Fungal endophytes from saline-adapted shrubs induce salinity stress tolerance in tomato seedlings.

To meet the food and feed demands of the growing population, global food production needs to double by 2050. Climate change-induced challenges to food crops, especially soil salinization, remain a major threat to food production. We hypothesize that endophytic fungi isolated from salt-adapted host plants can confer salinity stress tolerance to salt-sensitive crops. Therefore, we isolated fungal endophytes from shrubs along the shores of saline alkaline Lake Magadi and evaluated their ability to induce salinity stress tolerance in tomato seeds and seedlings. Of 60 endophytic fungal isolates, 95% and 5% were from Ascomycetes and Basidiomycetes phyla, respectively. The highest number of isolates (48.3%) were from the roots. Amylase, protease and cellulase were produced by 25, 30 and 27 isolates, respectively; and 32 isolates solubilized phosphate. Only eight isolates grew at 1.5 M NaCl. Four fungal endophytes (Cephalotrichum cylindricum, Fusarium equiseti, Fusarium falciforme and Aspergilus puniceus) were tested under greenhouse conditions for their ability to induce salinity tolerance in tomato seedlings. All four endophytes successfully colonized tomato seedlings and grew in 1.5 M NaCl. The germination of endophyte-inoculated seeds was enhanced by 23%, whereas seedlings showed increased chlorophyll and biomass content and decreased hydrogen peroxide content under salinity stress, compared with controls. The results suggest that the the four isolates can potentially be used to mitigate salinity stress in tomato plants in salt-affected soils.

The soil microbiomes of forest ecosystems in Kenya: their diversity and environmental drivers.

Soil microbiomes in forest ecosystems act as both nutrient sources and sinks through a range of processes including organic matter decomposition, nutrient cycling, and humic compound incorporation into the soil. Most forest soil microbial diversity studies have been performed in the northern hemisphere, and very little has been done in forests within African continent. This study examined the composition, diversity and distribution of prokaryotes in Kenyan forests top soils using amplicon sequencing of V4-V5 hypervariable region of the 16S rRNA gene. Additionally, soil physicochemical characteristics were measured to identify abiotic drivers of prokaryotic distribution. Different forest soils were found to have statistically distinct microbiome compositions, with Proteobacteria and Crenarchaeota taxa being the most differentially abundant across regions within bacterial and archaeal phyla, respectively. Key bacterial community drivers included pH, Ca, K, Fe, and total N while archaeal diversity was shaped by Na, pH, Ca, total P and total N. To contextualize the prokaryote diversity of Kenyan forest soils on a global scale, the sample set was compared to amplicon data obtained from forest biomes across the globe; displaying them to harbor distinct microbiomes with an over-representation of uncultured taxa such as TK-10 and Ellin6067 genera.

Culturable Bacterial Endophytes Associated With Shrubs Growing Along the Draw-Down Zone of Lake Bogoria, Kenya: Assessment of Antifungal Potential Against Fusarium solani and Induction of Bean Root Rot Protection.

Vascular shrubs growing along the draw-down zones of saline lakes must develop adaptive mechanisms to cope with high salinity, erratic environmental conditions, and other biotic and abiotic stresses. Microbial endophytes from plants growing in these unique environments harbor diverse metabolic and genetic profiles that play an important role in plant growth, health, and survival under stressful conditions. A variety of bacterial endophytes have been isolated from salt tolerant plants but their potential applications in agriculture have not been fully explored. To further address this gap, the present study sought to isolate culturable bacterial endophytes from shrubs growing along the draw-down zone of Lake Bogoria, a saline alkaline lake, and examined their functional characteristics and potential in the biocontrol of the bean root rot pathogen, Fusarium solani. We collected shrubs growing within 5 m distance from the shoreline of Lake Bogoria and isolated 69 bacterial endophytes. The endophytic bacteria were affiliated to three different phyla (Firmicutes, Proteobacteria, and Actinobacteria) with a bias in the genera, Bacillus, and they showed no tissue or plant specificity. All selected isolates were positive for catalase enzyme grown in 1.5 M NaCl; three isolates (B23, B19, and B53) produced indole acetic acid (IAA) and only one isolate, B23 did not solubilize phosphate on Pikovskaya agar. Isolates, B19 and B53 exhibited more than 50% of mycelial inhibition in the dual culture assay and completely inhibited the germination of F. solani spores in co-culture assays while two isolates, B07 and B39 had delayed fungal spore germination after an overnight incubation. All isolates were able to establish endophytic association in the roots, stems, and leaves of been seedlings in both seed soaking and drenching methods. Colonization of bean seedlings by the bacterial endophytes, B19 and B53 resulted in the biocontrol of F. solani in planta, reduced disease severity and incidence, and significantly increased both root and shoot biomass compared to the control. Taxonomic identification using 16S rRNA revealed that the two isolates belong to Enterobacter hormaechei subsp., Xiangfangensis and Bacillus megaterium. Our results demonstrate the potential use of these two isolates in the biocontrol of the bean root rot pathogen, F. solani and plant growth promotion.

Isolation, characterization and analysis of bacteriophages from the haloalkaline lake Elmenteita, Kenya.

As a step towards better understanding of diversity and biology of phages and their hosts in haloalkaline Lake Elmenteita, phages were isolated from sediment samples and overlying water using indigenous bacteria as hosts. 17 seemingly different phages of diverse morphotypes with different dimensions and partly exhibiting remarkably unusual ultrastructures were revealed by transmission electron microscopy. 12 clonal phage isolates were further characterized. Infection capability of the phages was optimum at 30-35°C and in alkali condition with optimum at pH 10-12. Structural protein profiles and restriction fragment length polymorphism analyses patterns were distinct for each of the phage type. Complete nucleotide sequences of phages vB-VmeM-32, vB_EauS-123 and vB_BhaS-171 genomes varied in size from 30,926-199,912 bp and G + C content of between 36.25-47.73%. A range of 56-260 potential open reading frames were identified and annotated. The results showed that the 12 phages were distinct from each other and confirmed the presence and diversity of phages in extreme environment of haloalkaline Lake Elmenteita. The phages were deposited at the German Collection of Microorganisms and Cell Cultures and three of their genomes uploaded to NCBI GenBank.

Comparative genomic analysis of eight novel haloalkaliphilic bacteriophages from Lake Elmenteita, Kenya.

We report complete genome sequences of eight bacteriophages isolated from Haloalkaline Lake Elmenteita found on the floor of Kenyan Rift Valley. The bacteriophages were sequenced, annotated and a comparative genomic analysis using various Bioinformatics tools carried out to determine relatedness of the bacteriophages to each other, and to those in public databases. Basic genome properties like genome size, percentage coding density, number of open reading frames, percentage GC content and gene organizations revealed the bacteriophages had no relationship to each other. Comparison to other nucleotide sequences in GenBank database showed no significant similarities hence novel. At the amino acid level, phages of our study revealed mosaicism to genes with conserved domains to already described phages. Phylogenetic analyses of large terminase gene responsible for DNA packaging and DNA polymerase gene for replication further showed diversity among the bacteriophages. Our results give insight into diversity of bacteriophages in Lake Elmenteita and provide information on their evolution. By providing primary sequence information, this study not only provides novel sequences for biotechnological exploitation, but also sets stage for future studies aimed at better understanding of virus diversity and genomes from haloalkaline lakes in the Rift Valley.

Potential human pathogenic bacteria in five hot springs in Eritrea revealed by next generation sequencing.

Human pathogens can survive and grow in hot springs. For water quality assessment, Escherichia coli or Enterococci are the main thermotolerant enteric bacteria commonly used to estimate the load of pathogenic bacteria in water. However, most of the environmental bacteria are unculturable thus culture methods may cause bias in detection of most pathogens. Illumina sequencing can provide a more comprehensive and accurate insight into environmental bacterial pathogens, which can be used to develop better risk assessment methods and promote public health awareness. In this study, high-throughput Illumina sequencing was used to identify bacterial pathogens from five hot springs; Maiwooi, Akwar, Garbanabra, Elegedi and Gelti, in Eritrea. Water samples were collected from the five hot springs. Total community DNA was extracted from samples using the phenol-chloroform method. The 16S rRNA gene variable region (V4-V7) of the extracted DNA was amplified and library construction done according to Illumina sequencing protocol. The sequence reads (length >200 bp) from Illumina sequencing libraries ranged from 22,091 sequences in the wet sediment sample from Garbanabra to 155,789 sequences in the mat sample from Elegedi. Taxonomy was assigned to each OTU using BLASTn against a curated database derived from GreenGenes, RDPII, SILVA SSU Reference 119 and NCBI. The proportion of potential pathogens from the water samples was highest in Maiwooi (17.8%), followed by Gelti (16.7%), Akwar (13.6%) and Garbanabra (10.9%). Although the numbers of DNA sequence reads from Illumina sequencing were very high for the Elegedi (104,328), corresponding proportion of potential pathogens very low (3.6%). Most of the potential pathogenic bacterial sequences identified were from Proteobacteria and Firmicutes. Legionella and Clostridium were the most common detected genera with different species. Most of the potential pathogens were detected from the water samples. However, sequences belonging to Clostridium were observed more abundantly from the mat samples. This study employed high-throughput sequencing technologies to determine the presence of pathogenic bacteria in the five hot springs in Eritrea.

Bacteria and Archaea diversity within the hot springs of Lake Magadi and Little Magadi in Kenya.

BACKGROUND: Lake Magadi and little Magadi are hypersaline, alkaline lakes situated in the southern part of Kenyan Rift Valley. Solutes are supplied mainly by a series of alkaline hot springs with temperatures as high as 86 °C. Previous culture-dependent and culture-independent studies have revealed diverse groups of microorganisms thriving under these conditions. Previous culture independent studies were based on the analysis of 16S rDNA but were done on less saline lakes. For the first time, this study combined illumina sequencing and analysis of amplicons of both total community rDNA and 16S rRNA cDNA to determine the diversity and community structure of bacteria and archaea within 3 hot springs of L. Magadi and little Magadi. METHODS: Water, wet sediments and microbial mats were collected from springs in the main lake at a temperature of 45.1 °C and from Little Magadi "Nasikie eng'ida" (temperature of 81 °C and 83.6 °C). Total community DNA and RNA were extracted from samples using phenol-chloroform and Trizol RNA extraction protocols respectively. The 16S rRNA gene variable region (V4 - V7) of the extracted DNA and RNA were amplified and library construction performed following Illumina sequencing protocol. Sequences were analyzed done using QIIME while calculation of Bray-Curtis dissimilarities between datasets, hierarchical clustering, Non Metric Dimensional Scaling (NMDS) redundancy analysis (RDA) and diversity indices were carried out using the R programming language and the Vegan package. RESULTS: Three thousand four hundred twenty-six and one thousand nine hundred thirteen OTUs were recovered from 16S rDNA and 16S rRNA cDNA respectively. Uncultured diversity accounted for 89.35 % 16S rDNA and 87.61 % 16S rRNA cDNA reads. The most abundant phyla in both the 16S rDNA and 16S rRNA cDNA datasets included: Proteobacteria (8.33-50 %), Firmicutes 3.52-28.92 %, Bacteroidetes (3.45-26.44 %), Actinobacteria (0.98-28.57 %) and Euryarchaeota (3.55-34.48 %) in all samples. NMDS analyses of taxonomic composition clustered the taxa into three groups according to sample types (i.e. wet sediments, mats and water samples) with evident overlap of clusters between wet sediments and microbial mats from the three sample types in both DNA and cDNA datasets. The hot spring (45.1 °C) contained less diverse populations compared to those in Little Magadi (81-83 °C). CONCLUSION: There were significant differences in microbial community structure at 95 % level of confidence for both total diversity (P value, 0.009) based on 16S rDNA analysis and active microbial diversity (P value, 0.01) based on 16S rRNA cDNA analysis, within the three hot springs. Differences in microbial composition and structure were observed as a function of sample type and temperature, with wet sediments harboring the highest diversity.

Nocardiopsis mwathae sp. nov., isolated from the haloalkaline Lake Elmenteita in the African Rift Valley.

During a screening for novel and biotechnologically useful bacteria in haloalkaline lakes, strain No.156(T) was isolated from a sediment sample from lake Elmenteita in the African Rift Valley and studied by a polyphasic taxonomic approach. The strain was observed to form yellow aerial and substrate mycelia; optimal growth was found to be at 30-35 °C in salt concentrations of 6-9 % (w/v) and at pH 7-9. The DNA G+C content of the novel strain was 71 mol%. Analysis of 16S rRNA sequences indicated that the isolate belongs to the genus Nocardiopsis with sequence similarities below 98 % to the type strains of all other representatives of the genus. Mycolic acids were not detected in whole cell methanolysates. The peptidoglycan was found to contain meso-diaminopimelic acid as the diamino acid with no diagnostic sugars. The main polar lipids were identified as phosphatidylmethylethanolamine, phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol but no diphosphatidylglycerol. The predominant menaquinones were MK-11(H8), MK-11(H6), MK-10(H8) and MK-10(H6). Cellular fatty acids were found to consist of saturated and monounsaturated iso- and anteiso-branched acids with 16-18 C-length, tuberculostearic acid (Me18:0), and straight-chain saturated (16:0, 18:0) acids. These characteristics match those of the genus Nocardiopsis. Based on 16S rRNA gene sequence analysis and phenotypic characteristics, a novel species with the name Nocardiopsis mwathae is proposed. The type strain is No.156(T) (=DSM 46659(T) = CECT 8552(T)). The INSDC accession number for the 16S rRNA gene sequence of strain No.156(T) is KF976731.

Streptomyces alkaliphilus sp. nov., isolated from sediments of Lake Elmenteita in the Kenyan Rift Valley.

A novel strain, designated No. 7(T), was isolated from a sediment sample collected from the alkaline, saline Lake Elmenteita located in the Kenyan Rift Valley. The optimal growth for the strain was found to be at temperature 30-35 °C, at pH 8.0-12.0 in the presence of 7.0-10.0 % (w/v) NaCl. The strain was observed to form a light green beige abundant aerial mycelium on Horikoshi 1 agar and to have morphological and chemotaxonomic characteristics typical of members of the genus Streptomyces. The peptidoglycan was found to contain LL-diaminopimelic acid as the diamino acid, with no diagnostic sugars identified. The predominant menaquinone was identified as MK-9(H6). The main polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and an unknown phospholipid. Cellular fatty acids were found to consist of saturated branched-chain acids with iso-C(15:0), anteiso-C(15:0), iso-C(16:0) and anteiso-C(17:0) acids predominating. The type strain had a genomic DNA G+C content of 72.8 mol% and formed a distinct phyletic line within the genus Streptomyces. Based on the chemotaxonomic results, 16S rRNA gene sequence analysis and the low DNA-DNA hybridization value with the type strain of Streptomyces calidiresistens, it is proposed that strain No. 7(T) (= DSM 42118 = CECT 8549) represents a novel species, Streptomyces alkaliphilus. The INSDC accession number for the 16S rRNA gene sequence of strain No. 7(T) is KF976730.

Belliella kenyensis sp. nov., isolated from an alkaline lake.

A red-pigmented, Gram-reaction-negative, aerobic bacterial strain, designated No.164(T), was isolated from sediment sample from the alkaline Lake Elmenteita located in the Kenyan Rift Valley. Results of 16S rRNA gene sequence analysis indicated that the isolate represented a member of the genus Belliella, with the highest sequence similarity (97 %) to Belliella pelovolcani DSM 46698(T). Optimal growth temperature was 30-35 °C, at pH 7.0-12.0 in the presence of 0-4 % (w/v) NaCl. Flexirubins were absent. The respiratory menaquinone (MK-7), predominant cellular fatty acids (iso-C15 : 0, anteiso-C15 : 0 and a mixture of C16 : 1ω7c and/or iso-C15 : 0 2-OH) and DNA G+C content (38.1 mol%) of strain No.164(T) were consistent with those of other members of the genus Belliella. The polar lipids consisted of phosphatidylethanolamine, eight unspecified lipids and one unspecified phospholipid. Several phenotypic characteristics can be used to differentiate this isolate from those of other species of the genus Belliella. The results of polyphasic analyses presented in this study indicated that this isolate should be classified as representing a novel species of the genus Belliella. The name Belliella kenyensis sp. nov. is therefore proposed; the type strain is strain No.164(T) ( = DSM 46651(T) = CECT 8551(T)).

Efficacy of Metarhizium anisopliae in controlling the two-spotted spider mite Tetranychus urticae on common bean in screenhouse and field experiments.

The efficacy of aqueous and emulsifiable formulations of the fungus Metarhizium anisopliae isolate ICIPE78 was evaluated on the population density of Tetranychus urticae infesting common bean plants under screenhouse and field conditions. Synthetic acaricide abamectin was included as a check. Bean plants were artificially infested with T. urticae and allowed to multiply. Three treatments were applied in the screenhouse and 1 treatment in field trials. Mite density was recorded 2 d before spraying and weekly postspraying. The number of pods per plant, number of seeds per pod, and the dry weight of seeds per plant were recorded only in the screenhouse trials. In both screenhouse and field trials, fungal formulations applied at the concentration of 10(8) conidia/mL and the acaricide reduced the population density of mites as compared to the controls. There were significant differences in T. urticae population densities between the treatments at the various post-spraying sampling dates. In the screenhouse, the mite densities were near zero from 3-week postspraying in the treated leaves. At 4-week postspraying, there were no more leaves in the untreated control (T1) and in the control water + Silwet-L77 (T2). Fungal formulations were as effective as abamectin in reducing mite densities in both screenhouse and field experiments. There were significant differences in the production parameters during the 2 screenhouse trials, with fungal and abamectin treatments generally having the highest yield. Results of this study underline the potential of the M. anisopliae isolate ICIPE78 as an alternative to acaricides for T. urticae management.

Influence of temperature on virulence of fungal isolates of Metarhizium anisopliae and Beauveria bassiana to the two-spotted spider mite Tetranychus urticae.

Twenty-three isolates of Metarhizium anisopliae (Metschnikoff) Sokorin and three isolates of Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales: Clavicipitaceae) were assessed for their virulence against the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). Based on the screening results, nine isolates of M. anisopliae and two isolates of B. bassiana were tested for their virulence against young adult (1- to 2-day-old) female T. urticae at constant temperatures of 20, 25, 30 and 35 degrees C. At all temperatures tested, all the fungal isolates were pathogenic to T. urticae but mortality varied with isolates and temperatures. Fungal isolates were more virulent at 25, 30 and 35 degrees C than at 20 degrees C. The lethal time to 50% mortality (LT(50)) and lethal time to 90% mortality (LT(90)) values decreased with increased temperature. There were no significant differences in virulence between fungal isolates at 30 and 35 degrees C; however, significant differences were observed at 20 and 25 degrees C.