Biochemical characterization of two chitinases from Bacillus cereus sensu lato B25 with antifungal activity against Fusarium verticillioides P03.

Bacterial chitinases are a subject of intense scientific research due to their biotechnological applications, particularly their use as biological pesticides against phytopathogenic fungi as a green alternative to avoid the use of synthetic pesticides. Bacillus cereus sensu lato B25 is a rhizospheric bacterium that is a proven antagonist of Fusarium verticillioides, a major fungal pathogen of maize. This bacterium produces two chitinases that degrade the fungal cell wall and inhibit its growth. In this work, we used a heterologous expression system to purify both enzymes to investigate their biochemical traits in terms of Km, Vmax, optimal pH and temperature. ChiA and ChiB work as exochitinases, but ChiB exhibited a dual substrate activity and it is also an endochitinase. In this work, the direct addition of these chitinases inhibited fungal conidial germination and therefore they may play a major role in the antagonism against F. verticillioides.

Prevalence and characterization of Listeria monocytogenes isolated from pork meat and on inert surfaces.

This study focuses on the prevalence of Listeria monocytogenes (Lm) in pork meat and on inert surfaces from slaughterhouses in Sonora, Mexico. A total of 21 Lm were obtained from 103 samples, giving a prevalence of 20.3%. The prevalence of Lm in pork loin was 15.9% and 20.8% for inert surfaces in Federal Inspection Type (FIT) slaughterhouses. For non-FIT slaughterhouses, the prevalence was 25.7%. PCR amplification of genomic DNA from the Lm isolates revealed the presence of the hlyA gene, suggesting a pathogenic nature for these isolates. The isolates obtained in this work all clustered with Lm, according to our phylogenetic analysis based on the 16S rDNA sequence. This Lm cluster indicates that Lm isolates 7-2, 4, 2-1, 10B, 8, 3, 3-3, and 9 share 16S rRNA identity with other Lm isolates that have been reported as foodborne pathogens (rR2-502, J1817, J1816, J1926) and that are involved in foodborne outbreaks. The most commonly detected serotypes were 1/2a and 1/2b. All isolates displayed differential responses to the assayed antibiotics, and most isolates were able to grow in the presence of penicillin G, or both penicillin and penicillin-derived (oxacillin) antibiotics.

Rhizospheric bacteria of maize with potential for biocontrol of Fusarium verticillioides.

The stalk, ear and root rot (SERR) of maize caused by Fusarium verticillioides (Fv) severely impacts crop production in tropical and subtropical regions. The aim of the present work was to screen bacterial isolates in order to find novel native biocontrol agents against Fv. A culturable bacterial collection consisting of 11,520 isolates enriched in Firmicutes and Proteobacteria was created from rhizosphere samples taken from SERR symptomatic or asymptomatic maize plants. The complete collection was screened for potential activity against Fv using a liquid antagonism assay followed by dual cultures in solid medium, selecting for 42 bacteria (Bacillus, Pseudomonas and Paenibacillus) that inhibit Fv growth (>45 %). In planta assays demonstrated that three Bacillus isolates: B. megaterium (B5), B. cereus sensu lato (B25) and Bacillus sp. (B35) displayed the highest antagonistic activity against Fv. Pot experiments performed in a greenhouse with Bacillus cereus sensu lato B25 confirmed these findings and showed a reduction of Fv disease severity and incidence on plants. Antagonistic activity analysis revealed that these strains produce glucanases, proteases or chitinases, as well as siderophores and auxins and suggests these as possible control mechanisms against Fv.

A high-throughput screening assay to identify bacterial antagonists against Fusarium verticillioides.

A high-throughput antagonistic assay was developed to screen for bacterial isolates capable of controlling the maize fungal phytopathogen Fusarium verticillioides. This assay combines a straightforward methodology, in which the fungus is challenged with bacterial isolates in liquid medium, with a novel approach that uses the plant lectin wheat germ agglutinin (WGA) coupled to a fluorophore (Alexa-Fluor® 488) under the commercial name of WGA, Alexa Fluor® 488 conjugate. The assay is performed in a 96-well plate format, which reduces the required laboratory space and streamlines quantitation and automation of the process, making it fast and accurate. The basis of our assay is that fungal biomass can be assessed by WGA, Alexa Fluor® 488 conjugate staining, which recognizes the chitin in the fungal cell wall and thus permits the identification of potential antagonistic bacteria that inhibit fungal growth. This principle was validated by chitin-competition binding assays against WGA, Alexa Fluor® 488 conjugate; confocal laser microscopy confirmed that the fluorescent WGA, Alexa Fluor® 488 conjugate binds to the chitin of the fungal cell wall. The majority of bacterial isolates did not bind to the WGA, Alexa Fluor® 488 conjugate. Furthermore, including washing steps significantly reduced any bacterial staining to background levels, even in the rare cases where bacterial isolates were capable of binding to WGA. Confirmatory conventional agar plate antagonistic assays were also conducted to validate our technique. We are now successfully employing this large-scale antagonistic assay as a pre-screening step for potential fungal antagonists in extensive bacteria collections (on the order of thousands of isolates).

Native soil bacteria isolates in Mexico exhibit a promising antagonistic effect against Fusarium oxysporum f. sp. radicis-lycopersici.

Sinaloa state accounts for 23% of Mexico's tomato production. One constraint on this important crop is the Fusarium crown and root rot, caused by Fusarium oxysporum f. sp. radicis-lycopersici, which has been reported to reduce crop yield by up to 50%. In this study, we set out to identify bacterial populations which could be used to control this disease through natural antagonism. Five tomato rhizospheric soil samples were collected, dried for 1-week, and homogenized. Sub-samples were used to prepare an aqueous solution used to isolate microorganisms in pure cultures. Organisms were purified and grown separately, and used to generate a collection of 705 bacterial isolates. Thirty-four percent from this bank (254 strains) was screened against Forl, finding 27 bacteria displaying in vitro Forl growth inhibition levels from 5% to 60%. These isolates belonged to the genus Bacillus and their 16Sr DNA sequences showed that they are closely related to seven species and they were putatively designated as: B. subtilis, B. cereus, B. amyloliquefaciens, B. licheniformis, B. thuringiensis, B. megaterium, and B. pumilus. One isolate belonged to the genus Acinetobacter. Two B. subtilis isolates (144 and 151) and one B. cereus isolate (171) showed the best antagonistic potential against FCRRT when evaluated on seedlings. Plate and activity assays indicate that these isolates include a diverse repertoire of functional antagonistic traits that might explain their ability to control FCRRT. Moreover, bacteria showed partial hemolytic activity, and future research will be directed at ensuring that their application will be not harmful for humans and effective against Forl in greenhouse or field conditions.

Arsenate induces the expression of fungal genes involved in As transport in arbuscular mycorrhiza.

We utilized the two-compartment system to study the effect of arsenic (As) on the expression of the Glomus intraradices high-affinity phosphate transporter GiPT, and the GiArsA gene, a novel protein with a possible putative role as part of an arsenite efflux pump and similar to ArsA ATPase. Our results show that induction of GiPT expression correlates with As(V) uptake in the extra-radical mycelium of G. intraradices. We showed a time-concerted induction of transcript levels first of GiPT, followed by GiArsA, as well as the location of gene expression using laser microdissection of these two genes not only in the extra-radical mycelium but also in arbuscules. This work represents the first report showing the dissection of the molecular players involved in arbuscular mycorrhizal fungus (AMF)-mediated As tolerance in plants, and suggests that tolerance mediated by AMF may be caused by an As exclusion mechanism, where fungal structures such as the extra-radical mycelium and arbuscules may be playing an important role. Our results extend knowledge of the mechanisms underlying As efflux in arbuscular mycorrhizal fungi and mechanisms related to As tolerance.