Improvement of Medicago sativa Crops Productivity by the Co-inoculation of Sinorhizobium meliloti-Actinobacteria Under Salt Stress.

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.

Date Palm Trees Root-Derived Endophytes as Fungal Cell Factories for Diverse Bioactive Metabolites.

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 α,α-diphenyl-ß-picrylhydrazyl (DPPH), ß-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 µg/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 Chromatography­High Resolution Mass Spectrometry) and GC-MS (Gas Chromatography­Mass 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.

Pythium kandovanense sp. nov., a fungus-like eukaryotic micro-organism (Stramenopila, Pythiales) isolated from snow-covered ryegrass leaves.

Pythiumkandovanense sp. nov. (ex-type culture CCTU 1813T = OPU 1626T = CBS 139567T) is a novel oomycete species isolated from Lolium perenne with snow rot symptoms in a natural grassland in East-Azarbaijan province, Iran. Phylogenetic analyses based on sequence data from internal transcribed spacer (ITS)-rDNA, coxI and coxII mitochondrial genes clustered our isolates in Pythium group E as a unique, well supported clade. Pythium kandovanense sp. nov. is phylogenetically and morphologically distinct from the other closely related species in this clade, namely Pythium rostratifingens and Pythium rostratum. Pythium kandovanense sp. nov. can be distinguished from these two species by its cylindrical sporangia and lower temperatures for optimum and maximum growth rate. The development of zoospores released through a shorter discharge tube is an additional morphological feature which can be used to differentiate Pythium kandovanense sp. nov. from Pythium rostratifingens. Laboratory inoculation tests demonstrated the pathogenicity of Pythium kandovanense sp. nov. to L. perenne under wet cold (0-3 °C) conditions.

Examining Innovative Technologies: Nano-Chelated Fertilizers for Management of Wheat Aphid (Schizaphis graminum Rondani).

The use of nanofertilizers has both advantages and concerns. One benefit is that nano-fertilizers can enhance plant resistance against insect pests, making them a valuable strategy in integrated pest management (IPM). This study focused on the effect of wheat leaves treated with nano-chelated fertilizers and nitrogen (N) fertilizer on the wheat aphid (Schizaphis graminum Rondani), a harmful pest of wheat plants that transmits dangerous viruses. The nano-Cu treatment showed the longest pre-adult longevity. Additionally, the nano-Cu treatment resulted in the lowest adult longevity, fecundity, nymphoposition day number, intrinsic rate of population growth (r), finite rate of population increase (λ), and net reproductive rate (R0) and gross reproductive rate (GRR). Also, nano-Cu treatment led to the highest amount of (T). The N treatment led to the highest levels of fecundity, nymphoposition days, r, λ, and R0. Nano-Fe and nano-Zn demonstrated fewer negative effects on S. graminum life table parameters than nano-Cu. Our results indicate that N treatment yielded numerous advantageous effects on the wheat aphid while simultaneously impeding the efficacy of the aphid control program. Conversely, nano-Cu treatment exhibited a detrimental influence on various parameters of the aphid's life table, resulting in a reduction in the pest's fitness. Consequently, the integration of nano-Cu should be seriously considered as a viable option in the IPM of the wheat aphid.

High Occurrence Among Calves and Close Phylogenetic Relationships With Human Viruses Warrants Close Surveillance of Rotaviruses in Kuwaiti Dairy Farms.

Rotavirus, one of the main pathogens causing morbidity and mortality in neonatal dairy calves worldwide, is responsible for 30-44% of cattle deaths. It is considered to be the most common etiologic agent of diarrhea in neonatal dairy calves and children, the dominant type being group A. Two hundred seventy animals from 27 farms from 2 regions of Kuwait were tested for the presence of Rotavirus serogroup A (RVA) using latex agglutination test (LAT) and reverse transcription-polymerase chain (RT-PCR) testing. RVA non-structural proteins NSP1-2, NSP4-5 and capsid protein genes VP1-7 were characterized by next generation sequencing. LAT was positive in 15.56% of the animals, and RT-PCR in 28.89%. Using RT-PCR as a reference method, LAT was 100% specific but only 83.33% sensitive. ANOVA analysis showed correlation only with the location of the farms but no significant correlation with the age and sex of the animals. Although there was a tendency of clustering of RVA positive animals, it did not reach statistical significance (p = 0.035 for LAT). The phylogenetic analysis showed that Kuwaiti isolates of group A rotavirus clustered with human rotaviruses. Taken together, it seems that rotavirus was present in most of the dairy farms in Kuwait. The high occurrence of the virus in calves in Kuwaiti dairy farms and the close phylogenetic affinity with human isolates warrants urgent action to minimize and control its spread between calves in farms.

Response Surface Methodology Optimization of an Acidic Protease Produced by Penicillium bilaiae Isolate TDPEF30, a Newly Recovered Endophytic Fungus from Healthy Roots of Date Palm Trees (Phoenix dactylifera L.).

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 °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.

Screening of the High-Rhizosphere Competent Limoniastrum monopetalum' Culturable Endophyte Microbiota Allows the Recovery of Multifaceted and Versatile Biocontrol Agents.

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.

Screening for Fusarium Antagonistic Bacteria From Contrasting Niches Designated the Endophyte Bacillus halotolerans as Plant Warden Against Fusarium.

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.

Comparative Genomics of Bacillus amyloliquefaciens Strains Reveals a Core Genome with Traits for Habitat Adaptation and a Secondary Metabolites Rich Accessory Genome.

The Gram positive, non-pathogenic endospore-forming soil inhabiting prokaryote Bacillus amyloliquefaciens is a plant growth-promoting rhizobacterium. Bacillus amyloliquefaciens processes wide biocontrol abilities and numerous strains have been reported to suppress diverse bacterial, fungal and fungal-like pathogens. Knowledge about strain level biocontrol abilities is warranted to translate this knowledge into developing more efficient biocontrol agents and bio-fertilizers. Ever-expanding genome studies of B. amyloliquefaciens are showing tremendous increase in strain-specific new secondary metabolite clusters which play key roles in the suppression of pathogens and plant growth promotion. In this report, we have used genome mining of all sequenced B. amyloliquefaciens genomes to highlight species boundaries, the diverse strategies used by different strains to promote plant growth and the diversity of their secondary metabolites. Genome composition of the targeted strains suggest regions of genomic plasticity that shape the structure and function of these genomes and govern strain adaptation to different niches. Our results indicated that B. amyloliquefaciens: (i) suffer taxonomic imprecision that blurs the debate over inter-strain genome diversity and dynamics, (ii) have diverse strategies to promote plant growth and development, (iii) have an unlocked, yet to be delimited impressive arsenal of secondary metabolites and products, (iv) have large number of so-called orphan gene clusters, i.e., biosynthetic clusters for which the corresponding metabolites are yet unknown, and (v) have a dynamic pan genome with a secondary metabolite rich accessory genome.

Increased Biological Activity of Aneurinibacillus migulanus Strains Correlates with the Production of New Gramicidin Secondary Metabolites.

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.

Fungal Root Microbiome from Healthy and Brittle Leaf Diseased Date Palm Trees (Phoenix dactylifera L.) Reveals a Hidden Untapped Arsenal of Antibacterial and Broad Spectrum Antifungal Secondary Metabolites.

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.