Maurício Dutra Costa

Identification of differentially expressed genes of the fungus Hydnangium sp. during the pre-symbiotic phase of the ectomycorrhizal association with Eucalyptus grandis.

The pre-symbiotic phase that precedes physical contact between symbionts is a crucial phase in determining their compatibility, allowing the formation of the ectomycorrhiza. A subtractive cDNA library representing the differentially expressed genes of the fungus Hydnangium sp. in the pre-symbiotic phase was constructed using fungal mycelia obtained through the in vitro mycorrhization technique. The fungus was cultured in the presence of Eucalyptus grandis roots, but with no contact between the hyphae and the root system of the host plant. Genes that code for proteins related to carbohydrate, amino acid, and energy metabolisms, transcription, and protein synthesis, cellular communication, signal transduction, stress response, transposons, and proteins related to the biogenesis of cell components were identified among the 131 expressed sequence tags. Expression of the genes that code for acetyl-CoA acetyltransferase, pyruvate dehydrogenase, ATP synthase, a voltage-dependent protein from the selective ion channel, and hydrophobin was evaluated by the RT-qPCR technique, confirming the activation of these genes in this phase of the association.

Optimization of Aspergillus niger rock phosphate solubilization in solid-state fermentation and use of the resulting product as a P fertilizer.

A biotechnological strategy for the production of an alternative P fertilizer is described in this work. The fertilizer was produced through rock phosphate (RP) solubilization by Aspergillus niger in a solid‐state fermentation (SSF) with sugarcane bagasse as substrate. SSF conditions were optimized by the surface response methodology after an initial screening of factors with significant effect on RP solubilization. The optimized levels of the factors were 865 mg of biochar, 250 mg of RP, 270 mg of sucrose and 6.2 ml of water per gram of bagasse. At this optimal setting, 8.6 mg of water‐soluble P per gram of bagasse was achieved, representing an increase of 2.4 times over the non‐optimized condition. The optimized SSF product was partially incinerated at 350°C (SB‐350) and 500°C (SB‐500) to reduce its volume and, consequently, increase P concentration. The post‐processed formulations of the SSF product were evaluated in a soil–plant experiment. The formulations SB‐350 and SB‐500 increased the growth and P uptake of common bean plants (Phaseolus vulgaris L.) when compared with the non‐treated RP. Furthermore, these two formulations had a yield relative to triple superphosphate of 60% (on a dry mass basis). Besides increasing P concentration, incineration improved the SSF product performance probably by decreasing microbial immobilization of nutrients during the decomposition of the remaining SSF substrate. The process proposed is a promising alternative for the management of P fertilization since it enables the utilization of low‐solubility RPs and relies on the use of inexpensive materials.

Diversity of endophytic bacteria in the fruits of Coffea canephora

Endophytic bacteria colonize the internal tissues of plants without causing infection or negative effects on their hosts. This study investigates the occurrence and diversity of culturable endophytic bacteria in the fruits of Coffea canephora at three developmental stages. Isolation and quantification were performed in R2A culture medium, and the diversity was established using molecular methods and analysis of fatty acid methyl esters (FAME). α- and γ-Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes were identified in the investigated community. Kocuria turfanensis and Pantoea vagans were identified as endophytes for the first time. Of the 18 species that were found, the following seven had not been previously described as endophytic in coffee fruits: Bacillus thuringiensis, Bacillus licheniformis, Agrobacterium tumefaciens, Escherichia coli, Enterobacter hormaechei, Chryseobacterium sp., and Ochrobactrum sp. The diversity of endophytic bacteria varied during the three developmental stages that were investigated, and the diversity was greatest in fruits during the green stage, during which Bacillus subtilis predominated. The number of Gram-positive bacteria was larger than the number of Gram-negative bacteria during the two earliest developmental stages, whereas their numbers were similar during the ripe stage. The diversity was lowest during the ripe stage, and Klebsiella oxytoca was the predominant species at this stage, probably due to the higher caffeine and sugar contents in the fruits.

Soil microorganisms and their role in the interactions between weeds and crops

A competicao entre plantas daninhas e culturas e um topico de grande interesse, uma vez que essa interacao pode causar grandes perdas na agricultura. Apesar de alguns estudos nesse tema, pouco se sabe sobre a importância dos microrganismos do solo na modulacao dessa interacao. As plantas competem por agua e nutrientes presentes no solo e a capacidade das especies em absorver esses recursos pode ser diretamente afetada pela presenca de alguns grupos microbianos comumente encontrados no solo. Os fungos micorrizicos arbusculares (FMA) sao capazes de se associar com as raizes das plantas, o que afeta a capacidade das diferentes especies em absorver agua e nutrientes do solo, promovendo alteracoes no crescimento. Outros grupos podem promover alteracoes positivas ou negativas no crescimento das especies vegetais, a depender da identidade das especies microbianas e vegetais envolvidas nas diferentes interacoes, alterando a capacidade competitiva de cada uma delas. Estudos recentes tem demonstrado que as plantas daninhas sao capazes de se associar com fungos micorrizicos arbusculares nos ambientes agricolas, e que a colonizacao das raizes por esses fungos e afetada pela presenca de outras plantas daninhas ou de culturas. Alem disso, as plantas daninhas tendem a apresentar interacoes positivas com a microbiota do solo, ao passo que as culturas apresentam interacoes neutras ou negativas. A competicao entre plantas daninhas e culturas promove alteracoes na comunidade microbiana do solo, que se torna diferente daquelas observadas nas monoculturas, afetando a capacidade competitiva das especies vegetais. Quando colocadas em competicao, plantas daninhas e culturas apresentam diferentes comportamentos relacionados a microbiota do solo, sendo que as plantas daninhas apresentam maior dependencia de associacoes com microorganismos do solo, em relacao as culturas, para aumentar seu crescimento. Esses dados demonstram a importância dos microorganismos do solo na modulacao das interacoes entre plantas daninhas e culturas nos ambientes agricolas. Novas perspectivas e hipoteses sao apresentadas para guiar as futuras pesquisas nessa area.

Basidiosporogenesis, meiosis, and post-meiotic mitosis in the ectomycorrhizal fungus Pisolithus microcarpus

Pisolithus microcarpus (Cooke and Massee) G. Cunn. is a model organism for the studies on the ecology, physiology, and genetics of the ectomycorrhizal associations. However, little is known about the basidiosporogenesis in this species and, in particular, the nuclear behavior after karyogamy. In this work, the events involved in basidiosporogenesis and meiosis in P. microcarpus were analyzed using fluorescence and scanning electron microscopy. The basidia are formed inside peridioles by the differentiation of the cells along the whole hyphae. Basidial cells measure 12-18 microm in length and 6-7 microm in diameter. P. microcarpus produces eight basidiospores per basidium imbibed in a gelatinous matrix in the basidiocarp. The basidiospores are globose, equinate, with blunt spines, and measure 6-8 microm. Karyogamy can take place inside basidia as well as in undifferentiated hyphal cells followed by nuclear migration to a newly developed basidium where meiosis takes place. After the formation of the meiotic tetrad, one round of post-meiotic mitosis occurs, resulting in the production of eight nuclei per basidium. The newly-formed nuclei migrate into the basidiospores asynchronously, resulting in the production of eight uninucleate spores. This corresponds to pattern A of post-meiotic mitosis. This work is the first report on meiosis and post-meiotic mitosis during basidiosporogenesis in P. microcarpus and contributes to clarify some aspects of the biology and genetics of this ectomycorrhizal species.

In vitro ectomycorrhiza formation by monokaryotic and dikaryotic isolates of Pisolithus microcarpus in Eucalyptus grandis

The formation of ectomycorrhizas by monokaryotic and dikaryotic isolates of Pisolithus microcarpus (Cooke & Massee) G. Cunn. in Eucalyptus grandis W. Hill ex Maid. was studied by in vitro synthesis in Petri dishes. The formation of ectomycorrhizas was observed for all strains tested. Ectomycorrhizas formed by the monokaryotic strains presented a sheath of hyphae around the roots and a Hartig net limited to the epidermis layer, typical of the angiosperm ectomycorrhizas. Colonization rates, a measure of the number of ectomycorrhizas in relation to the total number of lateral root tips, varied from 23 to 62%. Some monokaryotic strains stimulated the formation of lateral roots, promoting increases of up to 109% above the control. The presence of some of the isolates in the in vitro synthesis medium stimulated the production of thicker lateral root tips. The dimensions of the lateral roots tips and ectomycorrhizas varied from one isolate to the next, indicating a variation in their capacity to provoke morphological changes in the host plant roots. The dikaryotic strain M5M11 presented higher values for lateral root yield, number of ectomycorrhizas, and colonization percentage than the corresponding monokaryotic strains, M5 and M11. This indicated the possibility of selecting compatible performing monokaryotic isolates for the yield of superior dikaryotic strains. The set of monokaryotic strains tested varied greatly in their ability to colonize E. grandis roots and cause secondary metabolism-related morphological changes in roots, providing a wealth of model systems for the study of genetic, physiological, and morphogenetic processes involved in Pisolithus-Eucalyptus ectomycorrhiza formation.

Formação de ectomicorrizas por monocários e dicários de Pisolithus sp. e interações nutricionais em Eucalyptus grandis

The germination of Pisolithus spp. basidiospores originates monokaryons, characterized by having a single haploid nucleus per cell. In the field, eucalypts are associated with dykaryons of Pisolithus spp., there being no reports on the capacity of monokaryons to establish the ectomycorrhizal association with the host plant under such conditions. Although Pisolithus spp. monokaryons have been shown to form ectomycorrhizas in vitro, there is no information on the ability of these strains to promote nutrient uptake and growth of eucalypts. The objective of this study was to investigate the formation of ectomycorrhizae by monokaryotic and dikaryotic isolates of Pisolithus sp. in Eucalyptus grandis under greenhouse conditions, and the relationships between the establishment of ectomycorrhizae and the host plant growth and uptake of P, Ca, Mg, K, Cu, and Zn. The fungal isolates were highly variable in dry mycelial mass production and in their ability to take up macro- and micronutrients. Generally, the nutrient use efficiency of monokaryons was higher than that of dikaryons. All tested monokaryotic and dikaryotic isolates were able to form typical ectomycorrhizae when associated with E. grandis. The presence of monokaryotic isolates associated with the lateral roots of E. grandis resulted in typical increases in the radial diameter of the root epidermal cells, characteristic of eucalypt ectomycorrhizae, indicating that monokaryons, similarly to dikaryons, are capable of producing growth regulator compounds. The mean root colonization percentage of the monokaryotic and dikaryotic isolates varied from 12 to 76 %. The Ca, K, and Mg uptake was highly stimulated by the presence of ectomycorrhizae, with up to 760-fold increases, suggesting that the association must play a significant role in supplying the host with these nutrients, especially Ca. Some monokaryotic isolates are as efficient as the dikaryons at promoting root colonization and nutrient uptake. The characterization of Pisolithus sp. monokaryons is a basic requirement for the selection and crossing of isolates with desirable traits, aiming at the genetic improvement of fungal strains and a higher efficiency of the ectomycorrhizal association. This is the first report on the nutritional interactions between Pisolithus sp. monokaryons and the host plant.

In vitro Scleroderma laeve and Eucalyptus grandis mycorrhization and analysis of atp6, 17S rDNA, and ras gene expression during ectomycorrhizal formation

The interaction between fungi and plants that form ectomycorrhizae (ECM) promotes alterations in the gene expression profiles of both organisms. Fungal genes expression related to metabolism were evaluated at the pre‐symbiotic stage and during the ECM development between Scleroderma laeve and Eucalyptus grandis. Partial sequences of ATP synthase (atp6), translation elongation factor (ef1α), the RAS protein (ras), and the 17S rDNA genes were isolated. The expression of the atp6 and 17S rDNA genes during the pre‐symbiotic stage showed an approximately threefold increase compared to the control. During ECM development, the expression of the 17S rDNA gene showed a 4.4‐fold increase after 3 days of contact, while the expression of the atp6 gene increased 7.23‐fold by the 15th day, suggesting that protein synthesis and respiratory chain activities are increased during the formation of the mantle and the Hartig net. The ras gene transcripts were only detected by RT‐PCR 30 days after fungus–plant contact, suggesting that RAS‐mediated signal transduction pathways are functional during the establishment of symbiosis. The present study demonstrates that alterations in gene expression occur in response to stimuli released by the plant during ECM association and increases the understanding of the association between S. laeve and E. grandis.

Electron microscopy studies of basidiosporogenesis in Agaricus brasiliensis

The objective of this work was to study the basidiosporogenesis and the intraspecific variation in the number of basidiospores produced per basidium in Agaricus brasiliensis with transmission (TEM) and scanning electron microscopy (SEM). A. brasiliensis produces predominantly tetrasporic basidia, but this trait may vary depending on the strain. For certain strains, such as CS2 and CS7, the frequency of bisporic and trisporic basidia was similar to, or greater than, that of tetrasporic strains. These results suggest that some strains of A. brasiliensis may be amphithallic; however, this behavior is variable and strain dependent. The development of basidia and basidiospores occurs asynchronously during basidiocarp production. The basidiospore cell wall is composed of three distinct layers and presents variable thickness. The conspicuous presence of lipid bodies also was observed in the basidiospores, while nuclei, mitochondria, vacuoles and dolipore septa could be visualized only in the basidia. The basidiospores generally are produced free but also may be enveloped by an extracellular matrix with unknown chemical composition. The presence of connection hyphae linking the basidia was observed for the first time in A. brasiliensis. This characteristic, so far not reported for other fungi, may represent a specific strategy of A. brasiliensis for exchanging nuclei and other cell material between basidial cells during basidiosporogenesis.

Production and regeneration of protoplasts from orchid Mycorrhizal Fungi Epulorhiza repens and Ceratorhiza sp.

The aim of this work was to study the standardization of conditions to obtain and regenerate Epulorhiza repens and Ceratorhiza sp. protoplasts. For E. repens, the largest number of protoplasts (8.0 × 106 protoplasts/mL) was obtained in 0.6 M KCl, using 15 mg/mL of Lysing Enzymes, and 2-day-old fungal mycelium. When 0.5 M sucrose was used as osmotic stabilizer, the highest frequency of regeneration was achieved (8.5 %); 80.0 % of protoplasts were nucleated, and 20.0 % anucleated. For Ceratorhiza sp., the largest number of protoplasts (4.0 × 107 protoplasts/mL) was achieved in 0.6 M NaCl, when 15 mg/mL of Lysing Enzymes and 15mg/mL of Glucanex, with 2-day-old fungal mycelium were used. The highest frequency of regeneration was 6.7 % using 0.5 M sucrose as osmotic stabilizer; 88.8 % of protoplasts were nucleated, and 11.2 % anucleated.