Mervat Hamza

Plant microbiota have co-evolved with their associated plants in the entire holobiont, and their assemblages support diversity and productivity on our planet. Of importance is in vitro cultivation and identification of their hub taxa for possible core microbiome modification. Recently, we introduced the in situ-similis culturing strategy, based on the use of plant leaves as a platform for in vitro growth of plant microbiota. Here, the strategy is further extended by exploring plant organ compatible cultivation of plant microbiota when grown on corresponding leaf/root-based culture media. Pooling the advantages of MPN enrichment methodology together with natural plant-only-based culture media, the introduced method efficiently constructed a nutritional milieu governed by vegan nutrients of plant origin, i.e., leaf strips/root segments, immersed in plain semi-solid water agar. MPN estimates exceeded log 7.0 and 4.0 g−1 of endo-rhizosphere and endo-phyllosphere, respectively, of maize ...

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 Chryseobacter...

In a field trail designed in sand soil of north Sinai, sweet pepper cv. J62-44 F1 was inoculated with 6 strains of the associative diazotrophs Agrobacterium radiobacter, Bacillus circulans and Bacillus polymyxa, as root dipping and/or seedlings spray. This was accompanied by a fertilization regime encompassing NPK and chicken manure. After 90 days of transplanting, no significant effect on fruit number per plant was attributed either to diazotroph strain or inoculation method. Shoot and root dry weights significantly increased due to inoculation, an effect that was strain-dependent. The endophytic B. polymyxa isolated from Retama raetam was very supportive for biomass yield, respective increases of 30.7 and 40.1% in shoot and root dry matter production were estimated over the control. In general, biomass yield increases due to root dipping (up to 104%) exceeded those resulted from seedling spray (14.1 – 48.0%). Regardless of inoculation method, the influence of strain type on total ...

Dinitrogen fixation associated with non-legumes has been well documented (Dobereiner, 1983; Hegazi, 1983; Fayez, 1990) and is now generally considered to make a significant contribution to the nitrogen economy of several ecosystems. This intensifies the use of field inoculation with various diazotrophs. Therefore, the need arises to monitor the populations and individuals of several associative diazotrophs inhabiting the plant-soil system. The ability of several diazotrophs to multiply and reduce acetylene on numerous carbon sources encourages the search for a single medium suitable for collective growth and biomass production of a number of diazotrophs. The present study evaluates a new medium modification based on basal salts and a mixture of five carbon sources (glucose, malic acid, mannitol, sodium lactate and sucrose) to fulfil the requirements for a single medium on which the majority of associative diazotrophs would exhibit good growth and biomass production.

This study was carried out to monitor the effect of introducing single and multiple strains of diazotrophs to plant systems (wheat and barley) under gnotobiotic conditions. The relative scarcity of nutrient supply and maintenance requirements under the present experimental conditions resulted in slower growth and much longer doubling time compared to those found in their respective batch cultures. Single inoculations significantly enhanced the efficiency of nitrogen fixation in both plant systems compared to non-inoculated controls. Among tested diazotrophic strains, cells of Azospirillum spp. and Azotobacter spp. were superior in their ARAs, being higher with barley than with wheat seedlings. Increases in total dry weight of plants between 16.8% and 78% were achieved in the wheat system and between 54.5% and 68% in the barley system, with the highest-value being for Azospirillum spp. and Pseudomonas spp. treatments, respectively. It was observed that the type of plant host has a pronounced effect on cell growth and the type of interactions between the two diazotrophic candidates inoculated jointly into wheat and barley systems. Similar to dual inoculation, the doubling time of each candidate increased when inoculated as a mixed inoculum of three or four diazotrophs into the wheat system, and decreased in the barley system.

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 ...

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.

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 ...

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...

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.

... Correspondence: Nabil A. Hegazi, Environmental Studies and Research Unit (ESRU), Faculty of Agriculture, Cairo Universiy, Giza, Egypt, 12613 ... 1999, the slurry inoculant (BIOGRAMINA) was distributed among barley and wheat farmers at Rafah and El-Sheikh Zowaid, north ...