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Cellulose represents the most abundant component of plant biomass on earth; it is degraded by cellulases, specific enzymes produced by microorganisms. However, cellulases of bacterial origin attract more interest due to their natural diversity and ability to inhabit a variety of niches, allowing the selection of cellulolytic strains resistant to environmental stresses. The screening of the cellulolytic activity of 398 bacteria isolated from various ecosystems in Algeria (cave, ruins, chott, thermal station, and rhizosphere of arid and semi-arid regions) was performed by the appearance of a hydrolysis zone on carboxymethylcellulose (CMC) medium. The cellulase activity on CMC (1%) broth allowed to select 26 strains among which 12 had the best activity (0.3 U/mL to 2.2 U/mL). Optimization of physicochemical parameters (salinity: 0–1 M NaCl; pH: 3, 4, 7, 9, and 11; temperature: 30, 45, and 50 °C; PEG8000: 0 and 30%) involved in growth and cellulose production showed that the majority of strains were mesophilic, neutrophilic, or alkali- tolerant and tolerant to 30% of PEG8000. The cellulase activity and stability under different stress allowed to retain five strains, which the most efficient. Based on the 16S-rRNA sequencing results, they belonged to the genus Bacillus. The physicochemical properties of cellulases (crude extract) showed a CMCase active over a wide range of pH (4 to 11), optimal at 50 °C and 60 °C. The inhibiting salinity effect on the activity was not detected and was negligible on the enzymatic stability. The residual CMCase activity remained between 40 and 70% in a temperature range between 40 and 70 °C, was stable over a wide range of saline concentrations (0–2000 mM), and was weakly affected at 30% of PEG8000. The crude enzyme extract was able to hydrolyze both soluble and insoluble cellulosic substrates. The evaluation of the hydrolysis capacity of lignocellulosic waste revealed the ability of tested strains to degrade wheat bran, barley bran, and corncob. In addition, the enzyme showed significant multi-stress resistance on solid and liquid media. By these characteristics, these cellulolytic bacteria could be attractive to be used in various industrial and biotechnology applications.

Biochar is known as a well-developed porous carbonaceous material with multifunctional abilities that can enhance the physical properties of soils. However, the lack of certainty about the consequences of biochar application to soils has limited its acceptability. Application of biochar can lead to a series of changes in the physical functions of soil, which are crucial in both agricultural and environmental management. The type of feedstock, pyrolysis conditions, size of particles, and rate of amendments are responsible for biochar effectiveness. Concurrently, the physical characteristics of soil, such as particle-size distribution, can intensify the impacts. Beside the physical attributes, the chemical components and interactions between biochar and the soil interface may play an important role. The chemical properties, such as the value of electrical conductivity, pH and zeta potential, are the remarkable parameters in the hydrophysical behavior. The summary proposes that biochar has a great contribution In enhancing the definite range of aggregation formation, reduction of compaction and shear strength frequency and/or intensity, improvement of microorganisms activity, and abundance. Simultaneously, biochar plays a devastating role by filling the pores, blocking the water flow pathways, and inhibiting macro fauna growth. Particle size of biochar as a major factor, and surface functional groups as a minor factor, affect the performance of biochar in improving the hydrophysical properties of amended soils. The increment in the dosage of biochar application is not promising to enhance the physical properties of soils. Therefore, it is necessary to find a balance between the consumption of biochar and promotion of the soil-water dynamic. This review provides an overview of fastidious perspectives on how to achieve an efficient and sustainable use of biochar in hydrophysical properties.

Biotic and abiotic stresses are severely limiting plant production and productivity. Of notable importance is salt stress that not only limits plant growth and survival, but affects the soil fertility and threatens agricultural ecosystems sustainability. The problem is exacerbated in fragile arid and semi-arid areas where high evaporation, low precipitation and the use of salty water for irrigation is accelerating soil salinization. Legumes, considered very nutritious foods for people and providing essential nutrients for ecosystems are a fundamental element of sustainable agriculture. They can restore soil health by their ability to fix nitrogen in a symbiotic interaction with the rhizobia of the soil. However, salt stress is severely limiting productivity and nitrogen fixation ability in legumes. Plant growth-promoting rhizobacteria (PGPR) and mainly actinobacteria promote plant growth by producing phytohormones, siderophores, antibiotics and antifungal compounds, solubilizing phosphate and providing antagonism to phytopathogenic microorganisms. In addition, actinobacteria have beneficial effects on nodulation and growth of legumes. In this study, actinobacteria isolated from different niches and having PGP activities were used in co-inoculation experiments with rhizobia in Medicago sativa plants rhizosphere submitted to salt stress. The results indicate that drought- and salinity-tolerant Actinobacteria with multiple PGP traits can potentially increase alfalfa growth under saline conditions, in the presence or absence of symbiotic rhizobial bacteria. Actinobacteria discovered in this study can, therefore, be suitable biofertilizers in the formulation of agricultural products improving plant development, health and productivity in saline soils, a necessary alternative for modern agriculture and sustainable development. Supplementary Information The online version contains supplementary material available at 10.1007/s00284-021-02394-z.

Abiotic stress significantly limits plant growth and production. Drought, in particular, is a severe constraint that affects growth and limits agricultural productivity on a global scale. Water stress induces in plants a set of morpho-anatomical (modification of root and leaf structure), physiological, and biochemical (relative water content, membrane stability, photosynthesis, hormonal balance, antioxidant systems, and osmolyte accumulation) changes mainly employed to cope with the drought stress. These strategies allow the plant to overcome the unfavorable period of limited water availability. Currently, a promising alternative is available to improve plant growth and tolerance under drought conditions. The use of osmotolerant plant growth-promoting rhizobacteria (PGPR) as inoculants can alleviate water stress by increasing the water use efficiency of the plant. The PGPR improve the tolerance of plants to drought, through changes in the morphology and architecture of the root system, production of phytohormones, extracellular polysaccharides, ACC 1-(aminocyclopropane-1-carboxylate) deaminase, volatile chemicals, and osmolyte accumulation. They may also enhance the antioxidant defense system and induce transcriptional regulation of stress response genes. This review addresses the effects of stress on plant growth, adaptation, and response to drought conditions and discusses the significant potential of PGPR to modulate the physiological response against water scarcity, ensuring plant survival and improving the resistance and growth of agricultural crops.

World population growth and modernization have engendered multiple environmental problems: the propagation of humans and crop diseases and the development of multi-drug-resistant fungi, bacteria and viruses. Thus, a considerable shift towards eco-friendly products has been seen in medicine, pharmacy, agriculture and several other vital sectors. Nowadays, studies on endophytic fungi and their biotechnological potentials are in high demand due to their substantial, cost-effective and eco-friendly contributions in the discovery of an array of secondary metabolites. For this review, we provide a brief overview of plant–endophytic fungi interactions and we also state the history of the discovery of the untapped potentialities of fungal secondary metabolites. Then, we highlight the huge importance of the discovered metabolites and their versatile applications in several vital fields including medicine, pharmacy, agriculture, industry and bioremediation. We then focus on the challenges and on the possible methods and techniques that can be used to help in the discovery of novel secondary metabolites. The latter range from endophytic selection and culture media optimization to more in-depth strategies such as omics, ribosome engineering and epigenetic remodeling.

Plant growth promoting bacteria (PGPB) have been the target of intensive research studies toward their efficient use in the field as biofertilizers, biocontrol, and bioremediation agents among numerous other applications. Recent trends in the field of PGPB research led to the development of versatile multifaceted PGPB that can be used in different field conditions such as biocontrol of plant pathogens in metal contaminated soils. Unfortunately, all these research efforts lead to the development of PGPB that failed to perform in salty environments. Therefore, it is urgently needed to address this drawback of these PGPB toward their efficient performance in salinity context. In this paper we provide a review of state-of-the-art research in the field of PGPB and propose a road map for the development of next generation versatile and multifaceted PGPB that can perform in salinity. Beyond soil desalinization, our study paves the way towards the development of PGPB able to provide services in diverse salty environments such as heavy metal contaminated, or pathogen threatened. Smart development of salinity adapted next generation biofertilizers will inevitably allow for mitigation and alleviation of biotic and abiotic threats to plant productivity in salty environments.

Endophytes have been known for more than a century and recent studies highlighted their endless potentialities in plant growth promotion through several direct and indirect mechanisms. Competent microbial endophytes have been acknowledged in several fields including medicine, industry, pharmacology, bioremediation, and phytoremediation of pollutants due to their safe handling and environment-friendly effects. Modern genomic approaches are considered an effective tool to get a better knowledge of the microbial modes of action. This chapter focuses on endophytes taxonomic affinities and lifestyle, their plant growth-promoting mechanisms, their applications as well as the potential of emerging “omic” tools including genomic, transcriptomic, metabolomics, and proteomics to shed the light on the wealth of their genomic and metabolic potentialities.

Halotolerant plant-growth-promoting rhizobacteria (PGPR) could not only promote plant growth, but also help in counteracting the detrimental effects of salt stress. In the present study, a total of 76 bacteria were isolated from the rhizosphere, non-rhizospheric soil and endophytes of the halophyte Salsola tetrandra, collected from natural saline soils in Algeria. Phylogenetic analysis based on the 16S rDNA sequence of Gram-negative bacteria (n = 51) identified, showed seventeen representative isolates grouped into four genera (Pseudomonas, Acinetobacter, Enterobacter, and Providencia). These bacterial isolates that exhibited different PGPR traits were selected and tested for their ability to tolerate different abiotic stress (NaCl, PEG8000, and pH). The majority of isolates were drought tolerant (60% of PEG8000) and had an optimal growth at high pH values (pH 9 and 11) and some strains tolerated 2 M of NaCl. Strains identified as Enterobacter xiangfangensis BE1, Providencia rettgeri BR5 and Pseudomonas stutzeri MLR6 showed high capacity of adaptation on their PGP traits. The salt-tolerant isolates were finally chosen to promote growth and enhance salt tolerance, separately or combined, of Arabidopsis thaliana (Col-0) exposed or not to 0.1 M NaCl, by following fresh and root weight, primary root elongation and lateral root number. The best bacterial effect was recorded for the MLR6 strain in increasing shoot fresh weight and for BE1 in terms of root fresh weight in the absence of salt stress. At stressed conditions, all growth parameters declined. However, inoculation of Arabidopsis thaliana with the three bacterial strains (MLR6, BE1 and BR5), single or in co-culture, conferred an increase in the shoot weight, primary root length and lateral root number. The use of these strains separately or combined as biofertilizers seems to be a powerful tool in the development of sustainable agriculture in saline soils.

The soil-borne species Aneurinibacillus migulanus (A. migulanus) strains Nagano and NCTC 7096 were shown to be potent biocontrol agents active against several plant diseases in agricultural and forest ecosystems. Both strains produce the cyclic peptide gramicidin S (GS) that was described as the main weapon inhibiting some gram-negative and gram-positive bacteria and fungus-like organisms along with the production of biosurfactant and hemolysis activities. However, the contribution of the cyclic peptide gramicidin S (GS) to the biocontrol ability of A. migulanus has never been studied experimentally. In this paper, using a mutant of the A. migulanus Nagano strain (E1 mutant) impaired in GS biosynthesis we evaluated the contribution of GS in the biocontrol potential of A. migulanus against Phytophthora spp. The two strains of A. migulanus, Nagano and NCTC 7096, were tested in a pilot study for the inhibition of the growth of 13 Phytophthora species in dual culture assays. A. migulanus Nagano was significantly more inhibitory than NCTC 7096 to all species. Additionally, using apple infection assays, P. rosacearum MKDF-148 and P. cryptogea E2 were shown to be the most aggressive on apple fruits displaying clear infection halos. Therefore, the three A. migulanus strains, Nagano, NCTC 7096, and E1, were used in apple infection experiments to check their effect on infection ability of these two Phytophthora species. Treatment with A. migulanus Nagano significantly reduced the severity of symptoms in apple fruits compared with NCTC 7096. A. migulanus E1 mutant showed total loss of biocontrol ability suggesting that GS is a major actor in the biocontrol ability of A. migulanus Nagano strain.

Bacillus velezensis gram-positive bacterium, is frequently isolated from diverse niches mainly soil, water, plant roots, and fermented foods. B. velezensis is ubiquitous, non-pathogenic and endospore forming. Being frequently isolated from diverse plant holobionts it is considered host adapted microorganism and recognized of high economic importance given its ability to promote plant growth under diverse biotic and abiotic stress conditions. Additionally, the species suppress many plant diseases, including bacterial, oomycete, and fungal diseases. It is also able after plant host root colonization to induce unique physiological situation of host plant called primed state. Primed host plants are able to respond more rapidly and/or effectively to biotic or abiotic stress. Moreover, B. velezenis have the ability to resist diverse environmental stresses and help host plants to cope with, including metal and xenobiotic stresses. Within species B. velezensis strains have unique abilities allowing them to adopt different life styles. Strain level abilities knowledge is warranted and could be inferred using the ever-expanding new genomes list available in genomes databases. Pangenome analysis and subsequent identification of core, accessory and unique genomes is actually of paramount importance to decipher species full metabolic capacities and fitness across diverse environmental conditions shaping its life style. Despite the crucial importance of the pan genome, its assessment among large number of strains remains sparse and systematic studies still needed. Extensive knowledge of the pan genome is needed to translate genome sequencing efforts into developing more efficient biocontrol agents and bio-fertilizers. In this study, a genome survey of B. velezensis allowed us to (a) highlight B. velezensis species boundaries and show that Bacillus suffers taxonomic imprecision that blurs the debate over species pangenome; (b) identify drivers of their successful acquisition of specific life styles and colonization of new niches; (c) describe strategies they use to promote plant growth and development; (d) reveal the unlocked strain specific orphan secondary metabolite gene clusters (biosynthetic clusters with corresponding metabolites unknown) that product identification is still awaiting to amend our knowledge of their putative role in suppression of pathogens and plant growth promotion, and (e) to describe a dynamic pangenome with a secondary metabolite rich accessory genome.

Natural dyes have been used from ancient times for multiple purposes, most importantly in the field of textile dying. The increasing demand and excessive costs of natural dye extraction engendered the discovery of synthetic dyes from petrochemical compounds. Nowadays, they are dominating the textile market, with nearly 8 × 105 tons produced per year due to their wide range of color pigments and consistent coloration. Textile industries consume huge amounts of water in the dyeing processes, making it hard to treat the enormous quantities of this hazardous wastewater. Thus, they have harmful impacts when discharged in non-treated or partially treated forms in the environment (air, soil, plants and water), causing several human diseases. In the present work we focused on synthetic dyes. We started by studying their classification which depended on the nature of the manufactured fiber (cellulose, protein and synthetic fiber dyes). Then, we mentioned the characteristics of synthetic dyes, however, we focused more on their negative impacts on the ecosystem (soil, plants, water and air) and on humans. Lastly, we discussed the applied physical, chemical and biological strategies solely or in combination for textile dye wastewater treatments. Additionally, we described the newly established nanotechnology which achieves complete discharge decontamination.

The phosphogypsum (PG) endogenous bacterial community and endophytic bacterial communities of four plants growing in phosphogypsum-contaminated sites, Suaeda fruticosa (SF), Suaeda mollis (SM), Mesembryanthmum nodiflorum (MN) and Arthrocnemum indicum (AI) were investigated by amplicon sequencing. Results highlight a more diverse community of phosphogypsum than plants associated endophytic communities. Additionally, the bacterial culturable communities of phosphogypsum and associated plant endophytes were isolated and their plant-growth promotion capabilities, bioremediation potential and stress tolerance studied. Most of plant endophytes were endowed with plant growth-promoting (PGP) activities and phosphogypsum communities and associated plants endophytes proved highly resistant to salt, metal and antibiotic stress. They also proved very active in bioremediation of phosphogypsum and other organic and inorganic environmental pollutants. Genome sequencing of five members of the phosphogypsum endogenous community showed that they belong to the recently described species Bacillus albus (BA). Genome mining of BA allowed the description of pollutant degradation and stress tolerance mechanisms. Prevalence of this tool box in the core, accessory and unique genome allowed to conclude that accessory and unique genomes are critical for the dynamics of strain acquisition of bioremediation abilities. Additionally, secondary metabolites (SM) active in bioremediation such as petrobactin have been characterized. Taken together, our results reveal hidden untapped valuable bacterial actors for waste remediation.

Oomycete and fungal pathogens, mainly Phytophthora and Fusarium species, are notorious causal agents of huge economic losses and environmental damages. For instance, Phytophthora ramorum, Phytophthora cryptogea, Phytophthora plurivora and Fusarium solani cause significant losses in nurseries and in forest ecosystems. Chemical treatments, while harmful to the environment and human health, have been proved to have little or no impact on these species. Recently, biocontrol bacterial species were used to cope with these pathogens and have shown promising prospects towards sustainable and eco-friendly agricultural practices. Olive trees prone to Phytophthora and Fusarium disease outbreaks are suitable for habitat-adapted symbiotic strategies, to recover oomycetes and fungal pathogen biocontrol agents. Using this strategy, we showed that olive trees-associated microbiome represents a valuable source for microorganisms, promoting plant growth and healthy benefits in addition to being biocontrol agents against oomycete and fungal diseases. Isolation, characterization and screening of root microbiome of olive trees against numerous Phytophthora and other fungal pathogens have led to the identification of the Bacillus velezensis OEE1, with plant growth promotion (PGP) abilities and strong activity against major oomycete and fungal pathogens. Phylogenomic analysis of the strain OEE1 showed that B. velezensis suffers taxonomic imprecision that blurs species delimitation, impacting their biofertilizers’ practical use. Genome mining of several B. velezensis strains available in the GenBank have highlighted a wide array of plant growth promoting rhizobacteria (PGPR) features, metals and antibiotics resistance and the degradation ability of phytotoxic aromatic compounds. Strain OEE1 harbours a large repertoire of secreted and volatile secondary metabolites. Rarefaction analysis of secondary metabolites richness in the B. velezenis genomes, unambiguously documented new secondary metabolites from ongoing genome sequencing efforts that warrants more efforts in order to assess the huge diversity in the species. Comparative genomics indicated that B. velezensis harbours a core genome endowed with PGP features and accessory genome encoding diverse secondary metabolites. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of OEE1 Volatile Organic Compounds (VOCs) and Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS) analysis of secondary metabolites identified numerous molecules with PGP abilities that are known to interfere with pathogen development. Moreover, B. velezensis OEE1 proved effective in protecting olive trees against F. solani in greenhouse experiments and are able to inhabit olive tree roots. Our strategy provides an effective means for isolation of biocontrol agents against recalcitrant pathogens. Their genomic analysis provides necessary clues towards their efficient implementation as biofertilizers.

In the arid region Bou-Sa&acirc, da at the South of Algeria, durum wheat Triticum durum L. cv Waha production is severely threatened by abiotic stresses, mainly drought and salinity. Plant growth-promoting rhizobacteria (PGPR) hold promising prospects towards sustainable and environmentally-friendly agriculture. Using habitat-adapted symbiosis strategy, the PGPR Pantoea agglomerans strain Pa was recovered from wheat roots sampled in Bou-Sa&acirc, da, conferred alleviation of salt stress in durum wheat plants and allowed considerable growth in this unhostile environment. Strain Pa showed growth up to 35 &deg, C temperature, 5&ndash, 10 pH range, and up to 30% polyethylene glycol (PEG), as well as 1 M salt concentration tolerance. Pa strain displayed pertinent plant growth promotion (PGP) features (direct and indirect) such as hormone auxin biosynthesis, production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase, and ammonia and phosphate solubilization. PGPR features were stable over wide salt concentrations (0&ndash, 400 mM). Pa strain was also able to survive in seeds, in the non-sterile and sterile wheat rhizosphere, and was shown to have an endophytic life style. Phylogenomic analysis of strain Pa indicated that Pantoea genus suffers taxonomic imprecision which blurs species delimitation and may have impacted their practical use as biofertilizers. When applied to plants, strain Pa promoted considerable growth of wheat seedlings, high chlorophyll content, lower accumulation of proline, and favored K+ accumulation in the inoculated plants when compared to Na+ in control non-inoculated plants. Metabolomic profiling of strain Pa under one strain many compounds (OSMAC) conditions revealed a wide diversity of secondary metabolites (SM) with interesting salt stress alleviation and PGP activities. All these findings strongly promote the implementation of Pantoea agglomerans strain Pa as an efficient biofertilizer in wheat plants culture in arid and salinity-impacted regions.

Halophyte Limoniastrum monopetalum, an evergreen shrub inhabiting the Mediterranean region, has well-documented phytoremediation potential for metal removal from polluted sites. It is also considered to be a medicinal halophyte with potent activity against plant pathogens. Therefore, L. monopetalum may be a suitable candidate for isolating endophytic microbiota members that provide plant growth promotion (PGP) and resistance to abiotic stresses. Selected for biocontrol abilities, these endophytes may represent multifaceted and versatile biocontrol agents, combining pathogen biocontrol in addition to PGP and plant protection against abiotic stresses. In this study 117 root culturable bacterial endophytes, including Gram-positive (Bacillus and Brevibacillus), Gram-negative (Proteus, Providencia, Serratia, Pantoea, Klebsiella, Enterobacter and Pectobacterium) and actinomycete Nocardiopsis genera have been recovered from L. monopetalum. The collection exhibited high levels of biocontrol abilities against bacterial (Agrobacterium tumefaciens MAT2 and Pectobacterium carotovorum MAT3) and fungal (Alternaria alternata XSZJY-1, Rhizoctonia bataticola MAT1 and Fusarium oxysporum f. sp. radicis lycopersici FORL) pathogens. Several bacteria also showed PGP capacity and resistance to antibiotics and metals. A highly promising candidate Bacillus licheniformis LMRE 36 with high PGP, biocontrol, metal and antibiotic, resistance was subsequently tested in planta (potato and olive trees) for biocontrol of a collection of 14 highly damaging Fusarium species. LMRE 36 proved very effective against the collection in both species and against an emerging Fusarium sp. threatening olive trees culture in nurseries. These findings provide a demonstration of our pyramiding strategy. Our strategy was effective in combining desirable traits in biocontrol agents towards broad-spectrum resistance against pathogens and protection of crops from abiotic stresses. Stacking multiple desirable traits into a single biocontrol agent is achieved by first, careful selection of a host for endophytic microbiota recovery, second, stringent in vitro selection of candidates from the collection, and third, application of the selected biocontrol agents in planta experiments. That pyramiding strategy could be successfully used to mitigate effects of diverse biotic and abiotic stresses on plant growth and productivity. It is anticipated that the strategy will provide a new generation of biocontrol agents by targeting the microbiota of plants in hostile environments.

The sap of the date palm "Lagmi" is a clear liquid, rich in sugars and minerals, with a pleasant flavour. Folk remedies based on the use of "Lagmi" for wound healing are still practiced. However, no studies investigated the relevance of "Lagmi" for wound healing. Therefore, the aim of this study was to identify the in vivo healing properties of "lagmi" on mechanically wounded wistar rats. Injured rats were divided into three groups: a first group treated by "lagmi", a second reference group processed by CICAFLORA(®) and a third untreated control group. On the 12th day of the experiment, total healing in the first group was reached, while healing was incomplete in the other groups. The sap seems to accelerate cell proliferation and contribute to faster healing with a gain of more than 30% as compared to CICAFLORA(®). Chemical Analysis of "Lagmi" showed important radical scavenging activity and high total antioxidant capacity. Features reported to help healing process and/or provides a favourable environment for tissue healing in wound sites. Extensive characterization of "Lagmi" phenolic and flavonoid compounds by High Resolution LC-MS (LC-HRESIMS) analysis indicates "Lagmi" is an important source of known anti-inflammatory compounds as well as promising wound healing candidates.

Aneurinibacillus migulanus strains Nagano and NCTC 7096 show potential in biocontrol against fungal and fungus-like plant pathogens, including a wide range of Ascomycota, Basidiomycota, and Oomycetes. Differences in terms of the range of pathogens that each strain inhibits, however, suggested that production of a single antibiotic cyclic peptide, gramicidin S (GS), by the two strains, is not the sole mechanism of inhibition. The availability of four sequenced genomes of Aneurinibacillus prompted us to apply genome mining techniques to identify the bioactive potential of A. migulanus and to provide insights into the secondary metabolite arsenal of the genus Aneurinibacillus. Up to eleven secondary metabolite biosynthetic gene clusters were present in the three Aneurinibacillus species. Biosynthetic gene clusters specifying bacteriocins, microcins, non-ribosomal peptides, polyketides, terpenes, phosphonates, lasso peptides and linaridins were identified. Chitinolytic potential and iron metabolism regulation were also investigated. With increasing numbers of biocontrol bacterial genomes being sequenced and mined, the use of approaches similar to those described in this paper will lead to an increase in the numbers of environmentally friendly natural products available to use against plant diseases.

Endophytic fungi of healthy and brittle leaf diseased (BLD) date palm trees (Phoenix dactylifera L.) represent a promising source of bioactive compounds with biomedical, industrial, and pharmaceutical applications. The fungal endophytes Penicillium citrinum isolate TDPEF34, and Geotrichum candidum isolate TDPEF20 from healthy and BLD date palm trees, respectively, proved very effective in confrontation assays against three pathogenic bacteria, including two Gram-positive bacteria Bacillus thuringiensis (Bt) and Enterococcus faecalis (Ef), and one Gram-negative bacterium Salmonella enterica (St). They also inhibited the growth of three fungi Trichoderma sp. (Ti), Fusarium sporotrichioides (Fs), Trichoderma sp. (Ts). Additionally, their volatile organic compounds (VOCs) were shown to be in part responsible for the inhibition of Ti and Ts and could account for the full inhibition of Fs. Therefore, we have explored their potential as fungal cell factories for bioactive metabolites production. Four extracts of each endophyte were prepared using different solvent polarities, ethanol (EtOH), ethyl acetate (EtOAc), hexane (Hex), and methanol (MetOH). Both endophyte species showed varying degrees of inhibition of the bacterial and fungal pathogens according to the solvent used. These results suggest a good relationship between fungal bioactivities and their produced secondary metabolites. Targeting the discovery of potential anti-diabetic, anti-hemolysis, anti-inflammatory, anti-obesity, and cytotoxic activities, endophytic extracts showed promising results. The EtOAc extract of G. candidum displayed IC50 value comparable to the positive control diclofenac sodium in the anti-inflammatory assays. Antioxidant activity was evaluated using &alpha, &alpha, diphenyl-&beta, picrylhydrazyl (DPPH), &beta, carotene bleaching, reducing power (RP), and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonique) (ABTS) radical scavenging assays. The findings revealed strong anti-oxidant power with an IC50 of 177.55 &micro, g/mL for G. candidum EtOAc extract using DPPH assay, probably due to high polyphenol and flavonoid content in both fungal extracts. Finally, LC-HRMS (Liquid Chromatography&ndash, High Resolution Mass Spectrometry) and GC-MS (Gas Chromatography&ndash, Mass Spectrometry) analysis of G. candidum and P. citrinum extracts revealed an impressive arsenal of compounds with previously reported biological activities, partly explaining the obtained results. Finally, LC-HRMS analysis indicated the presence of new fungal metabolites that have never been reported, which represent good candidates to follow for the discovery of new bioactive molecules.

Date palm (Phoenix dactylifera L.) plantations in North Africa are nowadays threatened with the spread of the Bayoud disease caused by Fusarium oxysporum f. sp. albedinis, already responsible for destroying date production in other infected areas, mainly in Morocco. Biological control holds great promise for sustainable and environmental-friendly management of the disease. In this study, the additional benefits to agricultural ecosystems of using plant growth promoting rhizobacteria (PGPR) or endophytes are addressed. First, PGPR or endophytes can offer an interesting bio-fertilization, meaning that it can add another layer to the sustainability of the approach. Additionally, screening of contrasting niches can yield bacterial actors that could represent wardens against whole genera or groups of plant pathogenic agents thriving in semi-arid to arid ecosystems. Using this strategy, we recovered four bacterial isolates, designated BFOA1, BFOA2, BFOA3 and BFOA4, that proved very active against F. oxysporum f. sp. albedinis. BFOA1–BFOA4 proved also active against 16 Fusarium isolates belonging to four species: F. oxysporum (with strains phytopathogenic of Olea europaea and tomato), F. solani (with different strains attacking O. europaea and potato), F. acuminatum (pathogenic on O. europaea) and F. chlamydosporum (phytopathogenic of O. europaea). BFOA1–BFOA4 bacterial isolates exhibited strong activities against another four major phytopathogens: Botrytis cinerea, Alternaria alternata, Phytophthora infestans, and Rhizoctonia bataticola. Isolates BFOA1–BFOA4 had the ability to grow at temperatures up to 35°C, pH range of 5–10, and tolerate high concentrations of NaCl and up to 30% PEG. The isolates also showed relevant direct and indirect PGP features, including growth on nitrogen-free medium, phosphate solubilization and auxin biosynthesis, as well as resistance to metal and xenobiotic stress. Phylogenomic analysis of BFOA1–BFOA4 isolates indicated that they all belong to Bacillus halotolerans, which could therefore considered as a warden against Fusarium infection in plants. Comparative genomics allowed us to functionally describe the open pan genome of B. halotolerans and LC-HRMS and GCMS analyses, enabling the description of diverse secondary metabolites including pulegone, 2-undecanone, and germacrene D, with important antimicrobial and insecticidal properties. In conclusion, B. halotolerans could be used as an efficient bio-fertilizer and bio-control agent in semi-arid and arid ecosystems.

The soil-borne gram-positive bacteria Aneurinibacillus migulanus strain Nagano shows considerable potential as a biocontrol agent against plant diseases. In contrast, A. migulanus NCTC 7096 proved less effective for inhibition of plant pathogens. Nagano strain exerts biocontrol activity against some gram-positive and gram-negative bacteria, fungi and oomycetes through the production of gramicidin S (GS). Apart from the antibiotic effects, GS increases the rate of evaporation from the plant surface, reducing periods of surface wetness and thereby indirectly inhibiting spore germination. To elucidate the molecular basis of differential biocontrol abilities of Nagano and NCTC 7096, we compared GS production and biosurfactant secretion in addition to genome mining of the genomes. Our results proved that: (i) Using oil spreading, blood agar lysis, surface tension and tomato leaves wetness assays, Nagano showed increased biosurfactant secretion in comparison with NCTC 7096, (ii) Genome mining indicated the presence of GS genes in both Nagano and NCTC 7096 with two amino acid units difference between the strains: T342I and P419S. Using 3D models and the DUET server, T342I and P419S were predicted to decrease the stability of the NCTC 7096 GS synthase, (iii) Nagano produced two additional GS-like molecules GS-1155 (molecular weight 1155) and GS-1169 (molecular weight 1169), where one or two ornithine residues replace lysine in the peptide. There was also a negative correlation between surface tension and the quantity of GS-1169 present in Nagano, and (iv) the Nagano genome had a full protein network of exopolysaccharide biosynthesis in contrast to NCTC 7096 which lacked the first enzyme of the network. NCTC 7096 is unable to form biofilms as observed for Nagano. Different molecular layers, mainly gramicidin secondary metabolite production, account for differential biocontrol abilities of Nagano and NCTC 7096. This work highlighted the basis of differential biological control abilities between strains belonging to the same species and demonstrates techniques useful to the screening of effective biocontrol strains for environmentally friendly secondary metabolites that can be used to manage plant pathogens in the field.

In this study, we aimed to explore and compare the composition, metabolic diversity and antimicrobial potential of endophytic fungi colonizing internal tissues of healthy and brittle leaf diseased (BLD) date palm trees (Phoenix dactylifera L.) widely cultivated in arid zones of Tunisia. A total of 52 endophytic fungi were isolated from healthy and BLD roots of date palm trees, identified based on internal transcribed spacer-rDNA sequence analysis and shown to represent 13 species belonging to five genera. About 36.8% of isolates were shared between healthy and diseased root fungal microbiomes, whereas 18.4 and 44.7% of isolates were specific to healthy and BLD root fungal microbiomes, respectively. All isolates were able to produce at least two of the screened enzymes including amylase, cellulase, chitinase, pectinase, protease, laccase and lipase. A preliminary screening of the isolates using disk diffusion method for antibacterial activity against four Gram-positive and three Gram-negative bacteria and antifungal activities against three phytopathogenic fungi indicated that healthy and BLD root fungal microbiomes displayed interesting bioactivities against examined bacteria and broad spectrum bioactivity against fungal pathogens. Some of these endophytic fungi (17 isolates) were fermented and their extracts were evaluated for antimicrobial potential against bacterial and fungal isolates. Results revealed that fungal extracts exhibited antibacterial activities and were responsible for approximately half of antifungal activities against living fungi. These results suggest a strong link between fungal bioactivities and their secondary metabolite arsenal. EtOAc extracts of Geotrichum candidum and Thielaviopsis punctulata originating from BLD microbiome gave best results against Micrococcus luteus and Bacillus subtilis with minimum inhibitory concentration (MIC, 0.78 mg/mL) and minimum bactericidal concentration (6.25 mg/mL). G. candidum gave the best result against Rhizoctonia solani with MIC 0.78 mg/mL and minimum fungicidal concentration (MFC, 6.25 mg/mL). In conclusion, using plant microbiomes subjected to biotic stresses offers new endophytes with different bioactivities than those of healthy plants. Therefore, date palm endophytic fungi represent a hidden untapped arsenal of antibacterial and broad spectrum antifungal secondary metabolites and could be considered promising source of bioactive compounds with industrial and pharmaceutical applications.

The soil-borne gram-positive bacteria Aneurinibacillus migulanus strain Nagano shows considerable potential as a biocontrol agent against plant diseases. In contrast, A. migulanus NCTC 7096 proved less effective for inhibition of plant pathogens. Nagano strain exerts biocontrol activity against some gram-positive and gram-negative bacteria, fungi and oomycetes through the production of gramicidin S (GS). Apart from the antibiotic effects, GS increases the rate of evaporation from the plant surface, reducing periods of surface wetness and thereby indirectly inhibiting spore germination. To elucidate the molecular basis of differential biocontrol abilities of Nagano and NCTC 7096, we compared GS production and biosurfactant secretion in addition to genome mining of the genomes. Our results proved that: (i) Using oil spreading, blood agar lysis, surface tension and tomato leaves wetness assays, Nagano showed increased biosurfactant secretion in comparison with NCTC 7096, (ii) Genome mining indicated the presence of GS genes in both Nagano and NCTC 7096 with two amino acid units difference between the strains: T342I and P419S. Using 3D models and the DUET server, T342I and P419S were predicted to decrease the stability of the NCTC 7096 GS synthase, (iii) Nagano produced two additional GS-like molecules GS-1155 (molecular weight 1155) and GS-1169 (molecular weight 1169), where one or two ornithine residues replace lysine in the peptide. There was also a negative correlation between surface tension and the quantity of GS-1169 present in Nagano, and (iv) the Nagano genome had a full protein network of exopolysaccharide biosynthesis in contrast to NCTC 7096 which lacked the first enzyme of the network. NCTC 7096 is unable to form biofilms as observed for Nagano. Different molecular layers, mainly gramicidin secondary metabolite production, account for differential biocontrol abilities of Nagano and NCTC 7096. This work highlighted the basis of differential biological control abilities between strains belonging to the same species and demonstrates techniques useful to the screening of effective biocontrol strains for environmentally friendly secondary metabolites that can be used to manage plant pathogens in the field.

In this study, we aimed to explore and compare the composition, metabolic diversity and antimicrobial potential of endophytic fungi colonizing internal tissues of healthy and brittle leaf diseased (BLD) date palm trees (Phoenix dactylifera L.) widely cultivated in arid zones of Tunisia. A total of 52 endophytic fungi were isolated from healthy and BLD roots of date palm trees, identified based on internal transcribed spacer-rDNA sequence analysis and shown to represent 13 species belonging to five genera. About 36.8% of isolates were shared between healthy and diseased root fungal microbiomes, whereas 18.4 and 44.7% of isolates were specific to healthy and BLD root fungal microbiomes, respectively. All isolates were able to produce at least two of the screened enzymes including amylase, cellulase, chitinase, pectinase, protease, laccase and lipase. A preliminary screening of the isolates using disk diffusion method for antibacterial activity against four Gram-positive and three Gram-negative bacteria and antifungal activities against three phytopathogenic fungi indicated that healthy and BLD root fungal microbiomes displayed interesting bioactivities against examined bacteria and broad spectrum bioactivity against fungal pathogens. Some of these endophytic fungi (17 isolates) were fermented and their extracts were evaluated for antimicrobial potential against bacterial and fungal isolates. Results revealed that fungal extracts exhibited antibacterial activities and were responsible for approximately half of antifungal activities against living fungi. These results suggest a strong link between fungal bioactivities and their secondary metabolite arsenal. EtOAc extracts of Geotrichum candidum and Thielaviopsis punctulata originating from BLD microbiome gave best results against Micrococcus luteus and Bacillus subtilis with minimum inhibitory concentration (MIC, 0.78 mg/mL) and minimum bactericidal concentration (6.25 mg/mL). G. candidum gave the best result against Rhizoctonia solani with MIC 0.78 mg/mL and minimum fungicidal concentration (MFC, 6.25 mg/mL). In conclusion, using plant microbiomes subjected to biotic stresses offers new endophytes with different bioactivities than those of healthy plants. Therefore, date palm endophytic fungi represent a hidden untapped arsenal of antibacterial and broad spectrum antifungal secondary metabolites and could be considered promising source of bioactive compounds with industrial and pharmaceutical applications.

To explore proteolytic activity of endophytic fungi inhabiting date palm roots, a Penicillium bilaiae isolate, displaying the highest level of protease production, has been recovered. Response surface methodology (RSM) was applied to optimize culture conditions for protease production by the fungus. Plackett-Burman design allowed for screening of variables effective in protease production. Results indicated that temperature, initial pH and glucose concentration dramatically affect protease yield. These factors were further optimized using a Box-Behnken design and RSM. A combination of initial pH (6.26), temperature (24.5 &deg, C), glucose (13.75 g/L), NaNO3 (1.5 g/L), MgSO4 (0.2 g/L), KH2PO4 (0.5 g/L) and KCl (0.5 g/L) were optimum for maximum production of protease. A 1086-fold enhancement of protease production was gained after optimization. Biochemical properties of fungal protease including the effect of pH and temperature on the activity and the stability of proteolytic enzyme were determined. Moreover, the influence of carbon and nitrogen sources, metal ions, detergents as well as enzyme inhibitors was investigated. Our results highlighted that protease of Penicillium bilaiae isolate TDPEF30 could be considered as a promising candidate for industrial applications.

Summary The ability of two strains of the antibiotic-producing bacillus Aneurinibacillus migulanus to control Dothistroma septosporum infection of pine was tested in artificial inoculation experiments on 2-year-old Pinus contorta plants. Disease severity (% needles with conidiomata) on control plants was 5.8 ± 1.7%, compared with 6.2 ± 2.0% on plants treated with A. migulanus NCTC 7096. In contrast, treatment with A. migulanus Nagano reduced Dothistroma needle blight (DNB) severity to 1.1 ± 0.4%. The conidial density and percentage germination of D. septosporum on needles were also greatly reduced on seedlings treated with A. migulanus Nagano compared with the A. migulanus NCTC 7096 and control treatments. The results suggest that A. migulanus Nagano has potential as a biological control agent for use against DNB in forest nurseries.

We report here the complete genome sequence of the Aneurinibacillus migulanus E1 mutant deficient in gramicidin S (GS) and d -phenylalanyl- l -propyl diketopiperazine (DKP) formation. The genome consists of a circular chromosome (6,301,904 bp, 43.20% G+C content) without any plasmid. The complete genome sequence enables further investigation of the biosynthetic mechanism and the biological function of gramicidin S.

Aneurinibacillus migulanus has biocontrol activities against fungal, fungus-like, and bacterial plant pathogens with different levels of efficacy depending on the target pathogens. Here, we report the high-quality draft genome sequence of A. migulanus NCTC 7096.

ACKNOWLEDGMENTS This project was funded by the Kuwait Government (to F.N.A.), the European Union’s Seventh Framework Programme under grant agreement no. 245268 (ISEFOR; to L.B. and S.W.). Further support came from the SwissBOL project, financed by the Swiss Federal Office for the Environment (grant holder, L.B.) and the Sciex–Scientific Exchange Programme NMS.CH (to L.B. and L.L.).