Bacterial endophytes inhabiting desert plants provide protection against seed rot caused by Fusarium verticillioides and promote growth in maize.

BACKGROUND: Fusarium maize ear and root rot disease caused by Fusarium verticillioides has become one of the most serious fungal diseases associated with maize production. Due to their abilities to promote plant development and manage diseases, bacterial endophytes provide a more promising approach for treating this vascular disease. RESULTS: This work was undertaken for the selection and identification of promising isolates as plant growth promoters and biocontrol agents against F. verticillioides in maize agroecosystems. A screening procedure consisting of in vitro and in situ tests was applied to 27 endophytic strains originating from desert plants: Euphorbia antiquorum, Calotropis procera, and Alcasia albida. In vitro studies indicated that the bacteria exhibited variable results in biocontrol, endophytism, and plant growth-promoting traits. In addition, in situ plant growth promotion and biocontrol experiments allowed the identification of the most promising bacterial endophytes. In vitro and in situ comparative study results indicated a low correlation. Our data revealed that in situ screening must be used as the method of selection of biocontrol agents against Fusarium ear and root rot disease. Based on in situ results, seven potent strains were selected and identified as Bacillus subtilis, Bacillus velezensis, Bacillus tequilensis, Stenotrophomonas maltophilia, and Klebsiella pneumoniae. CONCLUSION: The results of this study showed that the selected strains seem to be promising candidates to be exploited as biofertilizers and biocontrol agents against Fusarium maize ear and root rot disease. © 2023 Society of Chemical Industry.

Two Novel Bacillus Strains (subtilis and simplex Species) with Promising Potential for the Biocontrol of Zymoseptoria tritici, the Causal Agent of Septoria Tritici Blotch of Wheat.

Two novel Algerian field-collected isolates were selected for their antifungal activity against Zymoseptoria tritici (teleomorph Mycosphaerella graminicola). The novel strains, termed Alg.24B1 and Alg.24B2, were identified as Bacillus subtilis and Bacillus simplex since their respective nucleotide sequences of the 16S rRNA gene were 100% and 99.93% identical to those of B. subtilis and B. simplex, respectively. The antifungal activities of Alg.24B1 and Alg.24B2 were evaluated by the well diffusion method and compared to those of other Bacillus species. The maximum activity was obtained after two days of confrontation of the bacterial strain supernatants with the fungus for Alg.24B1 and three days for Alg.24B2. Furthermore, the metabolites responsible for the antifungal activity of both strains were detected by the investigation of either gene presence (PCR) or molecule production (activity detection of lytic enzymes and HPLC detection of lipopeptides). Overall, this study showed that in addition to their ability to produce lytic enzymes (protease and ß-glucanase), both strains coproduce three types of lipopeptides viz. surfactin, iturin, and fengycin. Thus, the biofungicide activity of both strains may be a result of a combination of different mechanisms. Therefore, they had a great potential to be used as biocontrol agents to effectively manage septoria tritici blotch of wheat (STB).

Development of a cost-effective medium for Photorhabdus temperata bioinsecticide production from wastewater and exploration of performance kinetic.

This study investigates the optimization of the culture conditions for enhancing Photorhabdus temperata biopesticide production using wastewater (WS4) as a raw material. Box-Behnken design (BBD) was used to evaluate the effects of carbon to nitrogen ratio (C/N), sodium chloride concentration and inoculum size on P. temperata biomass production and insecticidal activity. For an enhanced biopesticide production, the optimum operating conditions were as follows: inoculum size = 4%; C/N ratio = 12.5 and [NaCl] = 4 g/L for two responses. 1.95 and 2.75 fold improvements in oral toxicity and biomass production were respectively obtained in the cost-effective medium developed in this study (WS4 I) using the three variables at their optimal values. Under the optimized conditions, WS4 I-grown cells exhibited higher membrane integrity according to flow cytometry analysis since dead cells presented only 9.2% compared to 29.2% in WS4. From batch fermentations carried out in WS4 I and WS4, P. temperata kinetic parameters in terms of biomass production and substrate consumption rates were modeled. The obtained results showed that the maximum specific growth rate in WS4 I was of 0.43 h-1 while that obtained in WS4 was of 0.14 h-1. In addition, the efficiency of P. temperata to metabolize organic carbon was enhanced by optimizing the culture conditions. It reached 72.66% instead of 46.18% in the control fermentation after 10 h of incubation. Under the optimized conditions, P. temperata cells showed the highest specific consumption rate resulting in a toxin synthesis improvement.

Molecular and structural characterization of a novel Cry1D toxin from Bacillus thuringiensis with high toxicity to Spodoptera littoralis (Lepidoptera: Noctuidae).

The investigation of new Bacillus thuringiensis (Bt) insecticidal proteins (Cry) with specific toxicity is one of the alternative measures used for Lepidopteran pest control. In the present study, a new Cry toxin was identified from a promising Bt strain BLB250 which was previously selected for its high toxicity against Spodoptera littoralis. The corresponding gene, designated cry1D-250, was cloned. It showed an ORF of 3498bp, encoding a protein of 1165 amino acid residues with a putative molecular mass of 132kDa which was confirmed by SDS-PAGE and Western blot analyses. The corresponding toxin named Cry1D-250 showed a higher insecticidal activity towards S. littoralis than Cry1D-133 (LC50 of 224.4ngcm-2) with an LC50 of only 166ngcm-2. Besides to the 65kDa active toxin, proteolysis activation of Cry1D-133 protein with S. littoralis midgut juice generated an extra form of 56kDa, which was the result of a second cleavage. Via activation study and 3D structure analysis, novel substitutions found in the Cry1D-250 protein compared to Cry1D-133 toxin were shown to be involved in the protein stability and toxicity. Therefore, the Cry1D-250 toxin can be considered to be an effective alternative for the control of S. littoralis.

Molecular characterization of Cry1D-133 toxin from Bacillus thuringiensis strain HD133 and its toxicity against Spodoptera littoralis.

Bacillus thuringiensis subsp. aizawai strain HD133, known by its effectiveness against Spodoptera species, produces bipyramidal crystals encompassing the insecticidal proteins Cry1Ab, Cry1Ca and Cry1D-133 in the proportions 60:37:3, respectively. In this study, we dealt with the relevance of the low rate of Cry1D-133. The cry1D-133 gene from HD133 was cloned and sequenced. Both nucleotide and amino acid sequence similarity analyses with the cry1D genes available in the GenBank database revealed that cry1D-133 is a new variant of cry1Da-type genes with 99% identity with cry1Da1. Molecular modeling of the Cry1D-133 toxin showed that its higher toxicity is correlated to a higher number of toxin-receptor interactions. Optimal culture conditions of 4 h post-induction time, 160 rpm agitation and 37 °C post-induction temperature were determined and adopted to overproduce Cry1D-133 toxin at adequate amounts to carryout bioassays. A gradual increase of the proportion of Cry1D-133 to the HD133 insecticidal proteins forming the crystal (Cry1D-133, Cry1Ca and Cry1Ab) showed an improvement of the toxicity against Spodoptera littoralis larvae. Therefore, the potential of Cry1D-133 to enhance HD133 toxicity could promote its combination with other B. thuringiensis insecticidal proteins toxins in order to increase target range or to delay the emergence of resistance.

A promising HD133-like strain of Bacillus thuringiensis with dual efficiency to the two Lepidopteran pests: Spodoptera littoralis (Noctuidae) and Ephestia kuehniella (Pyralidae).

Isolation and identification of new strains with wide variety of target pests is an ever growing field. In this paper, a screening of 260 strains from Tunisian soil samples was conducted by dot-blot and PCR-sequencing analysis. The screening was done on the basis of the possession of cry1D-type genes and was followed by the evaluation of the insecticidal activity against Spodoptera littoralis. BLB250 strain showed an LC50 value (56.2 µg g(-1)) against S. littoralis lower than those of the two Bacillus thuringiensis reference strains HD1 and HD133. An interesting LC50 (167.6 µg g(-1)) was also recorded against Ephestia kuehniella larvae. The strain was, thus, selected because of its qualification to be highly toxic, at once, for both Lepidopteran insects. In vitro time course of proteolytic processing of BLB250 and HD133 protoxins by the gut juices from the two insect larvae revealed that the differences in toxicity against E. kuehniella are most likely attributed to differences in the efficiency of the activation of the corresponding protoxins into toxins. An activation comparative study using commercial proteases suggested that the intestinal proteases of E. kuehniella contain trypsin-like activities. With its high efficiency and toxicity against, at once, two Lepidopteran insects having different susceptibilities towards kurstaki and aizawai subspecies, BLB250 could be useful when developing more efficient and economical B. thuringiensis-based pesticides.

Evaluation of the effect of c.2946+1G>T mutation on splicing in the SCN1A gene.

Mutations in the SCN1A gene have commonly been associated with a wide range of mild to severe epileptic syndromes. They generate a wide spectrum of phenotypes ranging from the relatively mild generalized epilepsy with febrile seizures plus (GEFS+) to other severe epileptic encephalopathies, including myoclonic epilepsy in infancy (SMEI), cryptogenic focal epilepsy (CFE), cryptogenic generalized epilepsy (CGE) and a distinctive subgroup termed as severe infantile multifocal epilepsy (SIMFE). The present study was undertaken to investigate the potential effects of a transition in the first nucleotide at the donor splice site of intron 15 of the SCN1A gene leading to CGES. Functional analyses using site-directed mutagenesis by PCR and subsequent ex-vivo splicing assays, revealed that the c.2946+1G>T mutation lead to a total skipping of exon 15. The exclusion of this exon did not alter the reading frame but induced the deletion of the amino acids (853 Leu -971 Val) which are a major part in the fourth, fifth and sixth transmembrane segments of the SCN1A protein. The theoretical implications of the splice site mutations predicted with the bioinformatic tool human splice finder were investigated and compared with the results obtained by the cellular assay.

First functional analysis of a novel splicing mutation in the B3GALTL gene by an ex vivo approach in Tunisian patients with typical Peters plus syndrome.

Peters plus syndrome is a rare recessive autosomal disorder comprising ocular anterior segment dysgenesis, short stature, hand abnormalities and distinctive facial features. It was related only to mutations in the B3GALTL gene in the 13q12.3 region. In this study, we undertook the first functional analysis of a novel c.597-2 A>G splicing mutation within the B3GALTL gene using an ex-vivo approach. The results showed a complete skipping of exon 8 in the B3GALTL cDNA, which altered the open reading frame of the mutant transcript and generated a PTC within exon 9. This finding potentially elicits the nonsense mRNA to degradation by NMD (nonsense-mediated mRNA decay). The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation to ex-vivo results. The findings confirmed the key role played by the B3GALTL gene in typical Peters-plus syndromes and the utility of mRNA analysis to understand the primary impacts of this mutation and the phenotype of the disease.

Relationship between plasmid loss and gene expression in Bacillus thuringiensis.

The large genome of Bacillus thuringiensis was shortened by provoking plasmid loss, and the effect of such loss on the expression of both chromosomal and plasmid-harboured genes was studied. It was evidenced that the genomic material shortening of B. thuringiensis allows for the improvement of the expression of some of the constructive genes. Indeed, it was shown that curing was an efficient tool to obtain mutant strains with better expression of chromosomal genes. In fact, totally cured strains, HD1Cry-B and BNS3Cry, showed higher chitinolytic and/or proteolytic activities than the corresponding wild and/or partially cured ones. Although total curing is drastic for plasmid-harboured gene expression, the authors obtained partially cured strains, Cur255 and CurHD1, with a doubled level of delta-endotoxin production, and a 1.5-fold improvement of the bacteriocin-specific activity, respectively. In addition, the latter strains showed better relative proteolytic activities.

Integration of a recombinant chitinase into Bacillus thuringiensis parasporal insecticidal crystal.

Chitinases have been successfully used in combination with Bacillus thuringiensis delta-endotoxins forming crystals in order to enhance their insecticidal activities. In this context, we opted for promoting the chitinase integration into these crystals. Thus, we engineered, for the first time, a fusion protein (CDF) consisting of the chitinase Chi255 and the carboxy-terminal half of Cry1Ac, both from B. thuringiensis subsp. kurstaki. The constructed transcriptional fusion (chi255Δsp-CTcry1Ac) was cloned into a shuttle vector (Escherichia coli/B. thuringiensis) downstream the sporulation-dependent promoters BtI-BtII and upstream the cry1Ac transcription terminator. The resulting plasmid, named pF, was transferred by electroporation to crystalliferous B. thuringiensis strain BNS3 and acrystalliferous strain BNS3Cry-. The functionality of the chimerical chitinase was demonstrated by an improvement of the relative chitinolytic activity of the recombinant strain BNS3/pF by 2.5 folds. Western blot analyses showed that, despite of the instability of CDF when expressed in the acrystalliferous strain, the C-terminal half of Cry1Ac succeeded to allow the integration of the chimerical chitinase into the crystal of BNS3. The recombinant strain BNS3/pF (LC(50) = 144.06 µg g(-1)) was 1.5 times more active against Ephestia kuehniella larvae than the wild strain (LC(50) = 212.10 µg g(-1)).