Performance of Metarhizium rileyi Nm017: nutritional supplementation to improve production and quality conidia.

This study aimed to analyze the effect of nutritional supplements on improving conidia production of Metarhizium rileyi Nm017 at laboratory scale (yields of conidia/substrate and biomass/substrate, and substrate consumption). Also, the influence on quality parameters were evaluated (germination at 36 and 48 h, enzymatic activity, and insecticidal activity on Helicoverpa zea). Six treatments (T1-T6) were assessed and all of them reached maximum conidia concentration after 7 days fermentation, a feasible production timetable. Yields from treatment T6 (yeast extract + V8 juice) were 1.5-threefold higher than the other treatments. Conidia from T6 reached germinations of 56% and 12% at 36 and 48 h, respectively, higher than T1 (without supplements), which had the lowest values found. M. rileyi conidia obtained from treatment T6 had the highest enzymatic activity (0.45 U chitinase g-1, 0.28 U lipase g-1, and 1.29 U protease g-1). However, treatments with the highest conidia yields and enzymatic activity were not positively correlated to the efficacy against H. zea. When M. rileyi was produced on T5 (yeast hydrolysate + V8 juice), conidia were 35% more virulent than treatment T6. The findings evidenced the noticeable impact of nutritional substrate amended for conidia production and quality. This work showed the relevance of insecticidal activity assessment as a selection criterion in the mass production development of a biocontrol agent.

Hydrogel capsules as new approach for increasing drying survival of plant biostimulant gram-negative consortium.

Several plant growth-promoting bacteria (PGPB) are gram-negative, and their cell viability is affected during the bio-inoculant production. Hence, formulation-drying processes provide challenges that limit the adoption of these beneficial microorganisms in sustainable agricultural production. Among delivery system strategies for gram-negative PGPB, the encapsulating cells in biopolymeric materials are emerging as a promising alternative. This research aims to evaluate the effect of additives and crosslinking agents on the survival of the consortium of Herbaspirillum frisingense AP21, Azospirillum brasilense D7, and Rhizobium leguminosarum T88 in hydrogel capsules. Three crosslinkers and diverse potential drying protectors were tested. Calcium gluconate provides notable consortium survival advantages regarding colony-forming units (CFUs) (losses of up to 4 log CFU) compared to calcium lactate and calcium chloride (up to 6 log CFU). Additives such as skimmed milk, whey protein, and Gelita® EC improve the recovery of viable cells after the drying process, demonstrating an increase in cell survival of the three bacteria by up to 4 log CFU. The combination of these substances into a capsule prototype extends the storage stability of bacterial consortium up to 3 months at 18 ± 2 °C. This study expands the knowledge for formulating gram-negative PGPB consortium, regarding the crosslinker and drying protector relationship on encapsulation processes with drying survival and further storage stability performance. KEY POINTS: • Hydrogel immobilization formulation approach for PGPB consortium • Enhancing drying survival of gram-negative PGPB consortium • Increasing storage stability of PGPB consortium at 18 °C.

Nematode predatory ability of the fungus Duddingtonia flagrans affected by in vitro sequential exposure to ovine gastrointestinal tract.

Duddingtonia flagrans is a nematophagous fungus employed as a biocontrol agent of gastrointestinal nematodes in ruminants. After oral ingestion and passage through the digestive tract of animals, this microorganism captures the nematodes in the feces. The drastic conditions of ruminant digestive tract could affect fungi chlamydospores and therefore biocontrol activity. The aim of this study was to evaluate in vitro the effect of four ruminant digestive segments on the concentration and nematode predatory ability of a Colombian native strain of D. flagrans. The sequential four-step methodology proposed evaluated conditions of the oral cavity, rumen, abomasum, and small intestine such as pH (2, 6, 8), enzymes (pepsin, pancreatin), temperature (39 °C), and anaerobiosis comparing short (7 h) and long (51 h) exposure times. The results showed that the nematode predatory ability of the fungi is affected by sequential exposure to gastrointestinal segments and this effect depends on the exposure time to those conditions. After short exposure (7 h) through the four ruminant digestive segments, the fungi had a nematode predatory ability of 62%, in contrast, after long exposure (51 h) the nematode predatory ability was lost (0%). Moreover, the number of broken chlamydospores was higher in the long-exposure assay.

Assessment of a potential bioproduct for controlling Cerotoma arcuata tingomariana (coleoptera: Chrysomelidae).

AIMS: The leaf-feeding pest Cerotoma arcuata tingomariana (Bechyné) (Coleoptera: Chrysomelidae) produces huge economic losses in different crops. This study aimed to produce conidia by semisolid-state fermentation and to establish the insecticidal activity of two formulation prototypes based on a native Beauveria bassiana isolate for controlling this pest. METHODS AND RESULTS: A novel fabric-based semisolid-state fermentation strategy for quick and large-scale conidia production was performed and characterized. Conidia were formulated as an emulsifiable concentrate (EC) and a water-dispersible granulate (WG). Afterwards, the mortality of C. a. tingomariana adults was assessed. A conidia concentration of 2.9 × 109 conidia cm-2 was obtained after 9 days-course fermentation and a yield of 33.4 g kg-1 dry-substrate. CONCLUSIONS: The polyester fabric-based fermentation is an efficient technique for producing and collecting B. bassiana spores. Regarding LC90 , the potency analysis showed that the EC was 21-fold more potent than the non-formulated conidia, and ~ 2.6-fold more potent than the WG. SIGNIFICANCE AND IMPACT OF STUDY: A high throughput fermentation based on polyester fabric as support for B. bassiana conidia production and subsequent formulation as an EC comprises a promising strategy for obtaining a bioproduct to control adults of C. a. tingomariana and other Chrysomelidae pests.

Formulation Process Analysis of a Virus-based Biopesticide to Control the Tomato leafminer Tuta absoluta

Abstract: Virus-based biopesticides are effective biocontrol agents of crop insect pests. Development of suitable formulations and production processes are necessary to obtain high-quality products easily adopted by farmers. A detailed unit operation study was carried out for the production process of a Phthorimaea operculella granulovirus-based biopesticide to control the tomato leafminer, Tuta absoluta, one of the most important pests affecting this crop. Physicochemical, microbiological, and insecticidal parameters were implemented in the process and applied to the finished product, and a scaling strategy was developed. A Quantitative Polymerase Chain Reaction (Q-PCR) technique was implemented to quantify viral concentrations in the active ingredient (5.34 ± 1.44 x109 Occlusion Bodies mL-1) and in the finished product (>1.6x109 OB mL-1), without contaminant interferences. The Q-PCR methodology was also useful to select the appropriate solid mixing time following Lacey´s mixing index (8 min). Factors and similarity principles influencing the liquid mixing process were identified in the scaling evaluation. Furthermore, the drying kinetics analysis enabled identifying a drying temperature of 35 °C, with an efficacy under controlled conditions higher than 97%. Contaminant concentration was lower than 1%, indicating controlled and aseptic formulation process conditions. A simple statistical method was used to estimate the reproducibility and repeatability of the parameters assessed in the finished product. These results enable to establish and extrapolate important parameters in the standardization, scale-up, and quality control for the granulovirus-based biopesticide.

In vitro evaluation of physicochemical variables on the nematophagous fungus Duddingtonia flagrans.

Duddingtonia flagrans is a biological alternative to the use of anthelmintic drugs in ruminants. This fungus must be ingested by the animal, pass through the cavities of the digestive tract and reach the feces where it develops traps that capture the nematodes. The severe conditions encountered in this process negatively affect the fungus, which is reflected in the low recovery rates compared to the amount administered. The aim of this study was to evaluate independently the in vitro effect of typical physical and chemical conditions of the gastrointestinal cavities of ruminants on the concentration, viability, and the in vitro nematode predatory ability of the chlamydospores of D. flagrans. The factors evaluated individually were pH (2, 6, and 8), temperature (28 ± 2°C and 39 ± 2°C), exposure to artificial saliva, and milling. The results showed that the concentration and viability of D. flagrans were not affected by the action of pH, temperature, milling, or exposure to artificial saliva. Regarding the in vitro nematode predatory ability, a reduction was observed after the milling process and the exposure for 24 h at different pH.

Scale-Up of Duddingtonia flagrans Chlamydospores Production from Laboratory to Pilot-Scale Using a Solid-State Fermentation System.

The fungus Duddingtonia flagrans is a biological control tool to reduce infective larvae of gastrointestinal nematode in pastures. To create a commercially available bioproduct based on a nematophagous fungus, an efficient mass production process should be developed that is able to guarantee a good predatory capacity and satisfactory production rates. In this work, solid-state fermentation (SSF) parameters were investigated to produce D. flagrans at pilot-scale. The results showed that the relative humidity was a critical factor to increase productivity and to reduce fermentation time. The best production conditions using a tray bioreactor were a relative humidity in the room at 90% for 2 days, and inoculation by sprinkling. The fermentation process was composed of 7 days under submerged fermentation to produce the inoculum and 7 more days of SSF in a tray bioreactor. The productivity reached was 4.96 × 106 chlamydospores g-1 of dry substrate day-1, which is the highest productivity reported to date. The predatory capacity of the chlamydospores produced using this process was 91%. Also, a statistical control process analysis was applied, finding that the process presents stability in the biological activity, yield, and final moisture content of the substrate between batches. Finally, the operational expenses (OPEX) based on the use of the heating and humidification system were estimated, given a final cost of 0.20 USD g-1 of the fermented substrate.

A Framework for the Selection of Plant Growth-Promoting Rhizobacteria Based on Bacterial Competence Mechanisms.

The use of plant growth-promoting rhizobacteria (PGPR) is increasingly meaningful for the development of more environmentally friendly agricultural practices. However, often the PGPR strains selected in the laboratory fail to confer the expected beneficial effects when evaluated in plant experiments. Insufficient rhizosphere colonization is pointed out as one of the causes. With the aim of minimizing this inconsistency, we propose that besides studying plant growth promotion traits (PGP), the screening strategy should include evaluation of the microbial phenotypes required for colonization and persistence. As a model, we carried out this strategy in three Rhizobium sp. strains that showed phosphorus solubilization ability and production of siderophores. All strains displayed colonization phenotypes like surface spreading, resistance to hydrogen peroxide, and formed biofilms. Regarding their ability to persist, biofilm formation was observed to be influenced by pH and the phosphorus nutrient provided in the growth media. Differences in the competence of the strains to use several carbon substrates were also detected. As part of our framework, we compared the phenotypic characteristics of the strains in a quantitative manner. The data analysis was integrated using a multicriteria decision analysis (MCDA). All our results were scored, weighted, and grouped as relevant for PGP, colonization, or persistence. MCDA demonstrated that, when the phenotypes related to PGP and colonization are weighted over those for persistence, strain B02 performs better than the other two Rhizobium sp. strains. The use of our framework could assist the selection of more competent strains to be tested in greenhouse and field trials.IMPORTANCE Numerous plant growth-promoting rhizobacteria (PGPR) have been inoculated into the soil with the aim of improving the supply of nutrients to crop plants and decreasing the requirement of chemical fertilizers. However, sometimes these microbes fail to competitively colonize the plant roots and rhizosphere. Hence, the plant growth promotion effect is not observed. Here, we describe a new screening strategy aiming at the selection of more competent PGPR. We evaluated bacterial phenotypes related to plant growth promotion, colonization, and persistence. Our results demonstrated that despite the fact that our Rhizobium sp. strains successfully solubilized phosphorus and produced siderophores, their abilities to spread over surfaces, resist hydrogen peroxide, and form biofilms varied. Additionally, a multicriteria decision analysis was used to analyze the data that originated from bacterial physiological characterizations. This analysis allowed us to innovatively evaluate each strain as a whole and compare the performances of the strains under hypothetical scenarios of bacterial-trait requirements.

Retraso diagnóstico de la infección por el Virus de Inmunodeficiencia Humana / Diagnostic delay of infection with Human Immunodeficiency Virus

Objetivo. Analizar el comportamiento de la oportunidad diagnóstica evaluando Retrasos Diagnósticos (RD) y diagnós-ticos con Presentación en Etapa Avanzada (PEA) en casos VIH/SIDA. Materiales y metodos. Estudio descriptivo retrospecti-vo de diagnósticos de la infección por el virus del VIH en insti-tuciones de atención en salud. Se incluyeron los casos nuevos o incidentes diagnosticados entre el 1 enero 2011 a 31 de diciembre de 2016. Se rotuló como Retraso Diagnóstico (RD) aquellos que obtuvieron CD4 menor de 350, Presentación con Enfermedad Avanzada (PEA) por CD4 menor de 200, y una categoría adicional de CD4 menor de 500 utilizada como patrón de referencia. Resultados. Se ingresaron 188 inciden-tes todos con resultados de recuento de linfocitos CD4; la me-diana de edad fue de 30 (Rango Intercuartil 23-40), mediana de CD4 de 230. En cuanto a la interpretación de los retrasos 43.6% de los casos presentó PEA, 68.6% clasificó para RD. La proporción de RD y PEA fue mayor entre los hombres y en los grupos etarios de mayor edad; según el modo de transmisión predominó el sexual, siendo el RD y PEA más frecuente entre Hombres que tienen Sexo con Hombres (HSH) contrastando con lo observado en otros estudios, situación que denota difi-cultades de focalización de los sujetos en riesgo. Conclusión. Los casos que están siendo diagnosticados en la fase final corresponden a individuos de mayor edad, de sexo masculino con antecedentes de prácticas de HSH; los hallazgos permi-ten identificar factores que influyen en la falta de oportunidad para el diagnóstico, falencias la gestión en salud pública en lo relacionado con la prevención y control del VIH en pobla-ciones claves.

Objective. To analyze the behavior of the diagnostic opportunity by evaluating Diagnostic Delays (RD) and diagnoses with Advanced Stage Presentation (PEA) in HIV / AIDS cases. Materials and methods. Retrospective descriptive study of diagnoses of infection by the HIV virus in health care institutions. New cases or incidents diagnosed between January 1, 2011 and December 31, 2016 were included. Diagnostic delay (RD) was labeled as those that obtained CD4 less than 350, Presentation with Advanced Disease (PEA) by CD4 less than 200, and an additional category of CD4 less than 500 used as reference standard. Results. 188 incidents were entered all with results of CD4 lymphocyte count; the median age was 30 (Interquartile range 23-40), median CD4 of 230. Regarding the interpretation of the delays 43.6% of the cases presented PEA, 68.6% classified for RD. The proportion of RD and PEA was higher among men and in the older age groups; according to the mode of transmission, sexuality predominated, with the RD and PEA being more frequent among Men who have Sex with Men (MSM), in contrast to what has been observed in other studies, a situation that denotes difficulties in targeting subjects at risk. Conclusion. The cases that are being diagnosed in the final phase correspond to older individuals, male with a history of MSM practices; the findings allow to identify factors that influence the lack of opportunity for diagnosis, failures in public health management in relation to the prevention and control of HIV in key populations

Microencapsulation of a Colombian Spodoptera frugiperda Nucleopolyhedrovirus with Eudragit® S100 by spray drying

A Colombian Spodoptera frugiperda nucleopolyhedrovirus NPV003 with high potential for the development of an efficient biopesticide was microencapsulated by spray drying with a pH dependent polymer (Eudragit® S100). Conditions for microparticles production were standardized and microencapsulation process was validated. Physical properties, insecticide activity and photo-stability of microencapsulated virus were determined. The microparticles were spherical and irregular shaped, with sizes between 17.64 and 19.47 µm. Moisture content was 10.38 ± 0.87%; encapsulation efficiency 84.61± 13.09% and process yield was 91.20 ± 6.40%. Microencapsulation process did not affect viral insecticidal activity and provided efficient protection against UVB radiation. Results demonstrated technological feasibility of spray drying process to be used in formulating a biopesticide based on NPV003.