Fourteen fungal entomopathogenic strains were isolated from soil samples and infected field-collected fall armyworm larvae, in Guanajuato, Mexico. Isolates were identified by morphology and internal transcribed spacers sequencing. Isolates Ma22, Ma41, and Mr8 showed 99% identity with reference strains (RS) of Metarhizium anisopliae. Isolates Bb9, Bb19, Bb21, Bb40, Bb27, Bb23, and Bb39 showed identity between 99 and 100% with RS of Beauveria bassiana. Isolates Nr1, Nr2, Nr3, and Nr4 showed identity between 98 and 100% with RS of Nomuraea rileyi. Qualitative selection used one concentration (1 × 108 conidia/ml) on fall armyworm eggs and neonate larvae. Strains Ma22, Ma41, and Mr8 showed 100%, and strains Bb39, Bb23, Bb9, Bb40, Bb19, and Bb21 showed 92, 89.2, 87.6, 82.8, 58, and 38% egg mortality, respectively. Bioassays on neonate larvae showed 100% mortality with strains Ma22, Ma41, Mr8, and Bb9. Strains Bb39, Bb19, Bb27, Bb23, Bb21, and Bb40 showed 74, 60, 54, 53, 28, and 19% mortality, respectively. Bioassay estimated LC50s for strains Ma41 at 7.4 × 104, Mr8 at 8.9 × 104, and Ma22 at 10 × 104 conidia/ml, on fall armyworm eggs. LC50s on neonate larvae were estimated at 2.8 × 105, 16 × 105, 26 × 105, and 36 × 105 conidia/ml for strains Ma41, Bb9, Ma22, and Mr8, respectively. Virulence genes mad1 and mad2 were found in Mr8, Ma22, and Ma41, whereas the gen gmact was found only in the strain Ma22. Genes hyd1 and hyd2 were identified in Bb9, Bb19, Bb21, and Bb27. No correlation was observed between the virulence gene detection and the estimated LC50s. Strain Ma41 showed the highest potential to be developed as a bioinsecticide.

Somatic embryogenesis (SE) is the most important plant biotechnology process for plant regeneration, propagation, genetic transformation and genome editing of coffee, Coffea arabica L. Somatic embryo (SEs) conversion to plantlets is the principal bottleneck for basic and applied use of this process. In this study we focus on the maturation of SEs of C. arabica var. Typica. SEs conversion to plantlet up to 95.9% was achieved under osmotic stress, using 9 g/L gelrite, as compared with only 39.34% in non-osmotic stress. Mature SEs induced in osmotic stress developed shoot and root apical meristems, while untreated SEs were unable to do it. C. arabica regenerated plants from osmotic stress were robust, with higher leaf and root area and internode length. To understand a possible regulatory mechanism, gene expression of key genes of C. arabica, homologous to sequences in the Arabidopsis thaliana genome, were analyzed. A set of two component system and cytokinin signaling-related coding genes (AHK1, AHK3, AHP4 and ARR1) which interact with WUSCHEL and WOX5 homedomains and morphogenic genes, BABY-BOOM, LEC1, FUS3 and AGL15, underwent significant changes during maturation of SEs of C. arabica var. Typica. This protocol is currently being applied in genetic transformation with high rate of success.

The effects of soil depth, soil type and temperature on the activity of the nematode Steinernema carpocapsae (Filipjev) were examined using larvae of the West Indian fruit fly, Anastrepha obliqua (Macquart). Bioassays involved applying infective juveniles (IJs) to the surface of sterilized sand in PVC tubes previously inoculated with fly larvae of two ages. The 50% lethal concentration (LC₅₀) values estimated for 6-day-old larvae were 9, 20 and 102 IJs/cm² in tubes containing 2, 5 and 8 cm depth of sand, respectively, whereas for 8-day-old larvae, LC₅₀ values were 16, 40 and 157 IJs/cm², respectively. The effect of soil texture on the activity of S. carpocapsae was tested by applying the corresponding LC₅₀ concentrations of nematodes to sand, sand–clay and loamy–sand soils. For 6-day-old larvae, soil type had a highly significant effect on infection with the highest percentages of infection observed in the sand–clay mixture (60–82% depending on depth) compared to 45–64% infection in sand and 23–30% infection in loamy–sand soil. A very similar pattern was observed in 8-day-old larvae except that infection rates were significantly lower than in younger larvae. There was a significant interaction between soil type and soil depth. The effect of three temperatures (19, 25 and 30°C) on infection was examined in sand–clay soil. The infectivity of S. carpocapsae was affected by temperature and soil depth and by the interaction of these two factors. Response surface analysis applied to second order multiple linear regression models indicated that the optimal temperature for infection of larvae of both ages was ~26°C, at a depth of 7.9 cm for 6-day-old larvae and <2 cm for 8-day-old larvae, resulting in a predicted 91.4% infection of 6-day-old larvae and 61.2% infection of 8-day-old larvae. These results suggest that S. carpocapsae may have the potential to control fruit fly pests in tropical ecosystems with warm temperatures and high soil moisture levels, although this assertion requires field testing.

The objective of the present work was to develop a method for the preservation of T. harzianum conidia at room temperature and the immobilised conidia propagation in submerged culture. This was accomplished by immobilising the strain in sodium alginate capsules (white capsules) and subsequently propagating them in a column bubble reactor (green capsules). Three capsule diameters were tested (micro, medium and large capsules), which were produced by emulsion internal gelation and dripping methods. Tested variables were the immobilised conidia propagation in submerged culture for free conidia production, the immobilised conidia viability throughout the time (two years), the resistance of the encapsulated conidia to the UV irradiation of short and long wavelength, and the antagonistic effect of the encapsulated T. harzianum against four phytopathogenic fungi. It was found that the medium capsules (1.5 ± 0.3 mm) favoured the massive production of released conidia in submerged culture and that the higher the density of conidia per capsule, the greater the protection against the ultraviolet irradiation. Regarding the conidia preservation in calcium alginate, a viability loss of around 30% was observed two years after storage at environmental temperature in both white and green capsules; along the two years that the viability of conidia was analyzed, the purity of the formulation was corroborated. The results presented here show the efficacy of the green and white capsules for T. harzianum preservation at room temperature for a long period of time.

Bacillus thuringiensis has been widely used as a biological control agent against insect pests. Additionally, nematicidal strains have been under investigation. In this report, 310 native strains of B. thuringiensis against Caenorhabditis elegans were tested. Only the LBIT-596 and LBIT-107 strains showed significant mortality. LC₅₀s of spore-crystal complexes were estimated at 37.18 and 31.89 μg/ml for LBIT-596 and LBIT-107 strains, respectively, while LC₅₀s of partially purified crystals was estimated at 23.76 and 20.25 μg/ml for LBIT-596 and LBIT-107, respectively. The flagellin gene sequence and plasmid patterns indicated that LBIT-596 and LBIT-107 are not related to each other. Sequences from internal regions of a cry5B and a cyt1A genes were found in the LBIT-596 strain, while a cry21A, a cry14A, and a cyt1A genes were found in the LBIT-107 strain. Genome sequence of the LBIT-107 strain showed new cry genes, along with other virulence factors, hence, total nematicidal activity of the LBIT-107 strain may be the result of a multifactorial effect. The highlight of this contribution is that translocation of spore-crystal suspensions of LBIT-107 into tomato plants inoculated at their rhizosphere decreased up to 90% the number of galls of Meloidogyne incognita, perhaps the most important nematode pest in the world.

This report presents an efficient protocol of the stable genetic transformation of coffee plants expressing the Cry10Aa protein of Bacillus thuringiensis. Embryogenic cell lines with a high potential of propagation, somatic embryo maturation, and germination were used. Gene expression analysis of cytokinin signaling, homedomains, auxin responsive factor, and the master regulators of somatic embryogenesis genes involved in somatic embryo maturation were evaluated. Plasmid pMDC85 containing the cry10Aa gene was introduced into a Typica cultivar of C. arabica L. by biobalistic transformation. Transformation efficiency of 16.7% was achieved, according to the number of embryogenic aggregates and transgenic lines developed. Stable transformation was proven by hygromycin-resistant embryogenic lines, green fluorescent protein (GFP) expression, quantitative analyses of Cry10Aa by mass spectrometry, Western blot, ELISA, and Southern blot analyses. Cry10Aa showed variable expression levels in somatic embryos and the leaf tissue of transgenic plants, ranging from 76% to 90% of coverage of the protein by mass spectrometry and from 3.25 to 13.88 μg/g fresh tissue, with ELISA. qPCR-based 2^−ΔΔCt trials revealed high transcription levels of cry10Aa in somatic embryos and leaf tissue. This is the first report about the stable transformation and expression of the Cry10Aa protein in coffee plants with the potential for controlling the coffee berry borer.