Evaluation of conidia production and mycelial growth in solid culture media from native strains of entomopathogenic fungi isolated from citrus-growing areas of México

It is important to know the ability of native strains to carry out the process of sporulation and growth in different conditions and to determine their possible potential as biological control of pests of agricultural importance, mainly in citrus areas. The objective of this study was to evaluate five different solid culture media for the determination of the production of conidia and mycelial growth of twenty-five native isolates from the Mexican states of Sinaloa, San Luis Potosí, Nuevo León and Tamaulipas, and three collection strains using five different solid culture media. The results showed no statistical significant difference between the five media tested for the production of conidia per milliliter and mycelial growth among different isolates. However between isolates a significant difference was found. For conidia per milliliter, the isolate with the highest production of conidia was HIB-4 with an average of 4.85 x 109 (spores/ml). With respect to mycelial growth strain Ma presented the highest value with 8.06 cm on average.Key words: Entomopathogenic fungi, esporulation, conidia, mycelial growth

Optimization of Pathogenicity Tests for Selection of Native Isolates of Entomopathogenic Fungi Isolated from Citrusgrowing Areas of México on Adults ofDiaphorina citriKuwayama (Hemiptera: Liviidae)

Huanglonbing (HLB), es considerado una de las más letales enfermedades de los cítricos alrededor del mundo, y ha alcanzado las principales áreas de producción de limón Mexicano (Citrus latifolia Tanaka) en la costa del pacifico de México. Los productores han iniciado el uso de insecticidas para controlar las poblaciones del psílido asiático de los cítricos, Diaphorina citri Kuwayama (Hemiptera: Liviidae), el vector del patógeno ‘Candidatus Liberibacter asiaticus’ asociado con el HLB. Actualmente los costos de los insecticidas y los efectos secundarios de su uso son las principales preocupaciones, ya que podrían perjudicar la estrategia de manejo contra el vector; y por lo tanto, alternativas ecológicas y económicamente viables a los insecticidas convencionales serian necesarias a corto plazo. Por tanto, el objetivo de este estudio fue evaluar la patogenicidad de 27 aislados nativos y 3 cepas de hongos entomopatógenos para determinar su potencial como agentes de control biológico sobre Diaphorina citri usando 2 diferentes métodos de bioensayo. Los bioensayos fueron realizados bajo condiciones de laboratorio (26 ± 2 °C, 60 ± 5% H.R y 16:8 h L:O) mediante la exposición de insectos adultos a una concentración de 1 × 108 conidios por mililitro utilizando 2 diferentes métodos de aplicación, es decir, por asperjado de esporas en las plántulas de cítricos y por asperjado directo a los psílidos adultos. Los resultados mostraron que para el asperjado directo a los adultos los aislados HIB-24 (B. bassiana) y HIB-32 (I. fumosorosea) mostraron el mayor porcentaje de mortalidad (60.66%). Respecto al asperjado de plántulas el aislado HIB-19 (I. fumosorosea) mostró el mayor porcentaje de mortalidad (62.02%). Los resultados de este estudio demuestran el potencial para el uso de hongos entomopatógenos en el manejo de D. citri en México. ABSTRACT Huanglongbing (HLB), considered one of the most lethal diseases of citrus worldwide, has reached the main areas of Mexican lime (Citrus latifolia Tanaka) fruit production on the Pacific coast of México. Growers have initiated intensive use of insecticides in order to control populations of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), the vector of the pathogen, ‘Candidatus Liberibacter asiaticus’ associated with huanglongbing. Presently, costs of insecticides and the side effects of their use are major concerns, because they could impair the management strategy against the vector; and thus, ecologically and economically viable alternatives to conventional insecticides are required in the short term. Therefore the goal of this study was to evaluate the pathogenicity of 27 native isolates and 3 strains of entomopathogenic fungi and determine their potential as biological control agents of D. citri by using 2 different bioassay methods. Bioassays were performed under laboratory conditions (26 ± 2 °C, 60 ± 5% RH and 16:8 h L:D) by exposing adult insects to a concentration of 1 × 108 conidia per milliliter using 2 different application methods, i.e., spraying the spores onto the citrus seedlings and spraying the spores directly onto the adult psyllids. The results showed that by direct spraying the adults, HIB-24 (B. bassiana) and HIB-32 (I. fumosorosea) isolates showed the highest mortality (60.66%). Regarding spraying of the seedlings, HIB-19 (I. fumosorosea) showed the highest percentage of mortality (62.02%). The results from this study demonstrate potential for using entomopathogenic fungi in the management of D. citri in México

Attaching and effacing (A/E) lesion formation by enteropathogenic E. coli on human intestinal mucosa is dependent on non-LEE effectors

Enteropathogenic E. coli (EPEC) is a human pathogen that causes acute and chronic pediatric diarrhea. The hallmark of EPEC infection is the formation of attaching and effacing (A/E) lesions in the intestinal epithelium. Formation of A/E lesions is mediated by genes located on the pathogenicity island locus of enterocyte effacement (LEE), which encode the adhesin intimin, a type III secretion system (T3SS) and six effectors, including the essential translocated intimin receptor (Tir). Seventeen additional effectors are encoded by genes located outside the LEE, in insertion elements and prophages. Here, using a stepwise approach, we generated an EPEC mutant lacking the entire effector genes (EPEC0) and intermediate mutants. We show that EPEC0 contains a functional T3SS. An EPEC mutant expressing intimin but lacking all the LEE effectors but Tir (EPEC1) was able to trigger robust actin polymerization in HeLa cells and mucin-producing intestinal LS174T cells. However, EPEC1 was unable to form A/E lesions on human intestinal in vitro organ cultures (IVOC). Screening the intermediate mutants for genes involved in A/E lesion formation on IVOC revealed that strains lacking non-LEE effector/s have a marginal ability to form A/E lesions. Furthermore, we found that Efa1/LifA proteins are important for A/E lesion formation efficiency in EPEC strains lacking multiple effectors. Taken together, these results demonstrate the intricate relationships between T3SS effectors and the essential role non-LEE effectors play in A/E lesion formation on mucosal surfaces

Anticancer potential of Thevetia peruviana fruit methanolic extract

Abstract Background: Thevetia peruviana (Pers.) K. Schum or Cascabela peruviana (L.) Lippold (commonly known as ayoyote, codo de fraile, lucky nut, or yellow oleander), native to Mexico and Central America, is a medicinal plant used traditionally to cure diseases like ulcers, scabies, hemorrhoids and dissolve tumors. The purpose of this study was to evaluate the cytotoxic, antiproliferative and apoptotic activity of methanolic extract of T. peruviana fruits on human cancer cell lines. Methods: The cytotoxic activity of T. peruviana methanolic extract was carried out on human breast, colorectal, prostate and lung cancer cell lines and non-tumorigenic control cells (fibroblast and Vero), using the MTT assay. For proliferation and motility, clonogenic and wound-healing assays were performed. Morphological alterations were monitored by trypan blue exclusion, as well as DNA fragmentation and AO/EB double staining was performed to evaluate apoptosis. The extract was separated using flash chromatography, and the resulting fractions were evaluated on colorectal cancer cells for their cytotoxic activity. The active fractions were further analyzed through mass spectrometry. Results: The T. peruviana methanolic extract exhibited cytotoxic activity on four human cancer cell lines: prostate, breast, colorectal and lung, with values of IC50 1.91 ± 0.76, 5.78 ± 2.12, 6.30 ± 4.45 and 12.04 ± 3.43 μg/mL, respectively. The extract caused a significant reduction of cell motility and colony formation on all evaluated cancer cell lines. In addition, morphological examination displayed cell size reduction, membrane blebbing and detachment of cells, compared to non-treated cancer cell lines. The T. peruviana extract induced apoptotic cell death, which was confirmed by DNA fragmentation and AO/EB double staining. Fractions 4 and 5 showed the most effective cytotoxic activity and their MS analysis revealed the presence of the secondary metabolites: thevetiaflavone and cardiac glycosides. Conclusion: T. peruviana extract has potential as natural anti-cancer product with critical effects in the proliferation, motility, and adhesion of human breast and colorectal cancer cells, and apoptosis induction in human prostate and lung cancer cell lines, with minimal effects on non-tumorigenic cell lines. Keywords: Cytotoxic activity, Anti-proliferative activity, Motility, Apoptosis, Human cancer cells, Flavonoid, Cardiac glycoside

Draft Genome Sequence of Bacillus toyonensis Strain GM18, Isolated from Agricultural Soil

Bacillus toyonensis is a recently described species related to Bacillus cereus and Bacillus thuringiensis. The GM18 strain previously identified as B. thuringiensis is now classified as B. toyonensis based on the RNA 16S sequence and whole-genome average nucleotide identity. The genome analysis revealed the presence of insecticide, nemati�cide, and antitumoral protein

In vitro anticancer activity of methanolic extract of Granulocystopsis sp., a microalgae from an oligotrophic oasis in the Chihuahuan desert

With the purpose of discovering new anticancer molecules that might have fewer side effects or reduce resistance to current antitumor drugs, a bioprospecting study of the microalgae of the Cuatro Cienegas Basin (CCB), an oasis in the Chihuahuan desert in Mexico was conducted. A microalgae was identified as Granulocystopsis sp. through sequencing the rbcL gene and reconstruction of a phylogenetic tree, and its anticancer activities were assessed using various in vitro assays and different cell lines of human cancers, including lung, skin melanoma, colorectal, breast and prostatic cancers, as well as a normal cell line. The values of IC50 of the microalgae methanolic extract using the MTT assay were lower than 20 μg/ml, except that in the lung cancer line and the normal cell line. In vitro, the microalgae extract caused the loss of membrane integrity, monitored by the trypan blue exclusion test and exhibited marked inhibition of adhesion and cell proliferation in cancer cell lines, through the evaluation of the clonogenic assay. Also, typical nuclear changes of apoptotic processes were observed under the microscope, using the dual acridine orange/ethidium bromide fluorescent staining. Finally, the microalgae extract increased the activity of caspases 3 and 7 in skin melanoma, colon, breast and prostate cancer cells, in the same way as the apoptotic inductor and powerful antitumoral drug, doxorubicin. This study shows the anticancer activity from Granulocystopsis sp., a microalgae isolated from the CCB

Bioinsecticidas: su empleo, producción y comercialización en México

Una de las ventajas de la biotecnología agrícola ha sido el impulso del empleo de bioinsecticidas, los cuales tienen como objetivo primordial proteger al ambiente y mejorar la calidad de los alimentos. La industria de los bioinsecticidas incluye agentes microbianos de control (bacterias, hongos, virus, protozoarios y nemátodos), organismos entomófagos (depredadores y parasitoides), compuestos orgánicos y organismos y plantas transgénicas. Se tiene estimado que en México el empleo de Bacillus thuringiensis para control de plagas en campo es de un 4% al 10% del total de insecticidas, y la producción y el empleo de diferentes hongos entomopatógenos y organismos entomófagos es cada vez mayor, principalmente en las regiones agrícolas. Además, se ha iniciado la producción de otro tipo de bacterias, baculovirus, nemátodos y extractos de plantas, aunque con un mercado nacional muy pequeño. Para incrementar el consumo de bioinsecticidas en México se requiere de mayor investigación científica y de factibilidad económica, que se enfoquen a mejorar la producción y calidad de los mismos, con el fin de garantizar la efectividad del producto final

Elaboración de un bioinsecticida contra el gusano barrenador de la caña de azucar

La elaboración de una formulación a base de Bacillus thuringiensis, que incluya polímeros biodegradables y fagoestimulantes, se platea como una alternativa para el control del barrenador de la caña de azúcar Diatraea saccharalis. La formulación óptima fue: almidón- gelatina-caña de azúcar en polvo a la cual se le adicionó la cepa GM34 seleccionada por su alta toxicidad. Los formulados fueron evaluados a nivel de laboratorio, donde dieron óptimos resultados, y a nivel de campo, donde el mejor formulado resultó ser el asperjable con una concentración menor que los formulados comerciales, dando esto una ventaja para su posible comercialización

Electrospun Polyvinylpyrrolidone-Gelatin and Cellulose Acetate Bi-Layer Scaffold Loaded with Gentamicin as Possible Wound Dressing

Acceleration of wound healing can be achieved with the use of wound dressings. Through the electrospinning technique, a polymeric scaffold composed of two layers was processed: a gelatin and polyvinylpyrrolidone layer with gentamicin, and a second layer of cellulose acetate. The conditions for the electrospinning process were standardized for voltage parameters, feed flow and the distance from the injector to the collector. Once the values of the main variables for the electrospinning were optimized, a three-hour processing time was established to allow the separation of the material from the collector. The obtained material was characterized by observations on scanning electron microscopy, Fourier transform infrared spectroscopy and thermal analysis; contact angle measurement was performed to evaluate wettability properties, and antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus were evaluated using the Kirby–Bauer test. The obtained fibers that form the bi-layer scaffold present diameters from 100 to 300 nm. The scaffold presents chemical composition, thermal stability, wettability characteristics and antibacterial activity that fulfill the proposal from this study, based on obtaining a scaffold that could be used as a drug delivery vehicle and a wound dressing material