Mohamed Fayez Fouad

The recent introduction of plant-only-based culture media enabled cultivation of not-yet-cultured bacteria that exceed 90% of the plant microbiota communities. Here, we further prove the competence and challenge of such culture media, and further introduce “the inoculum-dependent culturing strategy, IDC”. The strategy depends on direct inoculating plant serial dilutions onto plain water agar plates, allowing bacteria to grow only on the expense of natural nutrients contained in the administered inoculum. Developed colonies are successively transferred/subcultured onto plant-only-based culture media, which contains natural nutrients very much alike to those found in the prepared plant inocula. Because of its simplicity, the method is recommended as a powerful tool in screening programs that require microbial isolation from a large number of diverse plants. Here, the method comfortably and successfully recovered several isolates of endophytic Actinobacteria represented by the six genera of Curtobacterium spp., Plantibacter spp., Agreia spp., Herbiconiux spp., Rhodococcus spp., and Nocardioides spp. Furthermore, two of the isolates are most likely novel species belonging to Agreia spp. and Herbiconiux spp. © 2019, The Author(s).

Plant microbiota support the diversity and productivity of plants. Thus, cultivation-dependent approaches are indispensable for in vitro manipulation of hub taxa. Despite recent advances in high-throughput methods, cultivability is lagging behind other environmental microbiomes, notably the human microbiome. As a plant-based culturing strategy, we developed culture media based on a broth of cooked aqueous mixtures of host plants. This improved the in vitro growth of representative isolates of plant microbiota and extended the in situ recovery of plant microbiota. With clover, 16S rRNA gene sequencing of representative isolates confirmed the predominance of Firmicutes, Alphaproteobacteria and Gammaproteobacteria, and less frequently Bacteroidetes and Actinobacteria. Whereas bovine-based culture media (modified R2A) confined the diversity to Firmicutes, the plant broth-based culture media revealed a wider scope of endophytes beyond rhizobia, i.e., multiple genera such as Chryseobacterium, Cronobacter, Kosakonia, Tsukamurella, and a potentially/presumptive novel species. Matrix-assisted laser desorption/ionization time-of-flight (MADI-TOF) analysis clustered isolates according to their plant niches, the endo-phyllosphere/endo-rhizosphere. We recommend the plant broth for simplicity, reproducibility and perdurable storage, supporting future culturomics applications, good laboratory practice (GLP) and good manufacturing practice (GMP). The strategy creates an “in-situ-similis” vegan nutritional matrix to analyze microbial diversity and reveal novel microbial resources pertinent to biotechnological and environmental applications. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

Although plant-based culture media enhances in vitro cultivation of rhizobacteria, studies assessing their biomass potential for large-scale applications are lacking. Here, we advance plant pellets (PPs) as a novel technology to unlock the potential of such vegan culture media for biomass production of Rhizobium leguminosarum. PP formulations were based on mixtures of Egyptian clover powder and the agro-byproducts glycerol and molasses. These mixtures were either contained or not contained in teabags during culture media preparation. Metrics of biomass included colony forming units, optical density (OD600nm), and cell dry weight (DW). Biomass comparisons between culture media based on PPs and standard yeast extract mannitol (YEM) revealed that the following PPs composition, contained in teabags, cultivated rhizobia at levels comparable to YEM: 16 g clover powder, 5% molasses, and 0.8% glycerol. This PPs composition enabled shorter generation times of rhizobia (PP: 3.83 h, YEM: 4.28 h). Strikingly, PPs mixtures supplemented with 10% molasses and not contained in teabags promoted rhizobia without apparent lag phases and produced 25% greater DW than YEM. PPs potentiate the use of dehydrated vegan feedstocks for both plant microbiota cultivation and biomass production and appear as cost-and labor-effective tools, easy to handle and store for plant-based culture media preparation. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

High-throughput cultivation methods have recently been developed to accelerate the recovery of microorganisms reluctant to cultivation. They simulate in situ environmental conditions for the isolation of environmental microbiota through the exchange of growth substrates during cultivation. Here, we introduce leaf-based culture media adopting the concept of the plant being the master architect of the composition of its microbial community. Pre-physical treatments of sunflower plant leaves, namely punching, freezing, and/or autoclavation, allowed the diffusion of electrolytes and other nutrients to configure the leaf surface as a natural pad, i.e., creating an “in situ similis” environment suitable for the growth of rarely isolated microbiota. We used surface inoculation and membrane-filtration methods to assess the culturability of endophytic bacteria from the sunflower phyllosphere and rhizosphere. Both methods supported excellent colony-forming unit (CFU) development when compared to standard R2A medium, with a special affinity to support better growth of epiphytic and endophytic populations of the phyllosphere compared with the rhizosphere. A 16S rRNA gene analysis of >122 representative isolates indicated the cultivation of a diverse set of microorganisms by application of the new methods. It indicated the predominance of 13 genera of >30 potential species, belonging to Firmicutes, Proteobacteria, and Actinobacteria, and especially genera not commonly reported for sunflower, e.g., Rhizobium, Aureimonas, Sphingomonas, Paracoccus, Stenotrophomonas, Pantoea, Kosakonia, and Erwinia. The strategy successfully extended diversity and richness in the endophyllosphere compared to the endorhizosphere, while CFUs grown on the standard R2A medium mainly pertain to Firmicutes, especially Bacillus spp. MALDI-TOF MS analysis clustered the isolates according to their niche and potential functions, where the majority of isolates of the endorhizosphere were clustered away from those of the endophyllosphere. Isolates identified as Gammaproteobacteria and Alphaproteobacteria were distinguishably sub-clustered, which was in contrast to the heterogeneous isolates of Firmicutes (Bacillus spp.). In conclusion, leaf in situ similis cultivation is an effective strategy to support the future application of culturomics of plant microbiota. This is an effort to access novel isolates that are more adapted and competitive in their natural environments, especially those subjected to abiotic stresses like those prevailing in arid/semi-arid zones, and, consequently, to support the application of agro-biotechnologies, among other technologies, to improving agriculture in such zones. © Copyright © 2020 Nemr, Khalil, Sarhan, Abbas, Elsawey, Youssef, Hamza, Morsi, El-Tahan, Fayez, Patz, Witzel, Ruppel, El-Sahhar and Hegazi.

Improving cultivability of a wider range of bacterial and archaeal community members, living natively in natural environments and within plants, is a prerequisite to better understanding plant-microbiota interactions and their functions in such very complex systems. Sequencing, assembling, and annotation of pure microbial strain genomes provide higher quality data compared to environmental metagenome analyses, and can substantially improve gene and protein database information. Despite the comprehensive knowledge which already was gained using metagenomic and metatranscriptomic methods, there still exists a big gap in understanding in vivo microbial gene functioning in planta, since many differentially expressed genes or gene families are not yet annotated. Here, the progress in culturing procedures for plant microbiota depending on plant-based culture media, and their proficiency in obtaining single prokaryotic isolates of novel and rapidly increasing candidate phyla are reviewed. As well, the great success of culturomics of the human microbiota is considered with the main objective of encouraging microbiologists to continue minimizing the gap between the microbial richness in nature and the number of species in culture, for the benefit of both basic and applied microbiology. The clear message to fellow plant microbiologists is to apply plant-tailored culturomic techniques that might open up novel procedures to obtain not-yet-cultured organisms and extend the known plant microbiota repertoire to unprecedented levels. © 2019

Aim: Egypt is the habitat for a large number of bird species and serves as a vital stopover for millions of migratory birds during their annual migration between the Palearctic and Afrotropical ecozones. Surveillance for avian influenza viruses (AIVs) is critical to assessing risks for potential spreading of these viruses among domestic poultry. Surveillance for AIV among hunted and captured wild birds in Egypt was conducted in order to understand the characteristics of circulating viruses. Methods: Sampling of wild bird species occurred in two locations along the Mediterranean Coast of Egypt in the period from 2014 to 2016. A total of 1316 samples (cloacal and oropharyngeal swabs) were collected from 20 different species of hunted or captured resident and migratory birds sold at live bird markets. Viruses were propagated then sequenced. Phylogenetic analysis and receptor binding affinities were studied. Results: Eighteen AIVs (1.37%) were isolated from migratory Anseriformes at live bird markets. Further characterization of the viral isolates identified five hemagglutinin (H3, H5, H7, H9, and H10) and five neuraminidase (N1, N2, N3, N6, and N9) subtypes, which were related to isolates reported in the Eurasian region. Two of the 18 isolates were highly pathogenic H5N1 viruses related to clade 2.2.1, while three isolates were G1-like H9N2 viruses. Conclusions: Our data show significant diversity of AIVs in Anserifromes sold at live bird markets in Egypt. This allows for genetic exchanges between imported and enzootic viruses and put the exposed humans at a higher risk of infection. © 2019 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

The rapid development of high-throughput techniques and expansion of bacterial databases have accelerated efforts to bring plant microbiomes into cultivation. We introduced plant-only-based culture media as a successful candidate to mimic the nutritional matrices of plant roots. We herein employed a G3 PhyloChip microarray to meticulously characterize the culture-dependent and-independent bacterial communities of the maize root compartments, the endo-and ecto-rhizospheres. An emphasis was placed on the preference of the growth of unculturable candidate divisions/phyla on plant-only-based culture media over standard culture media (nutrient agar). A total of 1,818 different operational taxonomic units (OTUs) were resolved representing 67 bacterial phyla. Plant-only-based culture media displayed particular affinity towards recovering endophytic over ectophytic rhizobacteria. This was shown by the slightly higher recovery of CFUs for endophytes on plant-only-based culture media (26%) than on standard culture media (10%) as well as the higher taxa richness and numbers of exclusive families of unculturable divisions/phyla. Out of 30 bacterial phyla (comprising >95% of the whole population), 13 were of a significantly higher incidence on plant-only-based culture media, 6 phyla of which were not-yet-cultured (Atribacteria, OP9; Dependentiae, TM6; Latescibacteria, WS3; Marinimicrobia, SAR406; Omnitrophica, OP3; BRC1). Furthermore, plant-only-based culture media significantly enriched less abundant and/or hard-to-culture bacterial phyla (Acidobacteria, Gemmatimonadetes, and Tenericutes). These results present conclusive evidence of the ability of plant-only-based culture media to bring the plant-fed in situ microbiome into the status of plant-fed in vitro cultures, and to widen the scope of cultivation of heretofore-unculturable bacterial divisions/phyla. © 2018, Japanese Society of Microbial Ecology. All rights reserved.

In the present study, juice of water hyacinth (Eichhorina crassipes), either crude or from its successive dilutions (1:1, 1:10, 1:30, 1:50 v/v) supported the in vitro development of Bacillus megaterium, Bacillus subtilis, Azotobacter chroococcum and Rhizobium leguminosarum biovar Phaseoli with doubling time (23.1–63.0 min) which was comparable if not shorter, to that calculated using the standard laboratory -synthetic media (nutrient, N-deficient mannitol and yeast extract agar media; 48.0–64.8 min). Rhizospheric microorganisms of legume and non-legume plants successfully grew on surface-inoculated agar plates of crude and diluted juices of the macrophyte. Tea bags filled with the dehydrated powders (5 and 10 g l−1) of water hyacinth supported the in situ recoverability of total rhizobacteria in population densities (3 × 107 - >108 cfu.g−1), which were found to be comparable, if not excessive, to those enumerated on the recommended culture media. Morpho-physiological identification of some isolates that had developed on the plant juice and tea bag culture media, revealed that they are not akin to those cultured on the chemically-synthetic culture media; they possibly represent a portion of recommended media - unculturables. © 2018 National Institute of Oceanography and Fisheries

This study was planned to examine the contribution of the plant growth promoting Rhizobium leguminosarum biovar vicea (ICARDA 441) along with five strains of endophytic bacteria isolated from saline soil to support growth of salt affected wheat plants. Phylogentic analysis based on the 16S rDNA sequences belonged these endophytes to Pantoea agglomernas HP2-MG738254, Pseudomonas stutzeri H1-MG738255, Klebsiella sp. H3-MG738256, Brevundimonas diminuta H4-MG738257 and Bacillus cereus H5- MG738258. In a field trial, inocula of these endophytes supported wheat plant growth in salt affected soil. The obtained results refer to improved germination, increases in dry weight, grain yield and protein concentration scored (2 g plant-1, 3799 Kg ha-1, 9.9%) respectively compared with control and increased nutrient uptake by wheat as a result of inoculation with the examined strains. © 2017 author (s).

In order to improve the culturability and biomass production of rhizobacteria, we previously introduced plant-only-based culture media. We herein attempted to widen the scope of plant materials suitable for the preparation of plant-only-based culture media. We chemically analyzed the refuse of turfgrass, cactus, and clover. They were sufficiently rich to support good in vitro growth by rhizobacteria isolates representing Proteobacteria and Firmicutes. They were also adequate and efficient to produce a cell biomass in liquid batch cultures. These culture media were as sufficient as artificial culture media for the cultivation and recovery of the in situ rhizobacteria of barley (Hordeum murinum L.). Based on culture-dependent (CFU plate counting) and culture-independent analyses (qPCR), mowed turfgrass, in particular, supported the highest culturable population of barley endophytes, representing >16% of the total bacterial number quantified with qPCR. This accurately reflected the endophytic community composition, in terms of diversity indices (S’, H’, and D’) based on PCR-DGGE, and clustered the plant culture media together with the qPCR root populations away from the artificial culture media. Despite the promiscuous nature of the plant materials tested to culture the plant microbiome, our results indicated that plant materials of a homologous nature to the tested host plant, at least at the family level, and/or of the same environment were more likely to be selected. Plant-only-based culture media require further refinements in order to provide selectivity for the in vitro growth of members of the plant microbiome, particularly difficult-to-culture bacteria. This will provide insights into their hidden roles in the environment and support future culturomic studies. © 2018, Japanese Society of Microbial Ecology. All rights reserved.

The plant-based-sea water culture medium is introduced to in vitro cultivation and in situ recovery of the microbiome of halophytes. The ice plant (Mesembryanthemum crystallinum) was used, in the form of juice and/or dehydrated plant powder packed in teabags, to supplement the natural sea water. The resulting culture medium enjoys the combinations of plant materials as rich source of nutrients and sea water exercising the required salt stress. As such without any supplements, the culture medium was sufficient and efficient to support very good in vitro growth of halotolerant bacteria. It was also capable to recover their in situ culturable populations in the phyllosphere, ecto-rhizosphere and endo-rhizosphere of halophytes prevailing in Lake Mariout, Egypt. When related to the total bacterial numbers measured for Suaeda pruinosa roots by quantitative-PCR, the proposed culture medium increased culturability (15.3–19.5%) compared to the conventional chemically-synthetic culture medium supplemented with (11.2%) or without (3.8%) NaCl. Based on 16S rRNA gene sequencing, representative isolates of halotolerant bacteria prevailed on such culture medium were closely related to Bacillus spp., Halomonas spp., and Kocuria spp. Seed germination tests on 25–50% sea water agar indicated positive interaction of such bacterial isolates with the germination and seedlings’ growth of barley seeds. © 2017

In an effort to axenically culture the previously uncultivable populations of the rhizobacteria of Lucerne (Medicago sativa L.), we propose plant-only teabags culture media to mimic the nutritional matrix available in the rhizosphere. Here, we show that culture media prepared from Lucerne powder teabags substantially increased the cultivability of Lucerne rhizobacteria compared with a standard nutrient agar, where we found that the cultivable populations significantly increased by up to 60% of the total bacterial numbers as estimated by Quantitative Real-time Polymerase Chain Reaction (qRT-PCR). Cluster analysis of 16S rDNA Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) of cultivable Colony-Forming Units (CFUs) revealed a more distinct composition and separation of bacterial populations recovered on the plant-only teabags culture media than those developed on a standard nutrient agar. Further, the new plant medium gave preference to the micro-symbiont Sinorhizobium meliloti, and succeeded in isolating a number of not-yet-cultured bacteria, most closely matched to Novosphingobium sp., Lysobacter sp. and Pedobacter sp. The present study may encourage other researchers to consider moving from the well-established standard culture media to the challenging new plant-only culture media. Such a move may reveal previously hidden members of rhizobacteria, and help to further explore their potential environmental impacts. © 2017 Hegazi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

We have developed teabags packed with dehydrated plant powders, without any supplements, for preparation of plant infusions necessary to develop media for culturing rhizobacteria. These bacteria are efficiently cultivated on such plant teabag culture media, with better progressive in situ recoverability compared to standard chemically synthetic culture media. Combining various plant-based culture media and incubation conditions enabled us to resolve unique denaturing gradient gel electrophoresis (DGGE) bands that were not resolved by tested standard culture media. Based on polymerase chain reaction PCR-DGGE of 16S rDNA fingerprints and sequencing, the plant teabag culture media supported higher diversity and significant increases in the richness of endo-rhizobacteria, namely Gammaproteobacteria (Enterobacteriaceae) and predominantly Alphaproteobacteria (Rhizobiaceae). This culminated in greater retrieval of the rhizobacteria taxa associated with the plant roots. We conclude that the plant teabag culture medium by itself, without any nutritional supplements, is sufficient and efficient for recovering and mirroring the complex and diverse communities of rhizobacteria. Our message to fellow microbial ecologists is: simply dehydrate your plant canopy, teabag it and soak it to prepare your culture media, with no need for any additional supplementary nutrients. © 2016 Scandinavian Plant Physiology Society.

Our previous publications and the data presented here provide evidences on the ability of plant-based culture media to optimize the cultivability of rhizobacteria and to support their recovery from plant-soil environments. Compared to the tested chemically-synthetic culture media (e.g. nutrient agar and N-deficient combined-carbon sources media), slurry homogenates, crude saps, juices and powders of cactus (Opuntia ficus-indica) and succulent plants (Aloe vera and Aloe arborescens) were rich enough to support growth of rhizobacteria. Representative isolates of Enterobacter spp., Klebsiella spp., Bacillus spp. and Azospirillum spp. exhibited good growth on agar plates of such plant-based culture media. Cell growth and biomass production in liquid batch cultures were comparable to those reported with the synthetic culture media. In addition, the tested plant-based culture media efficiently recovered populations of rhizobacteria associated to plant roots. Culturable populations of >106-108 cfu g-1 were recovered from the ecto- and endo-rhizospheres of tested host plants. More than 100 endophytic culture-dependent isolates were secured and subjected to morphophysiological identification. Factor and cluster analyses indicated the unique community structure, on species, genera, class and phyla levels, of the culturable population recovered with plant-based culture media, being distinct from that obtained with the chemically-synthetic culture media. Proteobacteria were the dominant (78.8%) on plant-based agar culture medium compared to only 31% on nutrient agar, while Firmicutes prevailed on nutrient agar (69%) compared to the plant-based agar culture media (18.2%). Bacteroidetes, represented by Chryseobacterium indologenes, was only reported (3%) among the culturable rhizobacteria community of the plant-based agar culture medium. © 2015.

Organic agriculture as well as good agricultural practices (GAPs) intrigues the concern of both consumers and producers of agricultural commodities. Bio-preparates of various rhizospheric microorganisms (RMOs) are potential sources of biological inputs supporting plant nutrition and health. The response of open-field potatoes to the application of RMO bio-preparates, the biofertilizer "Biofertile" and the bioagent "Biocontrol", were experimented over 5 successive years under N-hunger of north Sinai desert soils. Both vegetative and tuber yields of a number of tested cultivars were significantly improved due to rhizobacterial treatments. In the majority of cases, the biofertilizer "Biofertile" did successfully supply ca. 50% of plant N requirements, as the yield of full N-fertilized plants was comparable to those received 50% N simultaneously with bio-preparates treatment. The magnitude of inoculation was cultivar-dependent; cvs. Valor and Oceania were among the most responsive ones. Bio-preparate introduction to the plant-soil system was successful via soaking of tubers and/or spraying the plant canopy. The "Biocontrol" formulation was supportive in controlling plant pathogens and significantly increased the fruit yields. The cumulative effect of both bio-preparates resulted in tuber yield increases of ca. 25% over control. © 2014 .

North Sinai deserts were surveyed for the predominant plant cover and for the culturable bacteria nesting their roots and shoots. Among 43 plant species reported, 13 are perennial (e.g. Fagonia spp., Pancratium spp.) and 30 annuals (e.g. Bromus spp., Erodium spp.). Eleven species possessed rhizo-sheath, e.g. Cyperus capitatus, Panicum turgidum and Trisetaria koelerioides. Microbiological analyses demonstrated: the great diversity and richness of associated culturable bacteria, in particular nitrogen-fixing bacteria (diazotrophs); the majority of bacterial residents were of true and/or putative diazotrophic nature; the bacterial populations followed an increasing density gradient towards the root surfaces; sizeable populations were able to reside inside the root (endorhizosphere) and shoot (endophyllosphere) tissues. Three hundred bacterial isolates were secured from studied spheres. The majority of nitrogen-fixing bacilli isolates belonged to Bacillus megaterium, Bacillus pumilus, Bacillus polymexa, Bacillus macerans, Bacillus circulans and Bacillus licheniformis. The family Enterobacteriaceae represented by Enterobacter agglomerans, Enterobacter sackazakii, Enterobacter cloacae, Serratia adorifera, Serratia liquefaciens and Klebsiella oxytoca. The non-Enterobacteriaceae population was rich in Pantoae spp., Agrobacterium rdiobacter, Pseudomonas vesicularis, Pseudomonas putida, Stenotrophomonas maltophilia, Ochrobactrum anthropi, Sphingomonas paucimobilis and Chrysemonas luteola. Gluconacetobacter diazotrophicus were reported inside root and shoot tissues of a number of tested plants. The dense bacterial populations reported speak well to the very possible significant role played by the endophytic bacterial populations in the survival, in respect of nutrition and health, of existing plants. Such groups of diazotrophs are good candidates, as bio-preparates, to support the growth of future field crops grown in deserts of north Sinai and irrigated by the water of El-Salam canal. © 2011 .

The exclusive use of plant juices, not as a mere supplement to synthetic culture media, for culturing rhizospheric microorganisms (RMO) is introduced here. Juices were prepared from desert (Mesembryanthemum crystallinum L., Zygophyllum album L., Carpobrotus edulis L.) as well as cultivated (Trifolium alexandrinum L., Beta vulgaris L.) plants. Colonies of RMO (Azospirillum brasilense, Enterobacter agglomerans and Klebsiella pneumoniae) nicely developed on surface-inoculated agar plates prepared from crude and diluted juice of M. crystallinum (ice plant). Furthermore, hundreds of RMO colonies developed on various standard culture media were replicated (>90%) on agar plates of different plant juices. RMO cells grew nicely in liquid ice plant juice, with doubling times comparable to those grown in the reference culture medium. RMO populations resident in various host plants were able to develop on culture media prepared from homologous and heterologous juices. The application of a thin semi-solid overlay agar on the surfaces of inoculated agar plates significantly increased the recovery of micro-colonies on agar plates, particularly those prepared from plant juices. © 2011.

More than 12×10 9m 3/year of Nile Delta drainage water is annually discharged into the Mediterranean Sea. El-Salam (peace) canal, having a mixture of such drainage water and the Nile water (1:1 ratio), crosses the Suez canal eastward to the deserts of north Sinai. The suitability of the canal water for agriculture is reported here. Representative samples were obtained during two successive years to follow effects of seasonal and spatial distribution, along the first 55km course in north Sinai, on the water load of total bacteria, bacterial indicators of pollution, and chemical and heavy metals contents. In general, the canal water is acceptable for irrigation, with much concern directed towards the chemical contents of total salts (EC), Na and K, as well as the trace elements Cd and Fe. Extending the canal course further than 30km significantly lowered the fecal pollution rate to the permissible levels of drinking water. Results strongly emphasize the need for effective pre-treatment of the used drainage water resources prior mixing with the Nile water. © 2011.

Two strains (EBRI 2 and EBRI 26) belong to Rhizobium etli species and one strain (EBRI 32) belong to Rhizobium gallicum were identified among 12 Rhizobium isolates isolated from nodulated roots of common bean (Phaseolus vulgaris L.). Pot experiments were carried out to select the high efficient nitrogen fixing Rhizobium strains and these results revealed that there is a high degree of strain-cultivar compatibility whereas strain CIAT 899 and TAL 1367 were the best forming nodules with Giza 3, strains EBRI 2 and EBRI 32 were the superior nodule forming with Giza 6 and strain EBRI 32 gave the highest nodule record with Giza xbB 201. The addition of nitrogen fertilizer was associated with reduction in nodule numbers with the examined strains especially at high levels of N (60 kg N fed-1) while the low dosage (15 kg N fed-1) promoted nodule formation with cultivar Giza 6. On the contrary, the application of phosphorus fertilization was markedly increased nodule numbers and dry weight with higher dose of Phosphorus. A field trial was applied in a clay soil characterized by high pH at El-Shohada, in El Menoufia governorate in the middle of Delta Nile Valley as a major productive site for common bean as dry seeds or green vegetables. Under field conditions, there was a response to inoculation with Rhizobium strains and the native Egyptian strains were equal in its effect for forming nodules except strain EBRI 2, gave the highest record while these native strains produced seed yield higher than those obtained by reference strains.

The major agro-industrial effluents of sugarcane and starch industries pose a serious threat to surface waters. Their disposal in the River Nile around Cairo city transitionally affected the microbial load. In situ bacterial enrichment (50-180%) was reported and gradually diminished downstream; the lateral not vertical effect of the effluent disposal was evident. Disposed effluents increased BOD and COD, and then progressively decreased downstream. Ammoniacal N was elevated, indicating active biological ammonification and in situ biodegradability of the effluents. In vitro, the nitrogen-fixing rhizobacteria Crysomonas luteola, Azospirillum spp., Azomonas spp. and K. pneumoniae successfully grew in batch cultures prepared from the crude effluents. This was supported by adequate growth parameters and organic matter decomposition. Therefore, such biodegradability of the tested agro-industrial effluents strongly recommends their use for microbial biomass necessary for the production of bio-preparates. © 2010.

Effluent from the baker's yeast industry was experimented on as a culture medium for the growth and biomass production of diazotrophs. The effluent supported good growth of Azotobacter chroococcum, Enterobacter agglomerans and Klebsiella pneuomoniae, Azospirillum brasilense, Bacillus polymyxa and Pseudomonas putida and strongly proposed for biofertilizers production of associative diazotrophs. Slurry preparations containing natural polymers, e.g. Arabic gum (5%), pero-dextrin (20%), starch granules (10%) or gelatine (20%) were impregnated with cells of tested diazotrophs. With storage, entrapped cells of B. polymyxa were viable up to 160 days, while gradual decreases in Azospirillum numbers were recorded. Pero-dextrin, a by-product of the starch industry, was selected as the appropriate biocarrier accommodating diazotroph cells and maintaining prolonged survival rates and nitrogenase activity. Cell cultures of A. brasilense, A. chroococcum, B. polymyxa, E. agglomerans and P. putida were equally mixed and entrapped into pero-dextrin slurry biofertilizer formulation named as "BIOGRAMINA". Tested diazotrophs successfully survived (ca. 108 cfu ml-1) in such formulation up to 6 months at both ambient and cold temperatures. The response of wheat and barley to "BIOGRAMINA" in the presence or absence of N fertilizers was evaluated in greenhouse and field trials. Highest total biological yields were recorded for inoculated plants simultaneously supplemented with rational N fertilizer dose. © 2005 Taylor & Francis.

Industrial baker's yeast effluent (BYE) was experimented on as a culture medium for growth and biomass production of six fast-growing rhizobia strains. Diluting the effluent with distilled water was necessary to maximize bacterial biomass production. The addition of phosphate buffer, ammonium chloride or trace-elements did not improve the final biomass yield of tested micro-organisms. Rhizobial growth and biomass on the effluent were comparable to traditional yeast extract mannitol medium (YEM). The Rhizobium spp. biomass, produced using either YEM or BYE, was evaluated as inoculum for Leucaena leucocephala (Lam.) de Wit in a pot experiment. No significant differences were reported in respect of legume nodule and growth parameters. Simultaneous inoculation with rhizobia and a group of associative diazotrophs supported better nodulation and nitrogenase activity. © 2005 Taylor & Francis.