Sustainable production of biopesticides for common bean root rot control within the biorefinery approach: a Technology Readiness Level 3 experimental proof of concept.

Biopesticides are recognized as an efficient alternative to synthetic pesticides for pest and disease crop management. However, their commercial production processes use grains, generating large amounts of organic waste, even when agriculture waste or byproducts are the feedstock of choice. Frequently, these organic wastes are rich in nutrients that, after adequate treatment, can be used as nitrogen and carbohydrate sources for secondary metabolite production produced by microorganisms during submerged fermentation. In this sense, this study aimed to prove the concept that biopesticides could be produced under a full biorefinery process, using the entire biomass of an underexplored agroindustrial waste-damaged bean-as the main feedstock. A combination of sequential processes, including solid state fermentation, hydrolysis, and submerged fermentation, were designed for the production of two biopesticides (conventional-fungal conidia and second-generation secondary metabolite-cerulenin) from a high potential biological control agent strain Sarocladium oryzae BRM 59907. The combined processes, using damaged common bean grain as the main feedstock, provided biopesticides and organic fertilizer production that successfully controlled common bean root rot disease. This work proved to be possible the biopesticide production using a full biorefinery concept, inside the same productive chain, contributing to a sustainable environment and economy, together with animal and human health safety.

A Mad7 System for Genetic Engineering of Filamentous Fungi.

The introduction of CRISPR technologies has revolutionized strain engineering in filamentous fungi. However, its use in commercial applications has been hampered by concerns over intellectual property (IP) ownership, and there is a need for implementing Cas nucleases that are not limited by complex IP constraints. One promising candidate in this context is the Mad7 enzyme, and we here present a versatile Mad7-CRISPR vector-set that can be efficiently used for the genetic engineering of four different Aspergillus species: Aspergillus nidulans, A. niger, A. oryzae and A. campestris, the latter being a species that has never previously been genetically engineered. We successfully used Mad7 to introduce unspecific as well as specific template-directed mutations including gene disruptions, gene insertions and gene deletions. Moreover, we demonstrate that both single-stranded oligonucleotides and PCR fragments equipped with short and long targeting sequences can be used for efficient marker-free gene editing. Importantly, our CRISPR/Mad7 system was functional in both non-homologous end-joining (NHEJ) proficient and deficient strains. Therefore, the newly implemented CRISPR/Mad7 was efficient to promote gene deletions and integrations using different types of DNA repair in four different Aspergillus species, resulting in the expansion of CRISPR toolboxes in fungal cell factories.

Molecular identification of Trichoderma sp. isolates and biochemical characterization of antagonistic interaction against rice blast.

This study aimed to identify four isolates of Trichoderma sp. (Ufra.T06, Ufra.T09, Ufra.T12, and Ufra.T52) and characterize their interaction with Magnaporthe oryzae in vitro and in vivo conditions. The four isolates of Trichoderma sp. were sequenced, investigated as an antagonist against M. oryzae in five Petri plate assays, and as an inhibitor of conidial germination appressoria formation. Finally, were quantified the lytic activity of chitinase (CHI), glucanase (GLU), and protease (PRO) during co-cultivation of Trichoderma sp. and M. oryzae. In vivo, leaf blast suppression was evaluated in two assays: simultaneous and curative application. Both in vitro and in vivo assays were scanned by electron microscopy (SEM). All isolates were identified as Trichoderma asperellum. All in vitro Petri plates assays reduced M. oryzae colony growth (paired-91.18% by Ufra.T09, volatile metabolites-all isolates equally reduced, non-volatile-68.33% by Ufra.T06, thermostability-99.77% by Ufra.T52 and co-cultivate-64.25% by Ufra.T52). The filtrates and conidia suspensions for T. asperellum isolates inhibited the conidia germination and appressoria formation significantly. In co-cultivate (mycelial or cell wall), all enzymes (GLU, CHI, and PRO) and times (24, 48, and 72 h) showed increased activity. In vivo, reduced leaf blast severity until 94.64% (Ufra.T52cs) in a simultaneous and until 85% (Ufra.T09 24 and 48 hasi) in a curative application. T. asperellum isolates showed efficient control of M. oryzae by mycoparasitism, and antibiosis mechanisms were interfered with by the M. oryzae infection process.

Induction of resistance in rice plants using bioproducts produced from Burkholderia pyrrocinia BRM 32113.

Leaf blast is the main rice disease in the world causing significant losses in productivity. Blast integrate management (BIM) requires the use of genetic resistance, cultural practices, and chemical control, although for sustainable BIM, the insertion of biological agents may be the fourth component for. The objective of this work was to test three formulations of Burkholderia pyrrocinia (BRM32113) previously selected and to verify the effectiveness in resistance induction and blast control in rice. Two experiments were carried out, in a completely randomized design with three replications, in the greenhouse (E1 and E2). E1 aimed to select the best treatment for suppressing leaf blast severity and activating plant defense mechanisms. It was composed of 8 treatments: (1) formulated 11+ B. pyrrocina × Magnaporthe oryzae; (2) formulated 17+ B. pyrrocina × M. oryzae; (3) formulated 32+ B. pyrrocina × M. oryzae; (4) formulated 11 × M. oryzae; (5) B. pyrrocinia 17 × M. oryzae; (6) formulated 32 × M. oryzae; (7) B. pyrrocina × M. oryzae; (8) M. oryzae; (9) control (water). E2 aimed to investigate the effect of the best treatments, for the promotion of plant growth and suppression of leaf blast by calculating AUDPC. It was composed of 6 treatments: (1) formulated 11+ B. pyrrocina × M. oryzae; (2) formulated 32+ B. pyrrocina × M. oryzae; (3) formulated 11 × M. oryzae; (4) formulated 32 × M. oryzae; (5) B. pyrrocina × M. oryzae; (6) water. And after, we did two assays aimed to localize this biological agent after application at seed, soil, and rice plant. In E1, formulated 11+ B. pyrrocinia and 32+ formulated and B. pyrrocina were the best, suppressing leaf blast by up to 97% and providing the significant increase of the enzymes ß-1,3-glucanase, chitinase, phenylalanine ammonia lyase, lipoxygenase, and salicylic acid at 24 h and 48 h after inoculation with M. oryzae. In E2, treatments formulated 11+ B. pyrrocinia, formulated 32+ B. pyrrocinia, and B. pyrrocina provided more significant increases in growth promotion and reduced area under disease progress curve. B. pyrrocinia was detected in the rice plant for 18 days, predominantly in the root system (internal and external). The use of B. pyrrocinia formulations based on sugarcane molasses and glycerol can be an essential strategy for sustainable management. Although all the benefits come from these sustainable formulations, the adoption by commercial biological segment depends on an established formulation process. It seems that all the results showed here by this research will be readily assimilated by startups of the organic segment.

Bioagents and silicon promoting fast early upland rice growth.

Upland rice can overcome major challenges through the insertion of silicate fertilization and the presence of plant growth-promoting microorganisms (PGPMs) during its cultivation, as these factors promote an increase in vigor and plant disease resistance. Two consecutive experiments were conducted to evaluate the beneficial effects of silicon fertilization combined with the PGPM, Pseudomonas fluorensces, Burkholderia pyrrocinia, and a pool of Trichoderma asperellum, in upland rice seedlings, cultivar BRS Primavera CL: (a) E1, selecting PGPM type and Si doses for rice growth promotion and leaf blast supression, and (b) E2, evaluating physiological characteristics correlated with mechanisms involved in the higher vegetative growth in highlighted treatments from E1. In E1, 2 Si t ha-1 combined with the application of T. asperellum pool or PGPM mixture increased 54% in root dry matter biomass and 35 and 65% in shoot and root lengths, respectively; it also suppressed 99% of rice blast severity. In E2, shoot and root dry matter biomass and length, photosynthetic rate, water use efficiency, total soluble sugar, and chloroplastidic pigments were superior in BRS Primavera CL seedlings treated with 2 Si t ha-1 and T. asperellum pool or PGPM mixture. Higher salicilic and jasmonic acid levels were found in seedlings treated with Si and T. asperellum pool, individually. These physiological characteristics may explain, in part, the higher vigor of upland rice seedlings promoted by the synergistic effect between silicate fertilization and beneficial microorganisms.

Defence responses in rice plants in prior and simultaneous applications of Cladosporium sp. during leaf blast suppression.

An alternative method to control rice blast (Magnaporthe oryzae) is to include biological agent in the disease management strategy. The objective of this study was to assess the leaf blast-suppressing effects of rice phylloplane fungi. One Cladosporium sp. phylloplane fungus was shown to possess biocontrolling traits based on its morphological characteristics and an analysis of its 18S ribosomal DNA. Experiments aimed at determining the optimal time to apply the bioagent and the mechanisms involved in its rice blast-suppressing activities were performed under controlled greenhouse conditions. We used foliar spraying to apply the Cladosporium sp. 48 h prior to applying the pathogen, and we found that this increased the enzymatic activity. Furthermore, in vitro tests performed using isolate C24 showed that it possessed the ability to secrete endoxylanases and endoglucanases. When Cladosporium sp. was applied either prior to or simultaneous with the pathogen, we observed a significant increase in defence enzyme activity, and rice blast was suppressed by 84.0 and 78.6 %, respectively. However, some enzymes showed higher activity at 24 h while others did so at 48 h after the challenge inoculation. Cladosporium sp. is a biological agent that is capable of suppressing rice leaf blast by activating biochemical defence mechanisms in rice plants. It is highly adapted to natural field conditions and should be included in further studies aimed at developing strategies to support ecologically sustainable disease management and reduce environmental pollution by the judicious use of fungicidal sprays.

An approach on phytoalexins: function, characterization and biosynthesis in plants of the family Poaceae / Uma abordagem sobre fitoalexinas: função, caracterização e biossíntese em plantas da família Poaceae

ABSTRACT: Phytoalexins are compounds that have been studied a few years ago, which present mainly antimicrobial activity. The plants of the family Poaceae are the most geographically widespread and stand out for their economic importance, once they are cereals used as staple food. This family stands out for having a variety of phytoalexins, which can be synthesized via the shikimic acid (the phenylpropanoids), or mevalonic acid, being considered terpenoid phytoalexins. The characterization of these compounds with antimicrobial activity is carried out using chromatographic techniques, and the high-performance liquid chromatography (HPLC) coupled with mass spectrometry are the most efficient methods in this process. This research aimed to present an approach of the function, characterization and biosynthesis of phytoalexins in plants of the family Poaceae.

RESUMO: As fitoalexinas são compostos que vêm sendo estudados há alguns anos, apresentando principalmente atividade antimicrobiana. As plantas da família Poaceae são as geograficamente mais difundidas e se destacam por sua importância econômica, uma vez que são os cereais que compõe a base da alimentação, em todos os continentes. Essa família se destaca por apresentar uma variedade de fitoalexinas, sendo que as mais importantes são sintetizadas a partir da via do ácido chiquímico. A caracterização desses compostos com atividade antimicrobiana é realizada por meio de técnicas cromatográficas, sendo que a cromatografia líquida de alta eficiência (HPLC) acoplada com espectrometria de massas é a técnica mais eficiente nesse processo. O trabalho apresenta como objetivo uma abordagem da função, caracterização, biossíntese e aplicações das fitoalexinas em plantas da família Poaceae.