Thuringiensis Strains Research Articles

Biotoxicity comparison of Bacillus thuringiensis to control vector borne diseases against mosquito fauna

The current study was designed to evaluate the biotoxicity of screened echo-friendly Bacillus thuringiensis strains from different areas of Pakistan. Out of 50 samples, 36% Bt. isolates were quarantined from soil containing cattle waste after morphological, biochemical, and molecular characterization. The toxicity bioassays with Bt. spores and protein diet proved that 11 Bt. isolates were utmost noxious to 3rd instar larvae of mosquitoes Aedes aegypti, Anopheles stephensi, and Culex pipiens. The entopathogenic activity of first 4 Bt. toxins against A. aegypti was highly lethal as compared to the other dipteran larvae. The toxicity (LC50) of spore diet of Bt. strains GCU-DAB-NF4 (442.730 ± 0.38 μg/ml), NF6 (460.845 ± 0.29 μg/ml), NF3 (470.129 ± 0.28 μg/ml), and NF7 (493.637 ± 0.70 μg/ml) was quite high against A. aegypti as compared to the C. pipiens after 24 h of incubation. The highest toxicity of total cell protein was shown by GCU-DAB-NF4 (LC50 = 84.10 ± 50 μg/ml), NF6 (95.122 ± 0.40 μg/ml), NF3 (100.715 ± 06 μg/ml), and NF5 (103.40 ± 07 μg/ml) against A. aegypti after 24 h. So, these strains a have great potential to be used as biological control especially against A. aegypti as compared to the C. pipiens.

Utilization of a strong promoter combined with the knockout of protease genes to improve the yield of Vip3Aa in Bacillus thuringiensis BMB171.

Vip3Aa is an insecticidal protein secreted by some Bacillus thuringiensis strains during vegetative growth. It has excellent insecticidal activity, its mechanism of action is different from that of Cry protein, and it can delay the development of pest resistance. To date, Vip3Aa has been widely used in genetically modified Bt crops. However, the secretion of Vip3Aa by industrial production strains is usually very low. Moreover, most of the Vip3Aa in the medium is degraded by proteases, limiting its application as a biopesticide. We report a novel constitutive strong promoter from B. thuringiensis, Prsi , which directs the abundant expression of vip3Aa in B. thuringiensis BMB171. Furthermore, to reduce the degradation of Vip3Aa caused by proteases, we constructed B. thuringiensis mutants in which different protease genes were knocked out. We found that the degradation of Vip3Aa was greatly inhibited and its yield was significantly improved in a mutant that lacked all three protease genes. Our results provide a new strategy to enhance the production of Vip3Aa in B. thuringiensis and have reference value for the research and development of novel bioinsecticides. © 2023 Society of Chemical Industry.

Insecticidal activity of Bacillus thuringiensis strains isolated from tropical greenhouses towards Cydia pomonella and Spodoptera exigua larvae

Three Bacillus thuringiensis strains were isolated from a specific habitat of tropical greenhouses located in the Botanical Garden of Adam Mickiewicz University in Poznań, Poland. The BG11, BG12 and BG15 strains harbour genes encoding a specific set of insecticidal proteins (cry1Ba, cry1Ia, cry2Ab, vip3Aa)—entirely different from those found in commercial isolates, currently used as bioinsecticides. Despite high genetic similarity of the new strains, each of them produces unique Cry1Ba toxin as a main component of the parasporal crystals. Moreover, the tested entomopathogens contain genetic determinants encoding two types of chitinolytic enzymes ChiA and ChiB. The tested strains display insecticidal activity against two distinct, economically important pest insects, Cydia pomonella L. (Lepidoptera: Tortricidae) and Spodoptera exigua Hübner (Lepidoptera: Noctuidae). However, BG12 and BG15 strains are significantly more active than BG11 towards both pests. The BG12 and BG15 strains can be considered as candidates for the production of new lepidopteran-active bioinsecticides with high potential to augment the existing biocontrol strategies.

Screening of Anti-fungal Bacillus Strains and Influence of their Application on Cocoa Beans Fermentation and Final Bean Quality

Aims: Contamination with filamentous fungi reduces the quality of cocoa beans and poses a health risk for consumers due to the potential accumulation of mycotoxins. The aim of this study was to develop antifungal Bacillus cultures for cocoa beans fermentation.
 Methods and Results: Antifungal activity of 7 Bacillus isolates was investigated using overlay technique. Solid culture media inoculated with bacterial and fungal isolates were cultivated under aerobic conditions at 30 ºC for 5 days. The Bacillus strain with strong antifungal activity was studied and inoculated to 18 kg box fermentations cocoa beans in Cote d'Ivoire in three time-independent replications each including a spontaneous control fermentation. The comparison of inoculated and spontaneous fermentation processes revealed that the Bacillus thuringiensis strain ATCC 10792 don't affected the fermentation process and cocoa bean quality. The anti-fungal in vivo assays revealed that the Bacillus thuringiensis strain ATCC 10792 completely inhibited growth of all fungi on the surface of cocoa beans. The result showed that sensory evaluation of chocolates is not significant differences for all treatment. The sensory evaluation of control and chocolates made from the inoculated cocoa beans with Bacillus showed excellent sensory quality, demonstrating that Bacillus thuringiensis strain ATCC 10792 did not affect aroma, texture and appearance of chocolate.
 Conclusion: From this study, it is revealed that the addition of Bacillus thuringiensis strain ATCC 10792 during fermentation cocoa beans suppress fungal growth and improve the quality of cocoa beans. Therefore, the anti-fungal culture Bacillus thuringiensis strain ATCC 10792 is recommended for future applications and its capacity to limit fungal growth during industrial-scale cocoa bean fermentation should be investigated in further studies.

Use of pathogenic microorganisms in the control of the Colorado potato beetle (Leptinotarsa decemlineata Say.)

The focus of this article is the comprehensive study of the Colorado potato beetle population within potato agro-biocenosis. The research delves into the intricate processes that contribute to the formation and bioecological characteristics of this beetle population. The article also delves into a specific strain known as Beauveria bassiana VTq-28, which was isolated from the Colorado potato beetle. This strain was subjected to testing both in laboratory settings and in the field, targeting various developmental stages of the Colorado potato beetle. The objective was to evaluate the effectiveness of Beauveria bassiana VTq-28 as a biocontrol agent against the beetle. Additionally, the research assessed the insecticidal activity of locally sourced strains of B. thuringiensis against the Colorado potato beetle. This analysis provides insights into the potential of B. thuringiensis strains as another avenue for biocontrol. By thoroughly examining the population dynamics, bioecological characteristics, and the potential of specific microbial agents for controlling the Colorado potato beetle, this study contributes to the understanding of pest management strategies within potato agro-biocenosis. The findings have implications for sustainable agricultural practices and the effective control of this economically significant pest.

Identification of cry genes in Bacillus thuringiensis by multiplex real-time PCR

Bacillus thuringiensis is an important bacterium of the group Bacillus cereus sensu lato due to its insecticidal properties. This microorganism has high genetic variability and its strains produce different Cry toxins, known as δ-endotoxins, which are mainly responsible for its toxic effect on insects that are agricultural pests or vector human diseases. Each strain can express a variety of cry genes, out of a total of 789 cry genes described so far. The detection of these genes is very important to characterize strains, as they may indicate their toxic potential. Several methods have been used to characterize B. thuringiensis strains, but one of the most common techniques is Polymerase Chain Reaction (PCR) from primers that detect the presence of cry genes. This technique has been optimized to make real-time multiplex quantitative PCR (qPCR) assays faster, more efficient, and safer, because the presence of three genes can be detected in a single reaction. In this work, a multiplex assay was developed to identify the presence of genes from the cry1A, cry1C, and cry1F families whose respective toxins are present in both bioinsecticides, and commercial transgenic plants used to control caterpillars. Specific primers were designed to identify the families of the cited genes and the system was validated with samples that were sequenced by next-generation sequencing (NGS). The system was implemented and used to characterize 214 strains. Of these, eight were submitted to conventional PCR, and the results matched, again validating the system. Thus, the application of the proposed technique allows the reliable evaluation through this system to detect the presence of the genes of the families cry1A, cry1C, and cry1F in samples of B. thuringiensis.

Insecticidal Effects of Some Bacillus thuringiensis Commercial Biopreparats on the Larvae of the Tomato Leaf Miner, Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae)

Effects of 5 commercial biopreparats named; Dacron®, Florbac®, Dipel®, Delfin® and Rebound® containing different Bacillus thuringiensis (Bt) strains on the 3rd instar larvae of the tomato leaf miner, Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae) were determined under laboratory conditions. T. absoluta, tomato (Gusto F1) variety and some commercial biopreparats were used as materials in the study. Production of T. absoluta larvae were performed under the conditions of the growth chambers where the tomato variety was grown (25±1°C, 60±5% RH and 16:8 photoperiod). Under laboratory conditions, 1, 3 and 5% concentrations of commercial biopreparats were applied on the 3rd instar larvae of the pest, and the experiment was carried out with 5 replications according to a randomized parcels design. As a result of the application of 1% IU/mg concentrations of Dacron® and Delfin® commercial biopreparats, 100% mortality rate was determined in the 3rd instar larvae of T. absoluta on the 7th day counts. When 3 and 5% IU/mg concentrations of all commercial biopreparats were applied, a mortality rate of 100% was recorded on the 3rd instar larvae of T. absoluta on the 5th and 7th day counts. In-vitro, as the concentration of commercial biopreparats increased, mortality rate of the 3rd instar larvae of T. absoluta increased. However, it would be appropriate to conduct field trials with the same commercial biopreparats to recommend their use of them commercially.

Potential Management Options for the Invasive Moth Spodoptera frugiperda in Europe

We here review and discuss management options that growers in Europe could take in response to the expected invasion of the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae). The focus is put on maize but the information provided is also relevant for other crops potentially affected. A sound forecasting system for fall armyworm both on a regional as well as at local scale should be established to alert growers as early as possible. Whilst a number of cultural control methods are adopted by maize growers in different regions globally to fight fall armyworm, many of them may either not be highly effective, too laborious, or otherwise unfeasible within the mechanized crop production systems used in Europe. Potential is seen in the stimulation of natural enemies through conservation biocontrol approaches, e.g., the planting of flower strips or intermediate cover crops, reducing tillage intensity, and avoiding broad-spectrum insecticides. To manage fall armyworm infestations, several effective biologically-based products are available globally, and some in Europe, e.g., based on specific baculoviruses, certain Bacillus thuringiensis strains, few entomopathogenic nematodes, and a number of botanicals. These should be given priority to avoid a major influx of insecticides into the maize agro-ecosystem once the fall armyworm arrives and in case growers are not prepared. Plant protection companies, particularly biocontrol companies should act proactively in starting registration of ingredients and products against fall armyworm in Europe. European maize growers should be made aware, in time, of key features of this new invasive pest and appropriate control options.

Abundance, distribution, and expression of nematicidal crystal protein genes in Bacillus thuringiensis strains from diverse habitats

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that accumulates pesticidal proteins (Cry and Cyt) in parasporal crystals. Proteins from the Cry5, App6 (formerly Cry6), Cry12, Cry13, Cry14, Cry21, and Xpp55 (formerly Cry55) families have been identified as toxic to nematodes. In this study, a total of 846 Bt strains belonging to four collections were analyzed to determine the diversity and distribution of the Bt Cry nematicidal protein genes. We analyzed their presence by PCR, and positives were confirmed by sequencing. As a result, 164 Bt isolates (20%) contained at least one gene coding for nematicidal Cry proteins. The cry5 and cry21 genes were enriched in collection 1 and were often found together in the same strain. Differently, in collection 4, obtained from similar habitats but after 10 years, cry14 was the gene most frequently found. In collection 2, cry5 and app6 were the most abundant genes, and collection 3 had a low incidence of any of these genes. The results point to high variability in the frequencies of the studied genes depending on the timing, geographical origins, and sources. The occurrence of cry1A, cry2, and cry3 genes was also analyzed and showed that the nematicidal Cry protein genes were frequently accompanied by cry1A + cry2. The expression of the genes was assessed by mass spectrometry showing that only 14% of the positive strains produced nematicidal proteins. To our knowledge, this is the first comprehensive screening that examines the presence and expression of genes from the seven known Bt Cry nematicidal families.

Biological Activity of Indigenous Selected Plant Growth Promoting Rhizobacteria Isolates and their Ability to Improve the Growth Traits of Shallot (Allium ascalonicum L.)

The bacterial leaf blight is the primary disease and causes a significant loss of shallot yield in West Sumatera, Indonesia. Plant growth-promoting rhizobacteria (PGPR) is essential in suppressing diseases and improving shallots' growth and health. This study aimed to characterize and investigate the ability of PGPR isolates to control shallots bacterial leaf blight disease and improve its growth. Ten (10) isolates of PGPR designated as Bacillus thuringiensis strain MRSNRZ3.1, B. mycoides strain MRSNUMBE2.2, B. mycoides strain MRBPBT2.1, B. waihenstephanensis strain MRBTLL3.2, B. subtilis strain MRTDUMBE3.2.1, B. cereus strain MRDKBTE1.3, B. cereus MRPLUMBE1.3, Achromobacter insolitus strain MRBPUMBE1.3, Pseudomonas hibiscicola strain MRTLDRZ2.2, and Bacillus sp. strain MRSPRZ1.1 were collected from the rhizosphere and endosphere of shallots from Agam and Solok Regency. The variables observed were biocontrol characters (HCN production and siderophore) and biofertilizer characters (indole acetic acid production, ammonia, and phosphate dissolution). These isolates showed activity in controlling leaf blight caused by Xanthomonas axonopodis. The present study also suggests that PGPR isolates, viz. MRSNRZ3.1, MRSNUMBE2.2, MRBPBT2.1, MRBTLL3.2, MRTDUMMBE3.2.1, MRDKBTE1.3, MRPLUMBE1.3, MRBPUMBE1.3, MRTLDRZ2.2, and MRSPRZ1.1 may be used as biofertilizers to enhance the growth and productivity of shallots. This finding suggests that identified PGPR isolates can be used as biological control agents and biofertilizers to enhance the growth and productivity of shallot.

Efficiency of Bacillus thuringiensis and Bacillus cereus against Rhynchophorus ferrugineus.

Simple SummaryRynchophorus ferrugineus Olivier, the red palm weevil (RPW), is a damaging insect that often severely infests palm trees. Since insecticides have a detrimental environmental impact and may lead to pesticide resistance, new biological control methods are needed. Bacteria from many different species affect the RPW larval growth, health, and immunity. The work evaluates the crosstalk between the RPW and rhizosphere bacteria. Four bacterial isolates, three belonging to Bacillus cereus and one belonging to B. thuringiensis, had a significantly higher impact on the mortality of the larvae and adults of the RPW than other bacteria that were tested did. The results emphasize the significant potential of developing new microbial resource-based management techniques for this pest.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.

Eco-Friendly Solution Based on Rosmarinus officinalis Hydro-Alcoholic Extract to Prevent Biodeterioration of Cultural Heritage Objects and Buildings.

Biodeterioration of cultural heritage is caused by different organisms capable of inducing complex alteration processes. The present study aimed to evaluate the efficiency of Rosmarinus officinalis hydro-alcoholic extract to inhibit the growth of deteriogenic microbial strains. For this, the physico-chemical characterization of the vegetal extract by UHPLC–MS/MS, its antimicrobial and antibiofilm activity on a representative number of biodeteriogenic microbial strains, as well as the antioxidant activity determined by DPPH, CUPRAC, FRAP, TEAC methods, were performed. The extract had a total phenol content of 15.62 ± 0.97 mg GAE/mL of which approximately 8.53% were flavonoids. The polyphenolic profile included carnosic acid, carnosol, rosmarinic acid and hesperidin as major components. The extract exhibited good and wide spectrum antimicrobial activity, with low MIC (minimal inhibitory concentration) values against fungal strains such as Aspergillus clavatus (MIC = 1.2 mg/mL) and bacterial strains such as Arthrobacter globiformis (MIC = 0.78 mg/mL) or Bacillus cereus (MIC = 1.56 mg/mL). The rosemary extract inhibited the adherence capacity to the inert substrate of Penicillium chrysogenum strains isolated from wooden objects or textiles and B. thuringiensis strains. A potential mechanism of R. officinalis antimicrobial activity could be represented by the release of nitric oxide (NO), a universal signalling molecule for stress management. Moreover, the treatment of microbial cultures with subinhibitory concentrations has modulated the production of microbial enzymes and organic acids involved in biodeterioration, with the effect depending on the studied microbial strain, isolation source and the tested soluble factor. This paper reports for the first time the potential of R. officinalis hydro-alcoholic extract for the development of eco-friendly solutions dedicated to the conservation/safeguarding of tangible cultural heritage.

Biotoxicity assessment of cloned cry 11 protein gene from Bacillus thuringiensis 9NF

The current investigation describes the isolation and characterization of toxic Bt. local isolates harboring 99% homology with Bti. prototoxin Bacillus thuringiensis (AXJ97553.1 and novel OUB27301.1) which contains full length cry11 gene (1.9 kb). Initially, it was cloned in pTZ57R/T and then sub-cloned in pET30a(+) for expression. The optimized conditions for good expression were found 1 mM IPTG, 3.5–4 h incubation time, and 37 °C. Toxicological assays were determined against 3rd instar larvae of Aedes aegypti with expressed partially purified and crude recombinant protein using recombinant E. coli BL21, DE3 transformed with cry11 gene. It was found that partially purified Bt. protein is highly toxic against A. aegypti larvae with LC50 value of 42.883 ± 6 µg/ml. B. thuringiensis strains producing Cry 11 toxic protein can be used as biopesticide to control resistance in insects.

Semi-Arid-Habitat-Adapted Plant-Growth-Promoting Rhizobacteria Allows Efficient Wheat Growth Promotion

Plant-growth-promoting rhizobacteria (PGPR) introduced into agricultural ecosystems positively affect agricultural production and constitute an ecological method for sustainable agriculture. The present study demonstrated the effects of two PGPR, Pantoea agglomerans strain Pa and Bacillus thuringiensis strain B25, on seed germination, on the plant growth of two durum wheat varieties, Bousselam and Boutaleb, and on the frequency of the cultivable beneficial bacterial community. The bacterial strains were used as seed primers (individually or in consortia) by coating them with carboxymethyl cellulose (CMC 1%). The effect of PGPR was negligible on germinative ability but improved seed vigor in the Boutaleb variety after inoculation with the Pa strain alone or in combination with the B25 strain. The results showed that the germination capacity depends on the wheat variety. It seemed to be better in the Bousselam variety. Analysis of the results of morphological plant parameters in sterile compost after 75 days under controlled conditions (16 h light, 26/16 °C day/night) showed a significant improvement in plant growth in both wheat varieties with the Pa strain alone or in combination. Chlorophyll (a, and total), carotenoid, and total soluble sugars were significantly increased, while proline and MDA were strongly reduced by inoculation of the Bousselam variety. Bacterial survival of the Pa and B25 strains in the rhizosphere of sterile compost was appreciable (105–107 CFU/g) for both the Pa and B25 strains. Only the Pa strain was endophytic and able to colonize roots. Contrary to sterile compost, the different inoculation treatments in natural soil (after 114 days) significantly improved all morphological parameters and chlorophyll pigments in both wheat varieties. The reduction of proline contents at the leaf level was observed with Pa, mainly in Bousselam. Bacterial densities of the rhizosphere and endophyte cultivable communities did not differ significantly. However, the number of cultivable beneficial bacteria isolated from roots and rhizosphere with multiple PGP traits was significantly increased. Bacterial survival of CMC-coated seed inoculum was appreciable and remained constant, especially for the Pa strain, during 21 months at room temperature. Based on these results, the PGPR used after seed priming would be a feasible and sustainable strategy to improve soil fertility and promote growth of durum wheat in stressful and non-stressful environments.

Management of chromium(VI)-contaminated soils through synergistic application of vermicompost, chromate reducing rhizobacteria and Arbuscular mycorrhizal fungi (AMF) reduced plant toxicity and improved yield attributes in Ocimum basilicum L.

An integrated approach involving vermicompost, chromate reducing bacteria and AMF was tested to manage the toxic impacts of Cr(VI) on Ocimum basilicum as a model plant. Pot experiments were conducted on O. basilicum plants in an artificially Cr(VI)-contaminated soil in two phases of experiment as bioinoculants experiment and vermicompost experiment. In the first phase of the bioinoculants experiment the series of gradient concentrations of Cr(VI) (0, 25, 50 and 100 mg kg–1 in soil) were evaluated with previously isolated four efficient Cr(VI)-reducing rhizo-bacterial strains (Bacillus Cereus strain SUCR 44, BC; Microbacterium sp. strain SUCR 140, MB; Bacillus thuringiensis strain SUCR186, BT; and Bacillus subtilis strain SUCR188; BS) along with Arbuscular Mycorrhizal Fungus—Glomus fasciculatum (GF) in alone and in co-inoculation form. In the second experiment (vermicompost) the best performing strain (MB) was tested alone or in combination with GF along with different doses of vermicompost. It was observed that vermicompost by itself could be useful in decreasing the bioavailable Cr(VI), uptake of Cr besides improving the nutritional status of plants. The vermicompost also played an important and indirect role and improved herb yield by supporting the multiplication of MB (Microbacterium sp.), an efficient chromate reducing rhizobacteria, that further decreased the bioavailable and toxic form of Cr and improved population and colonization of GF too. The translocation of Cr(VI) was averted through improved colonization of GF, also prevented higher accumulation of Cr in aerial parts (leafy herb) of O. basilicum.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00203-022-03230-z.

Molecular Characterization of Native Bacillus thuringiensis Strains from Root Nodules with Toxicity Against the Fall Armyworm (FAW, Spodoptera frugiperda) and Brinjal Ash Weevil (Myllocerus subfasciatus).

The fall armyworm is an exotic pest which destroys a wide variety of crops Querywhereas the brinjal ash weevil is a serious pest of eggplant and other solanaceous vegetables. The goal of this research is to find a sustainable and ecologically friendly bio-control agent for managing FAW and brinjal ash weevils. Twelve natural Bacillus thuringiensis strains were isolated from cowpea root nodules, and the Gram-positive cells with characteristic Bt crystal structures were discovered using phase contrast and scanning electron microscopy. There were bipyramidal, cuboidal, rhombus, and spherical crystals. The Bt cry gene content was characterized by PCR analysis, which revealed the presence of cry1, cry1I, cry3, cry7, cry7,8, cry14, cry26, and cry55 genes. The identity of Bt was confirmed by cloning and sequencing the cry genes. In the nucleotide sequences, no pseudo genes or indels were found in cry sequences. SDS-PAGE examination indicated the presence of bands ranging in size from 13 to 130kDa, with 50-60kDa being the most common. When compared to the control, the new native Bt strains were lethal, with pathogenicity ranging from 93 to 100% against S. frugiperda larvae and M. subfasciatus adults. The studies revealed that the native strains with conserved regions of 16S rRNA genes were compared to NCBI database sequences and classified as native Bt strains with 99-100% similarity to known Bt strains. In conclusion, native Bt strains from cowpea root nodules were shown to have bio-insecticidal activity against fall armyworm larvae and brinjal ash weevil adults.

Toxicity of Bacillus thuringiensis Strains Derived from the Novel Crystal Protein Cry31Aa with High Nematicidal Activity against Rice Parasitic Nematode Aphelenchoides besseyi.

The plant parasitic nematode, Aphelenchoides besseyi, is a serious pest causing severe damage to various crop plants and vegetables. The Bacillus thuringiensis (Bt) strains, GBAC46 and NMTD81, and the biological strain, FZB42, showed higher nematicidal activity against A. besseyi, by up to 88.80, 82.65, and 75.87%, respectively, in a 96-well plate experiment. We screened the whole genomes of the selected strains by protein-nucleic acid alignment. It was found that the Bt strain GBAC46 showed three novel crystal proteins, namely, Cry31Aa, Cry73Aa, and Cry40ORF, which likely provide for the safe control of nematodes. The Cry31Aa protein was composed of 802 amino acids with a molecular weight of 90.257 kDa and contained a conserved delta-endotoxin insecticidal domain. The Cry31Aa exhibited significant nematicidal activity against A. besseyi with a lethal concentration (LC50) value of 131.80 μg/mL. Furthermore, the results of in vitro experiments (i.e., rhodamine and propidium iodide (PI) experiments) revealed that the Cry31Aa protein was taken up by A. besseyi, which caused damage to the nematode’s intestinal cell membrane, indicating that the Cry31Aa produced a pore-formation toxin. In pot experiments, the selected strains GBAC46, NMTD81, and FZB42 significantly reduced the lesions on leaves by up to 33.56%, 45.66, and 30.34% and also enhanced physiological growth parameters such as root length (65.10, 50.65, and 55.60%), shoot length (68.10, 55.60, and 59.45%), and plant fresh weight (60.71, 56.45, and 55.65%), respectively. The number of nematodes obtained from the plants treated with the selected strains (i.e., GBAC46, NMTD81, and FZB42) and A. besseyi was significantly reduced, with 0.56, 0.83., 1.11, and 5.04 seedling mL−1 nematodes were achieved, respectively. Moreover, the qRT-PCR analysis showed that the defense-related genes were upregulated, and the activity of hydrogen peroxide (H2O2) increased while malondialdehyde (MDA) decreased in rice leaves compared to the control. Therefore, it was concluded that the Bt strains GBAC46 and NMTD81 can promote rice growth, induce high expression of rice defense-related genes, and activate systemic resistance in rice. More importantly, the application of the novel Cry31Aa protein has high potential for the efficient and safe prevention and green control of plant parasitic nematodes.

Creation of an Industrial Bacillus thuringiensis Strain With High Melanin Production and UV Tolerance by Gene Editing.

Bacillus thuringiensis produces insecticidal crystal proteins (ICPs) which exhibit strong insecticidal toxicity. But when used in the field, ICPs would be destroyed by ultraviolet (UV) radiation in sunlight, thus decreasing the insecticidal activity and shortening the persistence. To improve the duration of B. thuringiensis preparations, we endowed a highly toxic industrial B. thuringiensis HD-1 with UV tolerance by making it produce melanin, a pigment that absorbs UV radiation. In B. thuringiensis, melanin is derived from homogentisate (HGA), an intermediate in the tyrosine pathway. And the absence of homogentisate-1,2-dioxygenase (HmgA) will lead to the formation of melanin. In this study, we used the CRISPR/Cas9 system to knock out the hmgA gene and obtained a melanin-producing mutant HD-1-ΔhmgA from strain HD-1. The melanin yield by mutant HD-1-ΔhmgA reached 3.60 mg/mL. And the anti-UV test showed that melanin serves as a protection to both the organism and the ICPs. After UV irradiation for 3 h, mutant HD-1-ΔhmgA still had an 80% insecticidal activity against the cotton bollworm, Helicoverpa armigera, while the control line only had about 20%. This study creates a light-stable biopesticide prototype based on a classic industrial strain that can be applied directly and takes the melanin-producing strain as a concept to improve the preparation validity.

Vegetative Insecticidal Protein Vip3Aa Is Transported via Membrane Vesicles in Bacillus thuringiensis BMB171.

Vegetative insecticidal protein Vip3Aa, secreted by many Bacillus thuringiensis (Bt) strains during the vegetative growth stage, represents the second-generation insecticidal toxin. In recent years, significant progress has been made on its structure and action mechanism. However, how it is translocated across the cytoplasmic membrane into the environment remains a mystery. This work demonstrates that Vip3Aa is not secreted by the General Secretion (Sec) System. To reveal the secretory pathway of Vip3A, we purified the membrane vesicles (MVs) of B. thuringiensis BMB171 and observed by TEM. The size of MVs was determined by the dynamic light scattering method, and their diameter was approximately 40–200 nm, which is consistent with the vesicles in Gram-negative bacteria. Moreover, Vip3A could be detected in the purified MVs by Western blot, and immunoelectron microscopy reveals Vip3A antibody-coated gold particles located in the MVs. After deleting its signal peptide, chitinase B (ChiB) failed to be secreted. However, the recombinant ChiB, whose signal peptide was substituted with the N-terminal 39 amino acids from Vip3A, was secreted successfully through MVs. Thus, this sequence is proposed as the signal region responsible for vesicle transport. Together, our results revealed for the first time that Vip3Aa is transported to the medium via MVs.

Molecular characterization of chitinase producing Bacillus thuringiensis

Aim: In the present study, twenty eight Bacillus thuringiensis (Bt) strains were studied for their potential to hydrolyze chitin. Methodology: The chitinolytic potential of Bt strains were determined using modified chitinase screening media (CSM) containing colloidal chitin as substrate. The potential Bt strains were screened for the presence of cry genes using PCR and further molecular characterized using 16S rDNA amplification and sequencing. Results: Among 28 Bt strains, 10 strains showed positive results by producing clear halo zone around bacterial colonies and the maximum chitinase solubilization index was observed in Bt-13 whereas the minimum solubilization index was observed in Bt-27 strain. Further, the chitinase assay revealed the minimum enzyme activity in Bt-26 (3.78 ± 0.101 U ml-1) whereas the maximum activity was observed in strain Bt-2 (10.19 ± 0.651 U ml-1). PCR based chitinase gene screening also revealed the presence of both endochitinase and exochitinase genes in these Bt strains. The potent Bt strains viz., Bt-7, Bt-10, Bt-11, Bt-13 and Bt-27 on the basis of chitinase production were molecular characterized based on 16S rDNA and sequencing results reveled their greatest sequence identity Bacillus thuringiensis. Interpretation: Results showed that the high chitinase activity of these Bt strains may be due to the presence of chitinase genes, which need to be explored for further biological applications.