Purpose of workA 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.

<p>COVID-19 has had an impact on the regional and worldwide agricultural value chain, jeopardizing food security. It is time to reassess the approach of the agri-food sector and to consider that the food supply and plant health, as agrosystemic services, must depend on strategies with a low impact on productive and environmental assets. One strategy is the use and optimization of microbial genetic resources (MGR) related to agroecosystems as a source of balance, functionality, productivity, inhibition of the impact of pests and pathogens, and contribution to the profitability of agri-food activity. It is necessary to strengthen and develop regional agricultural systems that are dynamic, that mitigate damages to the environment and produce nutritional and nutraceutical foods that ensure human health. Agricultural sciences are undergoing changes in scientific paradigms that will benefit the agri-food sector if we are able to learn from the impacts of an extensive technological agriculture. Approaching agriculture from an agro-systemic point of view in which the crop-community is the functional biological unit of study and to preserve the MGR diversity are the greatest challenges to create sustainable and resilient strategies and technologies that contribute towards human health and help prevent risks during health crises such as the ongoing COVID-19 pandemic.</p>

Zea mays and Sorghum bicolor are important crops for animal and human nutrition worldwide. In the Central Highland Valley of Mexico, both crops are extremely important, and research is aimed toward increasing yield, disease resistance, and crop adaptation from 1,900- to 2,700-m elevation. In a 3-year field breeding experiment (2004 to 2006), leaf blight and vascular wilt symptoms were frequently observed in contiguous plots of maize and sorghum crops in Montecillo, Mexico and maize plots in Tecamac, Mexico. To identify and characterize the causal agent of these symptoms, isolations were conducted on leaves from areas where healthy and diseased tissues converged. Leaf sections of 1 cm2 from both crops were disinfested, placed on casamino acid-peptone-glucose (CPG) medium, and incubated at 28°C. After 48 h, only yellow colonies were observed and 12 isolates were selected for further characterization. Physiological and biochemical tests indicated that the isolates were nonfluorescent on King's B medium, and API 50 CHE (bioMérieux, Marcy l'Etoile, France) revealed that they were negative for gelatin hydrolysis, indole production, acid production from raffinose and positive for utilization of glycerol, D-glucose, mannitol, arbutine, esculine, salicine, cellobiose, maltose, melibiose, D-fucose, and D-arabitol; all characteristics of Pantoea agglomerans. Further identification of these isolates was accomplished by DNA analysis. For DNA analysis, 1.4-kbp fragments of the 16S rRNA gene were amplified with primer set 8F/1492R (3) and sequenced with U514F/800R universal primers (2). Five sequences were obtained and deposited in GenBank (Accession Nos. EF050806 to EF050810). A phylogenetic tree was constructed using the UPGMA method (mega version 3.1). Results of the phylogenetic analysis grouped the species P. ananatis, P. stewartti, and P. agglomerans into three clusters. The five unknown sequences were grouped into the P. agglomerans cluster. There was a 98 to 99% similarity of the five 16S rRNA gene sequences with P. agglomerans strain type ATCC 27155. Pathogenicity of the 12 isolates was confirmed by injecting 108 CFU mL–1 of inoculum into stems of 3-week-old maize cv. Triunfo and sorghum cold tolerant hybrid (A1×B5)×R1 seedlings in the greenhouse at 28°C and 80% relative humidity. Also, seedlings were inoculated with water, nonpathogenic isolates of P. agglomerans from maize (GM13, and HLA1), and not inoculated as negative controls. Three replications were included for each isolate and control. All test strains developed water-soaked lesions on juvenile leaves at 8 days postinoculation and were followed by chlorotic to straw-colored leaf streaks and then leaf blight symptoms at 3 weeks postinoculation. All negative control seedlings did not develop symptoms. In addition, the 12 isolates were infiltrated at 107 CFU mL–1 into tobacco leaves that displayed a hypersensitive response at 4 days, indicating the presence of the type III secretion system (1). Isolates were reisolated, and the 16S rRNA gene fragments were 100% similar to their original isolate sequences. P. agglomerans has been reported to affect other crops, including chinese taro in Brazil (2007), onion in the United States (2006) and South Africa (1981), and pearl millet in Zimbabwe (1997); however, to our knowledge, this is the first report of P. agglomerans associated with leaf blight and vascular wilt symptoms in maize and sorghum in the Central Highland Valley of Mexico. References: (1) J. Alfano and A. Collmer. Annu. Rev. Phytopathol 42:385, 2004. (2) Y. Anzai et al. Int. J. Syst. Evol. Microbiol. 50:1563, 2000. (3) M. Sasoh et al. Appl. Environ. Microbiol. 72:1825, 2006.

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.

<p>Actualmente, los estudios sobre agentes de control biológico de origen microbiano (ACB-M) generalmente están enfocados en la caracterización taxonómica mediante el uso de marcadores moleculares convencionales y en evaluar la capacidad antagónica/mecanismos de acción <em>in vitro</em>, en invernadero y eventualmente bajo condiciones de campo. Los ACB-M se centran principalmente en cepas de <em>Trichoderma</em>, <em>Paecilomyces, Beauveria</em>, <em>Pseudomonas</em> y <em>Bacillus</em>. Aunque la investigación en México en este campo ha sido muy activa en los últimos años, el desarrollo e innovación de una mayor diversidad de bioplaguicidas registrados y comercializados puede ser potenciada. En este contexto, el uso de técnicas vanguardistas en la era de las ciencias ómicas (genómica, transcriptómica, y metabolómica) enfocadas a la correcta afiliación taxonómica de los ACB-M y en el estudio de mecanismos de acción y comportamiento agroecológico es determinante para la bioprospección y uso extensivo de estos ACB-M de manera eficaz, biosegura y costo-efectiva. En el marco de la celebración internacional de la sanidad vegetal, la presente revisión analiza críticamente el estado del conocimiento y de aquellos aspectos que limitan la bioprospección y el uso extensivo de ACB-M con énfasis en México, desde la aplicación de las ciencias ómicas para la identificación, selección y estudio de los mecanismos de acción de dichos agentes hasta la difusión y socialización del conocimiento científico generado. Se pretende promover la reflexión sobre este campo del conocimiento e incentivar la nueva generación ACB-M con una visión holística y sistémica en beneficio de una agricultura sustentable y resiliente.</p>

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.

During the spring of 2004, corn seedlings with symptoms of wilting and stunting were observed in corn fields with emergence problems in Madison and Brown counties, Ohio. Phytophthora isolates were recovered from sections of root tissue of diseased seedlings placed on dilute V8 media amended with pentachloronitrobenzene, iprodione, benlate, neomycin sulfate, and chloramphenicol. Colonies were rosaceous on potato dextrose agar, with a growth rate of 5 mm per day. Homothallic isolates with paragynous antheridia were observed on lima bean agar (LBA); oogonia were 35 to 50 μm in diameter. Sporangia were ovoid to obpyriform, nonpapillate, with an average size of 49 × 30 μm. Pathogenicity was tested on corn seeds using a petri dish assay with 3-day-old cultures on LBA and a sand-cornmeal cup test amended with inoculum from 7-day-old cultures on LBA (1). After 1 week in the petri dish assay, the seeds failed to germinate completely and were covered with white, fungal-like, aerial mycelia and the pathogen was recovered from brown discolored radicle roots. In the cup assay, 2-week-old seedlings developed the same symptoms observed in the field; the pathogen was also isolated from brown discolored roots. In both assays, no symptoms developed in the noninoculated controls. Both pathogenicity tests were repeated two times. Genomic DNA was extracted from mycelia of two isolates and the internal transcribed spacer (ITS) region was amplified and sequenced using ITS6/ITS4 primers (2). Both isolates had identical ITS sequences (GenBank Accession No. GQ853880). A BLAST search of the NCBI database showed 100% homology with the sequence of the haplotype isolate of Phytophthora sansomeana (Accession No. EU925375). P. sansomeana is a new species characterized principally by a large oogonial diameter (37 to 45 μm), rapid growth rate (7 to 10 mm/day), and an ITS sequence falling in Cooke's clade 8 (4). Pathogenicity tests, morphological characteristics, and the ITS sequence analysis indicate that P. samsomena is the causal agent of the symptoms observed on corn seedlings. P. sansomeana has been reported as a pathogen of soybean in Indiana, Douglas-fir in Oregon, and weeds in alfalfa fields in New York (4). To our knowledge, this is the first report of P. sansomeana infecting corn in Ohio, albeit other isolates have previously been recovered from soybean in the state. There are four previous reports of Phytophthora spp. affecting corn in the United States and Mexico (3). Crop rotation will have little effect in management of this pathogen since corn and soybean are produced in the same fields continuously throughout the state. References: (1) K. E. Broders et al. Plant. Dis. 91:727, 2007. (2) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (3) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN. 1989. (4) E. M. Hansen et al. Mycologia 101:129, 2009.

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.