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.

Trichoderma koningiopsis Survival on Coated Seeds and Effect on Plant Growth Promotion in Rice (Oryza sativa).

Seed coating is an alternative delivery system for beneficial plant microorganisms into the soil. Although seed coats are widely used for the application of agrochemicals, the incorporation of beneficial microorganisms has not been explored deeply and their survival on seeds while in storage is unknown. The study aimed to evaluate the effect of the coating process on microbial survival and on plant growth promotion. Two coating formulations were designed, and assessed by two coating processes: rotating drum and fluidized bed. The rotating drum process resulted in more uniform coatings than in the fluidized bed process. In addition, with this coating technique, lower viability losses over time were observed. The rotatory drum prototype containing a biopolymer and a clay mineral derivate (P90) showed the best behavior at the three temperatures evaluated, with superior viabilities compared to the other prototypes and the lowest loss of viability after 12 months. The formulation of this coating prototype may preserve the viability of Trichoderma koningiopsis Th003 up to 15 months at 8 °C, 9 months at 18 °C, and 3 months at 28 °C, which are very promising shelf-life results. Regarding the effect of seed coating on plant growth, prototypes showed higher yields > 16% than the control, comparable to the conventional use of Tricotec® WG, which may reduce the number of applications and water consumption for dissolution of the inoculant. The results demonstrated that the formulation composition, as well as the coating process may impact the microbial survival on seeds.

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.

Seed coating as a delivery system for the endophyte Trichoderma koningiopsis Th003 in rice (Oryza sativa).

Seed coating is a technique to cover seeds with external agents to upgrade their performance, handling, and plant establishment. Plant beneficial microbes (PBMs), such as plant growth-promoting bacteria, mycorrhizal fungi, and other fungi (e.g., Trichoderma spp.), decrease agrochemical inputs, enhance tolerance to biotic-abiotic stresses, and increase essential plant nutrition. The demand for pre-treated seeds as delivery systems for biological agents is advancing. Here, a seed coating formulation containing Trichoderma koningiopsis is presented. The physicochemical and biological characterization of the seed coating prototypes included drying protector screening, the effect of the inoculum concentration on survival, the assessment of microbial release profiles in soil extract, and plant tissue colonization capability under semi-controlled conditions. Gelatine and pectin, two of the tested drying protectors, maintained fungus germination after 60 days at 18 °C with significantly higher values of up to 38% compared with the control. The initial concentration of 106 colony-forming units (CFU) per seed undergoes a positive effect on survival over time. Regarding plant tissue colonization, the fungus establishes endophytically in rice. In conclusion, seed coating is a promising alternative for the formulation of beneficial microbial agents such as Trichoderma sp., maintaining cell survival and further promoting the establishment in rice systems.Key points• Enhancing drying survival of T. koningiopsis formulates• Seed coating formulation approach for T. koningiopsis in rice• Colonization capacity of formulated T. koningiopsis in rice tissue.

Effects of lipid formulations on clove extract spray dried powders: comparison of physicochemical properties, storage stability and in vitro intestinal permeation.

Clove is an aromatic plant spice with potent antioxidant and anti-inflammatory activity. Eugenol is the main compound which contributes to such medicinal and nutritional benefits. To date, the formulation of unstable, volatile and poorly water-soluble compounds remains a challenging task. Lipid formulations can be used to improve physicochemical and biopharmaceutical properties of poorly soluble compounds. The aim of this study is to investigate the effects of lipids, such as Gelucire and Compritol on physicochemical properties; stability and in vitro intestinal permeation of spray dried powdered formulations loaded with clove's bioactive compounds. Results showed that eugenol retention in spray-dried powders could be correlated with antioxidant activity and with mass recovery after spray drying. Adding Gelucire but not Compritol to clove extract formulations, improved solubility of spray dried powders. Stability test in high humidity environment (63.5% RH) suggested that formulations including both Gelucire and Compritol were significantly more stable compared to the formulation without any lipid at the two tested temperatures (25 °C and 40 °C). This suggests that lipid additions to clove (Syzygium aromaticum) extract formulations provide protective effects for the spray dried powders in high-humidity environments. In addition, results from in vitro intestinal permeation studies suggested that eugenol uptake, was not being hindered by transporters nor was the absorption being affected by lipid formulations.

Clove (Syzygium aromaticum): a precious spice.

Clove (Syzygium aromaticum) is one of the most valuable spices that has been used for centuries as food preservative and for many medicinal purposes. Clove is native of Indonesia but nowadays is cultured in several parts of the world including Brazil in the state of Bahia. This plant represents one of the richest source of phenolic compounds such as eugenol, eugenol acetate and gallic acid and posses great potential for pharmaceutical, cosmetic, food and agricultural applications. This review includes the main studies reporting the biological activities of clove and eugenol. The antioxidant and antimicrobial activity of clove is higher than many fruits, vegetables and other spices and should deserve special attention. A new application of clove as larvicidal agent is an interesting strategy to combat dengue which is a serious health problem in Brazil and other tropical countries. Pharmacokinetics and toxicological studies were also mentioned. The different studies reviewed in this work confirm the traditional use of clove as food preservative and medicinal plant standing out the importance of this plant for different applications.

Bioactive compounds in Bidens pilosa L. populations: a key step in the standardization of phytopharmaceutical preparations

The total flavonoid content (TFC), total polyphenols content (TPC), and in vitro antioxidant activity (AA) of six Bidens pilosa L., Asteraceae, populations harvested from different localities were evaluated in this work. The plants were separated in roots, stems, and leaves/flowers, and the influence of extraction methods was investigated.Areversed-phase high-performance liquid chromatography method (HPLC) was developed and employed to obtain characteristic HPLC fingerprints of the bioactive compounds present in the extractive solutions, which were correlated with the TFC, TPC, and AA. Extractive solutions of leaves/flowers presented a higher AA when compared with those obtained from other parts of the plant (IC50 of 35.35±0.10 µg/mL). The stem extracts presented the lowest AA (IC50 117.2±1.96 µg/mL). A direct correlation of AA with TFC and TPC was evidenced. The highest AA was obtained by dynamic maceration and was statistically different from the AA presented by the extractive solutions obtained by other extraction methods. The results of this work evidenced differences that can be found at different stages of development of phytopharmaceutical preparations from B. pilosa and highlighted the importance of using the concentration of marker compounds as well as HPLC fingerprints as quality control parameters.