SUMMARY: The genusTrichodermacontains fungi with high relevance for humans, with applications in enzyme production for plant cell wall degradation and use in biocontrol. Here, we provide a broad, comprehensive overview of the genomic content of these species for “hot topic” research aspects, including CAZymes, transport, transcription factors, and development, along with a detailed analysis and annotation of less-studied topics, such as signal transduction, genome integrity, chromatin, photobiology, or lipid, sulfur, and nitrogen metabolism inT. reesei,T. atroviride, andT. virens, and we open up new perspectives to those topics discussed previously. In total, we covered more than 2,000 of the predicted 9,000 to 11,000 genes of eachTrichodermaspecies discussed, which is >20% of the respective gene content. Additionally, we considered available transcriptome data for the annotated genes. Highlights of our analyses include overall carbohydrate cleavage preferences due to the different genomic contents and regulation of the respective genes. We found light regulation of many sulfur metabolic genes. Additionally, a new Golgi 1,2-mannosidase likely involved inN-linked glycosylation was detected, as were indications for the ability ofTrichodermaspp. to generate hybrid galactose-containingN-linked glycans. The genomic inventory of effector proteins revealed numerous compounds unique toTrichoderma, and these warrant further investigation. We found interesting expansions in theTrichodermagenus in several signaling pathways, such as G-protein-coupled receptors, RAS GTPases, and casein kinases. A particularly interesting feature absolutely unique toT. atrovirideis the duplication of the alternative sulfur amino acid synthesis pathway.

Trichoderma spp. can establish beneficial interactions with plants by promoting plant growth and defense systems, as well as, antagonizing fungal phytopathogens in mycoparasitic interactions. Such interactions depend on signal exchange between both participants and can be mediated by effector proteins that alter the host cell structure and function, allowing the establishment of the relationship. The main purpose of this work was to identify, using computational methods, candidates of effector proteins from T. virens, T. atroviride and T. reesei, validate the expression of some of the genes during a beneficial interaction and mycoparasitism and to define the biological function for one of them. We defined a catalogue of putative effector proteins from T. virens, T. atroviride and T. reesei. We further validated the expression of 16 genes encoding putative effector proteins from T. virens and T. atroviride during the interaction with the plant Arabidopsis thaliana, and with two anastomosis groups of the phytopathogenic fungus Rhizoctonia solani. We found genes which transcript levels are modified in response to the presence of both plant fungi, as well as genes that respond only to either a plant or a fungal host. Further, we show that overexpression of the gene tvhydii1, a Class II hydrophobin family member, enhances the antagonistic activity of T. virens against R. solani AG2. Further, deletion of tvhydii1 results in reduced colonization of plant roots, while its overexpression increases it. Our results show that Trichoderma is able to respond in different ways to the presence of a plant or a fungal host, and it can even distinguish between different strains of fungi of a given species. The putative effector proteins identified here may play roles in preventing perception of the fungus by its hosts, favoring host colonization or protecting it from the host’s defense response. Finally, the novel effector protein TVHYDII1 plays a role in plant root colonization by T, virens, and participates in its antagonistic activity against R. solani.

Cheeses are perishable foods that must fulfill sanitary and quality requirements according to the parameters established globally. Plasma as a nonthermal inactivation technique has been a current research topic for food preservation, so the objective of this work was to study the effect of plasma energy against microorganisms in Adobera cheese (traditional Mexican cheese) as well as evaluate the possible degradation of lipids and protein. 108CFU/mL of Escherichia coli ATCC 25922, Salmonella ATCC13076, and Staphylococcus aureus ATCC 6538 were inoculated at 0.5g of Adobera cheese and were subjected to an energy of 30 volts, in a dielectric barrier discharge reactor (DBDR) at intervals of times 1, 3, 5, 7, 10, and 15min. A flow of a mixture of air and helium at 96 purity was used. The decimal reduction time (D) was determined, and the oxidation of proteins and lipids was analyzed after each treatment. The results showed an annihilating effect of plasma on the indicator bacteria under study, and a reduction of 5 logarithmic cycles was obtained. The maximum degree of lipid oxidation was 23 acid degree values (ADV) after 7min of exposure to plasma. The oxidation of proteins showed a direct and proportional relationship between the formation of carbonyl groups with the percentage significant loss to the concentration of carbonyl groups with the concentration of protein oxidation, after 3min of exposure to cold plasma levels of 82 and 99 oxidation of Adobera cheese protein and free casein, respectively. We conclude that the plasma energy applied to Adobera cheese is an effective treatment to inactivate bacteria. However, there is the possibility of causing changes in taste and odor, due to the release of fatty acids and the oxidation of proteins.

The human milk microbiota (HMM) of healthy women can vary substantially, as demonstrated by recent advances in DNA sequencing technology. However, the method used to extract genomic DNA (gDNA) from these samples may impact the observed variations and potentially bias the microbiological reconstruction. Therefore, it is important to use a DNA extraction method that is able to effectively isolate gDNA from a diverse range of microorganisms. In this study, we improved and compared a DNA extraction method for gDNA isolation from human milk (HM) samples to commercial and standard protocols. We evaluated the extracted gDNA using spectrophotometric measurements, gel electrophoresis, and PCR amplifications to assess its quantity, quality, and amplifiability. Additionally, we tested the improved method’s ability to isolate amplifiable gDNA from fungi, Gram-positive and Gram-negative bacteria to validate its potential for reconstructing microbiological profiles. The improved DNA extraction method resulted in a higher quality and quantity of the extracted gDNA compared to the commercial and standard protocols and allowed for polymerase chain reaction (PCR) amplification of the V3–V4 regions of the 16S ribosomal gene in all the samples and the ITS-1 region of the fungal 18S ribosomal gene in 95% of the samples. These results suggest that the improved DNA extraction method demonstrates better performance for gDNA extraction from complex samples such as HM.

Recent advances in DNA sequencing technology have shown that the human milk microbiota of healthy women varies substantially. The gDNA extraction method may influence the observed variation, biasing the microbiological reconstruction after all. In this study, a genomic DNA extraction method for DNA isolation from human milk samples was standardized and compared with commercial and standard hose make methods. Spectrophotometric measurements, gel electrophoresis, and PCR amplifications were used as criteria for evaluating the quantity, quality, and functionality of the extracted DNA. Furthermore, the standardized method of extracting gDNA from human milk was evaluated for its ability to isolate functional DNA from gram-positive, and gram-negative bacteria and fungi, to improve the reconstruction of microbiological profiles. The novel DNA extraction method increased the quantity and quality of the gDNA extracted compared with commercial and standard house-make protocols. This method even allowed PCR amplification of the V3-V4 regions of the 16S ribosomal gene in all samples, and the ITS-1 region of the fungal 18S ribosomal gene in 95 % of the samples as well. It is concluded that the proposed method provides better performance for the extraction of gDNA from complex samples such as human milk.