Plant growth-promoting and heavy metal-resistant Priestia and Bacillus strains associated with pioneer plants from mine tailings.

Open mine tailings dams are extreme artificial environments containing sizeable potentially toxic elements (PTEs), including heavy metals (HMs), transition metals, and metalloids. Furthermore, these tailings have nutritional deficiencies, including assimilable phosphorus sources, organic carbon, and combined nitrogen, preventing plant colonization. Bacteria, that colonize these environments, have mechanisms to tolerate the selective pressures of PTEs. In this work, several Priestia megaterium (formerly Bacillus megaterium), Bacillus mojavensis, and Bacillus subtilis strains were isolated from bulk tailings, anthills, rhizosphere, and endosphere of pioneer plants from abandoned mine tailings in Zacatecas, Mexico. Bacillus spp. tolerated moderate HMs concentrations, produced siderophores and indole-3-acetic acid (IAA), solubilized phosphates, and reduced acetylene in the presence of HMs. The strains harbored different PIB-type ATPase genes encoding for efflux pumps and Cation Diffusion Facilitator (CDF) genes. Moreover, nifH and nifD nitrogenase genes were detected in P. megaterium and B. mojavensis genomic DNA. They showed similarity with sequences of the beta-Proteobacteria species, which may represent likely horizontal transfer events. These Bacillus species precede the colonization of mine tailings by plants. Their phenotypic and genotypic features could be essential in the natural recovery of the sites by reducing the oxidative stress of HMs, fixing nitrogen, solubilizing phosphate, and accumulating organic carbon. These traits of the strains reflect the adaptations of Bacillus species to the mine tailings environment and could contribute to the success of phytoremediation efforts.

Germination test for the evaluation of plant-growth promoting microorganisms.

There is an increased interest for finding strains able to contribute to plant nutrition and health, since these are desirable for the formulation of agricultural bioinoculants. Obtaining a safe and efficient product requires exhaustive evaluations from which most methods used for this purpose involve the use of substrates or are established under uncontrolled conditions, so that various factors can mask the results of the plant-microorganism interaction. In vitro methods mostly involve the use of Petri Dishes (PD) but limit the results to seed germination. Other methods of germination involve the use of acrylic boxes (GB) allowing for better plant development, but are little known. Methods such as ISTA are widely used to evaluate the physiological quality of seeds in productive terms. Despite their efficiency, these methods have not been previously used to evaluate the effect of plant-microorganism interaction on crops. In the present study, modifications were made to the germination between paper of ISTA (BP) method, and were compared to the PD anf GB methods to evaluate the impact of the bacterium Serratia liquefaciens 385 and the yeast Clavispora lusitaniae Y35 on maize, bean and squash. Through the evaluation of physiological parameters in seed and seedling, the results clearly showed the superiority of the BP method to evaluate the effect of microorganisms since it allows observing a better development in the seedlings in terms of growth of the plumule, a better architecture of the radical system in which the emergence of adventitious secondary roots and differentiated radical hairs is observed in comparison with seedlings obtained under the other methods. Similarly, it was possible to observe the different effects on each of the three crops with respect to the inoculation of the bacteria and yeast. These results were significantly better in seedlings obtained in the BP method independently of the type of crop evaluated, considering the BP method suitable to be applied in large-scale bioprospecting plant-growth-promoting microorganism studies.

Mycobiota of Mexican Maize Landraces with Auxin-Producing Yeasts That Improve Plant Growth and Root Development.

Compared to agrochemicals, bioinoculants based on plant microbiomes are a sustainable option for increasing crop yields and soil fertility. From the Mexican maize landrace "Raza cónico" (red and blue varieties), we identified yeasts and evaluated in vitro their ability to promote plant growth. Auxin production was detected from yeast isolates and confirmed using Arabidopsis thaliana plants. Inoculation tests were performed on maize, and morphological parameters were measured. Eighty-seven yeast strains were obtained (50 from blue corn and 37 from red corn). These were associated with three families of Ascomycota (Dothideaceae, Debaryomycetaceae, and Metschnikowiaceae) and five families of Basidiomycota (Sporidiobolaceae, Filobasidiaceae, Piskurozymaceae, Tremellaceae, and Rhynchogastremataceae), and, in turn, distributed in 10 genera (Clavispora, Rhodotorula, Papiliotrema, Candida, Suhomyces, Soliccocozyma, Saitozyma Holtermaniella, Naganishia, and Aeurobasidium). We identified strains that solubilized phosphate and produced siderophores, proteases, pectinases, and cellulases but did not produce amylases. Solicoccozyma sp. RY31, C. lusitaniae Y11, R. glutinis Y23, and Naganishia sp. Y52 produced auxins from L-Trp (11.9-52 µg/mL) and root exudates (1.3-22.5 µg/mL). Furthermore, they stimulated the root development of A. thaliana. Inoculation of auxin-producing yeasts caused a 1.5-fold increase in maize plant height, fresh weight, and root length compared to uninoculated controls. Overall, maize landraces harbor plant growth-promoting yeasts and have the potential for use as agricultural biofertilizers.

Functional characterization of culturable fungi from microbiomes of the "conical cobs" Mexican maize (Zea mays L.) landrace.

Mexican maize landraces, produced for local consumption, are adapted to different environmental conditions, and their yield is affected by abiotic and biotic factors, including the use of agrochemicals. The search for sustainable alternatives to agrochemicals includes the study of the culturable microbial communities. In this study, the fungal communities associated with 2 Mexican maize landraces reddish and bluish "conical cobs" were found to be comprised of Ascomycota fungi, represented by 89 strains within 6 orders (Pleosporales, Hypocreales, Onygenales, Capnodiales, Helotiales, and Eurotiales) and 16 genera. Cellulases and metallophores production were the primary enzymatic products and plant growth-promoting activities were detected among the isolates. Penicillium, Didymella, and Fusarium strains had the most active enzymatic and plant growth promoting activities, however, Aspergillus sp. HES2-2.2, Talaromyces sp. RS1-7, and Penicillium sp. HFS3-3 showed antagonistic activity against the four phytopathogenic Fusarium strains Fusarium oxysporum, Fusarium sambucinum, Fusarium fujikuroi and Fusarium incarnatum-equiseti and also a high and diverse production of enzymatic and plant growth promoting activities; here we identified fungal strains as candidates to promote maize growth.

Bacterial Succession through the Artisanal Process and Seasonal Effects Defining Bacterial Communities of Raw-Milk Adobera Cheese Revealed by High Throughput DNA Sequencing.

The bacterial community of the artisanal Adobera cheese from Los Altos de Jalisco was described through high-throughput sequencing of 16S rRNA gene libraries. Samples were collected in two different seasons (dry and rainy) during four key steps of the manufacturing process (raw milk, fresh curd, matured curd, and cheese). Bacterial diversity was higher in early steps in comparison with the final elaboration stages. Firmicutes and Proteobacteria were the most abundant phyla, strongly represented by the Streptococcaceae, Enterobacteriaceae and Lactobacillaceae families, and core bacteria genera such as Streptococcus spp., Lactococcus spp., and Lactobacillus spp. Undesirable bacteria, including Pseudomonas spp. and Acinetobacter spp., were also detected in raw milk but almost undetectable at the end of the cheese manufacturing process, and seemed to be displaced by lactic-acid bacteria-related genera. Seasonal effects were observed on the community structure but did not define the core microbiota composition. Predictive metabolism was related to membrane transport, and amino-acid, lipid, and carbohydrate metabolism pathways. Our results contribute to deduce the role of bacteria involved in Adobera cheese manufacturing in terms of the metabolism involved, cheese microbial safety, and how undesirable bacterial populations could be regulated by process standardization as a potential tool to improve safety.

Ammonia-Oligotrophic and Diazotrophic Heavy Metal-Resistant Serratia liquefaciens Strains from Pioneer Plants and Mine Tailings.

Mine tailings are man-made environments characterized by low levels of organic carbon and assimilable nitrogen, as well as moderate concentrations of heavy metals. For the introduction of nitrogen into these environments, a key role is played by ammonia-oligotrophic/diazotrophic heavy metal-resistant guilds. In mine tailings from Zacatecas, Mexico, Serratia liquefaciens was the dominant heterotrophic culturable species isolated in N-free media from bulk mine tailings as well as the rhizosphere, roots, and aerial parts of pioneer plants. S. liquefaciens strains proved to be a meta-population with high intraspecific genetic diversity and a potential to respond to these extreme conditions. The phenotypic and genotypic features of these strains reveal the potential adaptation of S. liquefaciens to oligotrophic and nitrogen-limited mine tailings with high concentrations of heavy metals. These features include ammonia-oligotrophic growth, nitrogen fixation, siderophore and indoleacetic acid production, phosphate solubilization, biofilm formation, moderate tolerance to heavy metals under conditions of diverse nitrogen availability, and the presence of zntA, amtB, and nifH genes. The acetylene reduction assay suggests low nitrogen-fixing activity. The nifH gene was harbored in a plasmid of ∼60 kb and probably was acquired by a horizontal gene transfer event from Klebsiella variicola.

Easy and efficient protocol for oomycete DNA extraction suitable for population genetic analysis.

PURPOSE OF WORK: A simple and rapid DNA extraction protocol capable of obtaining high-quality and quantity DNA from a large number of individuals is essential for assaying population and phylogenetic studies of plant pathogens. Most DNA extraction protocols used with oomycetes are relatively lengthy and cumbersome for high throughput analysis. Commercial kits are widely used, but low quantities of DNA are usually obtained, and with large scale analysis multiple isolations are required. A protocol for DNA isolation from Phytophthora and Pythium suitable for the evaluation of a large set of molecular markers was modified from one previously developed for soybean seed. There was a one to three fold increase in the amount of DNA that was extracted using the modified protocol compared to a commercial kit. The DNA obtained using the modified protocol was suitable for the amplification of microsatellite markers as well as the ITS region. This protocol is inexpensive, easy, quick, and efficient in terms of the volume of reagents and the number of steps involved in the procedure. The method may be applicable to other oomycetes and effectively implemented in other laboratories.