bacterium Bacillus thuringiensis (Bt). The relationship between topology and functions of this four-tiered phosphorylation signaling cascade, however, is an uncharted territory. Here, we carried out a genome-wide characterization of all the MAPK orthologs in P. xylostella to define their phylogenetic relationships and to confirm their evolutionary conserved modules. Results from quantitative phosphoproteomic analyses, combined with functional validations studies using specific inhibitors and dsRNAs lead us to establish a MAPK "road map", where p38 and ERK MAPK signaling pathways, in large part, mount a resistance response against Bt toxins through regulating the differential expression of multiple Cry toxin receptors and their non-receptor paralogs in P. xylostella midgut. These data not only advance our understanding of host-pathogen interactions in agricultural pests, but also inform the future development of biopesticides that could suppress Cry resistance phenotypes.

Effects of NF-kB Signaling Inhibitors on Bed Bug Resistance to Orally Provisioned Entomopathogenic Bacteria.

Simple SummaryBed bugs are prolific insect pests across the globe. Chemical control methods are failing at an increasing rate, creating a need for alternative methods. While the use of microbial agents for biological control of insects has had significant success against many pests and holds great promise, research in this area has only recently begun to be considered for bed bugs. Here, we show that chemical inhibition of pathways involved in bed bug immunity can boost mortality during infection with orally provisioned entomopathogenic bacteria by reducing the ability of bed bugs to clear bacterial infection. Our findings suggest that reducing resistance to infection through inhibition of immune signaling may be a viable strategy for biological control of bed bugs. From an applied perspective, this work is a critical step towards the optimization of ingestible biological control formulations.Bed bugs are globally important pests and there is an ongoing need for the development and improvement of bed bug control tools. Though promising against other insect pests, the exploration of biological methods for bed bug control is limited. Previously, we identified several species of bacteria that have entomopathogenic effects against bed bugs when ingested. We also described the conservation of several antibacterial responses in bed bugs, including the expression of immune effector genes regulated by NF-kB transcription factors through the Toll and immune deficiency (IMD) signaling pathways. Accordingly, we predicted that chemical inhibition of NF-kB signaling could reduce bed bug resistance to orally provisioned entomopathogenic bacteria, potentially improving their effectiveness as biological control agents. In the present study, we administered four small molecule inhibitors of NF-kB signaling (BMS345541, IKK16, IMD0354, Takinib) to bed bugs by feeding them in a blood meal. We then quantified basal mortality and mortality in response to oral infection with two different entomopathogenic bacteria (Pseudomonas entomophila and Bacillus thuringiensis israelensis). None of the NF-kB signaling inhibitors tested increased mortality above control levels when administered alone, suggesting a lack of direct toxicity. However, one inhibitor (IKK16) significantly enhanced the rate of mortality from oral infection with P. entomophila. Enhanced mortality was independent of direct effects of IKK16 on P. entomophila growth in vitro but was associated with higher bacterial loads in vivo (i.e., reduced resistance). Together, these results provide new insight into the regulation of the bed bug immune system and suggest that administration of entomopathogens in combination with inhibition of immune signaling pathways to reduce infection resistance may be effective for biological control of bed bugs.

Making 3D-Cry Toxin Mutants: Much More Than a Tool of Understanding Toxins Mechanism of Action.

3D-Cry toxins, produced by the entomopathogenic bacterium Bacillus thuringiensis, have been extensively mutated in order to elucidate their elegant and complex mechanism of action necessary to kill susceptible insects. Together with the study of the resistant insects, 3D-Cry toxin mutants represent one of the pillars to understanding how these toxins exert their activity on their host. The principle is simple, if an amino acid is involved and essential in the mechanism of action, when substituted, the activity of the toxin will be diminished. However, some of the constructed 3D-Cry toxin mutants have shown an enhanced activity against their target insects compared to the parental toxins, suggesting that it is possible to produce novel versions of the natural toxins with an improved performance in the laboratory. In this report, all mutants with an enhanced activity obtained by accident in mutagenesis studies, together with all the variants obtained by rational design or by directed mutagenesis, were compiled. A description of the improved mutants was made considering their historical context and the parallel development of the protein engineering techniques that have been used to obtain them. This report demonstrates that artificial 3D-Cry toxins made in laboratories are a real alternative to natural toxins.

Microbial biopesticides in agroecosystems

Microbial biopesticides include several microorganisms like bacteria, fungi, baculoviruses, and nematode-associated bacteria acting against invertebrate pests in agro-ecosystems. The biopesticide sector is experiencing a significant growth and many discoveries are being developed into new biopesticidal products that are fueling a growing global market offer. Following a few decades of successful use of the entomopathogenic bacterium Bacillus thuringiensis and a few other microbial species, recent academic and industrial efforts have led to the discovery of new microbial species and strains, and of their specific toxins and virulence factors. Many of these have, therefore, been developed into commercial products. Bacterial entomopathogens include several Bacillaceae, Serratia, Pseudomonas, Yersinia, Burkholderia, Chromobacterium, Streptomyces, and Saccharopolyspora species, while fungi comprise different strains of Beauveria bassiana, B. brongniartii, Metarhizium anisopliae, Verticillium, Lecanicillium, Hirsutella, Paecilomyces, and Isaria species. Baculoviruses are species-specific and refer to niche products active against chewing insects, especially Lepidopteran caterpillars. Entomopathogenic nematodes (EPNs) mainly include species in the genera Heterorhabditis and Steinernema associated with mutualistic symbiotic bacteria belonging to the genera Photorhabdus and Xenorhabdus. An updated representation of the current knowledge on microbial biopesticides and of the availability of active substances that can be used in integrated pest management programs in agro-ecosystems is reported her.

Toxicity of Bacillus thuringiensis Berl. KJ3P1 and DLM isolates towards pest of oil palm Oryctes rhinoceros [Coleoptera: Scarabaeidae

Oil palm plants are industrial plants that generate foreign exchange for Indonesia. Some obstacles are leading to production decrease. One of the important pests is Oryctes rhinoceros [Coleoptera: Scarabaeidae]. Both larval and imago stadia attack oil-palm in immature plants [IP] and mature plants [MP]. The method of controlling Oryctes often use chemical insecticides, in which continuously applications will cause residues in the environment and mortality of non-target insects. Therefore it is necessary to find an environmentally friendly control method that is easy, inexpensive and safe for the environment. The use of entomopathogenic bacteria Bacillus thuringiensis is an alternative. The study was aimed to study the toxicity of two B. thuringiensis isolates from the collection of Plant Protection Department, Faculty of Agriculture, Sriwijaya University [KJ3P1 and DLM isolate codes] on mortality of 3rd instar Oryctes larvae. Experiments were conducted at the Entomological Laboratory, Department of Plant Protection, Faculty of Agriculture, Sriwijaya University from March until May 2019. Bioinsecticide-based B. thuringiensis was made with 2 types of growth media, namely: 1] cow’s bio-urine enriched with 5% molasses, and 2]. Rice washing water enriched with 5% molasses. Treatments were included: 1] DLM5 isolate, 2] KJ3P1 isolate, 3] commercial B. thuringiensis and 4]. without treatment [as control]. The test insects used were 25 individuals of 1st instar Oryctes larvae in each treatment. The results showed KJ3PI isolate that propagated in bio-urine media showed the highest production of B. thuringiensis [5.69 x 106 spores/ml], the highest mortality [76% ] and the lowest LT50 value [5.28 days] as well. The use of agricultural and livestock waste as a medium to multiply B. thuringiensis bacteria may produce high cell and spore production and can be used as bioinsecticides to control O. rhinoceros.

Bacillus thuringiensis propagated in bio-urine media as a biological control of termite Coptotermes curvignathus and armyworm Spodoptera litura

Biological control using entomopathogenic bacteria Bacillus thuringiensis is in increasing demand because of its specific target toxicity, safe for natural enemies as well as pollinating insects. Propagation media for B. thuringiensis greatly determines the success of spores number produced and toxicity level to insect pests. This study aimed to record the spore density of indigenous isolates of South Sumatera propagated in bio-urine and 5% molasses media and their toxicity level against termites Coptotermes curvignathus and armyworm Spodoptera litura. The research was conducted at Biological Control Laboratory, Plant Protection Study Program, Faculty of Agriculture, UNSRI, from March to June 2019. The study was designed with Factorial Completely Randomized Design, using isolates as many as 3 isolates with 3 media treatments (bio-urine, bio-urine + 5% molasses, and Nutrient Broth) and 5 replications. The bio-assay used worker caste termites and 3rd instar armyworms. The results showed that a mixture of bio-urine and 5% molasses produced higher number of spores compared to bio-urine media only. The use of NB as a control showed higher spore production compared to the 2 other treatments. The mortality rate of Coptotermes termite was higher than the mortality rate of armyworms. Death symptoms of insect pests were indicated by the presence of wet rot and color change (blackish).

Successful and unexpected potential of entomopathogen Bacillus thuringiensis as plant growth promoting rhizobacterium

The microbiology of the rhizosphere has been investigated since 1904, revealing innumerable rhizomic microorganisms that promote growth in swamps (RPCPs), which have attracted a special interest to improve agroecological practices, crop productivity and protect soils from environmental contamination under a agricultural sustainability approach. This review addresses a topic as extensive as it is fascinating, beginning with the characteristics of soil microbiology. It also describes how RPCPs associated with plant roots offer greater crop growth through direct and indirect strategies. Also mention the complexity of plant growth and how it is done simultaneously through numerous metabolic activities. The bacterial genera of RPCP, the metabolic characteristics and the potential of entomopathogenic bacteria Bacillus thuringiensis (Bt) to be considered as RPCP are mentioned. Finally, the little known properties of Bt are exposed to benefit the growth of plants, their effectiveness in biocontrol of phytopathogens, as biofertilizers and / or biostimulants in crops. New perspectives are proposed to develop Bt products as a growth promoter in the near future. It is important to recognize that applied research of rhizosphere crops can be one of the key factors in achieving the sustainable yield of agriculture.

CRISPR/Cas9-mediated knockout of both the PxABCC2 and PxABCC3 genes confers high-level resistance to Bacillus thuringiensis Cry1Ac toxin in the diamondback moth, Plutella xylostella (L.)

Rapid evolution of resistance by insect pests severely jeopardizes the sustainable utilization of biopesticides and transgenic crops that produce insecticidal crystal proteins derived from the entomopathogenic bacterium Bacillus thuringiensis (Bt). Recently, high levels of resistance to Bt Cry1 toxins have been reported to be genetically linked to the mutation or down-regulation of ABC transporter subfamily C genes ABCC2 and ABCC3 in seven lepidopteran insects, including Plutella xylostella (L.). To further determine the causal relationship between alterations in the PxABCC2 and PxABCC3 genes and Cry1Ac resistance in P. xylostella, the novel CRISPR/Cas9 genome engineering system was utilized to successfully construct two knockout strains: the ABCC2KO strain is homozygous for a 4-bp deletion in exon 3 of the PxABCC2 gene, and the ABCC3KO strain is homozygous for a 5-bp deletion in exon 3 of the PxABCC3 gene, both of which can produce only truncated ABCC proteins. Bioassay results indicated that high levels of resistance to the Cry1Ac protoxin were observed in both the ABCC2KO (724-fold) and ABCC3KO (413-fold) strains compared to the original susceptible DBM1Ac-S strain. Subsequently, dominance degree and genetic complementation tests demonstrated that Cry1Ac resistance in both the knockout strains was incompletely recessive, and Cry1Ac resistance alleles were located in the classic BtR-1 resistance locus that harbored the PxABCC2 and PxABCC3 genes, similar to the near-isogenic resistant NIL-R strain. Moreover, qualitative toxin binding assays revealed that the binding of the Cry1Ac toxin to midgut brush border membrane vesicles (BBMVs) in both knockout strains was dramatically reduced compared to that in the susceptible DBM1Ac-S strain. In summary, our CRISPR/Cas9-mediated genome editing study presents, for the first time, in vivo reverse genetics evidence for both the ABCC2 and ABCC3 proteins as midgut functional receptors for Bt Cry1 toxins in insects, which provides new insight into the pivotal roles of both the ABCC2 and ABCC3 proteins in the complex molecular mechanism of insect resistance to Bt Cry1 toxins.

Microbial Biopesticides in Agroecosystems

Microbial biopesticides include several microorganisms like bacteria, fungi, baculoviruses, and nematode-associated bacteria acting against invertebrate pests in agro-ecosystems. The biopesticide sector is experiencing a significant growth and many discoveries are being developed into new biopesticidal products that are fueling a growing global market offer. Following a few decades of successful use of the entomopathogenic bacterium Bacillus thuringiensis and a few other microbial species, recent academic and industrial efforts have led to the discovery of new microbial species and strains, and of their specific toxins and virulence factors. Many of these have, therefore, been developed into commercial products. Bacterial entomopathogens include several Bacillaceae, Serratia, Pseudomonas, Yersinia, Burkholderia, Chromobacterium, Streptomyces, and Saccharopolyspora species, while fungi comprise different strains of Beauveria bassiana, B. brongniartii, Metarhizium anisopliae, Verticillium, Lecanicillium, Hirsutella, Paecilomyces, and Isaria species. Baculoviruses are species-specific and refer to niche products active against chewing insects, especially Lepidopteran caterpillars. Entomopathogenic nematodes (EPNs) mainly include species in the genera Heterorhabditis and Steinernema associated with mutualistic symbiotic bacteria belonging to the genera Photorhabdus and Xenorhabdus. An updated representation of the current knowledge on microbial biopesticides and of the availability of active substances that can be used in integrated pest management programs in agro-ecosystems is reported here.