André Narvaes da Rocha Campos

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.

Total lipid and fatty acid accumulation during basidiospore formation in the ectomycorrhizal fungus Pisolithus sp.

Os basidiosporos de Pisolithus sp. contem grande quantidade de lipideos, indicando provisao para futura germinacao na rizosfera da planta hospedeira. No entanto, pouco se conhece a respeito do acumulo, da composicao e da mobilizacao dos lipideos durante a formacao e germinacao dos esporos. Neste trabalho, o armazenamento de lipideos e a analise da composicao em acidos graxos durante a basidiosporogenese foram estudados utilizando-se tecnicas de microscopia de luz e cromatografia gasosa. O basidiocarpo fungico apresentou abundantes corpos lipidicos no interior de suas hifas, basidios e basidiosporos. Esse fato evidencia consideravel transporte de C dentro do basidiocarpo para suprir a demanda de C durante a formacao dos esporos. A composicao de acidos graxos revelou a presenca de 24 compostos com cadeias variando de 9 a 18 atomos de C, sendo tanto saturados ou contendo uma ou duas insaturacoes. A composicao de acidos graxos variou de acordo com o estadio de desenvolvimento dos peridiolos. Nos basidiosporos livres, os compostos predominantes foram 16:0, 16:1w5c, 18:1w9c e 18:2w6,9c/18:0ante, com concentracoes de 76, 46, 192 e 51 µg g-1 do acido graxo de materia seca, respectivamente. Esses resultados indicam que o acido oleico e o principal constituinte das reservas lipidicas nos basidiosporos de Pisolithus sp. Outros trabalhos estao sendo realizados para determinar os fatores que desencadeiam a mobilizacao de lipideos durante a germinacao de esporos.

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.

Fungos Micorrízicos Arbusculares em Euterpe edulis Martius (Palmeira Juçara) no Município de Rio Pomba/MG

This study aimed to evaluate the occurrence of arbuscular mycorrhizal association in Euterpe edulis (Palm Jucara) in the region of Rio Pomba, MG. Initially, soil was collected from the rhizosphere of E. edulis in five distinct areas. Following, there was extraction of spores, and the process of discoloration and staining of roots was established. Observation also showed presence of a characteristic structure of arbuscular mycorrhizal fungi in the roots to be analyzed, and presence of spores of mycorrhizal fungi in all areas. These results indicate the possibility for more comprehensive studies aiming at the efficient selection of fungal strains, and possible forms of this palm in nursery inoculation.

EVALUATION OF SUBSTRATES AND AMF SPORULATION IN THE PRODUCTION OF SEEDLINGS OF NATIVE FOREST SPECIES

The objective of this study was to evaluate organic substrates in the production of canafistula (Peltophorum dubium) (Spreng.) Taub, cutieira (Joannesiaprinceps Vell.), jatoba (Hymenaea courbaril L.) and rubber tree (Hevea brasiliensis M. Arg.) seedlings, native trees with potential use in forest restoration programs. The design was completely randomized with 10 substrate formulations with 4 repetitions of 3 plants for the four species. The evaluated substrates consisted of soil, bovine manure (BM), poultry manure (PM), chemical fertilizer (CF) and sand, in different proportions. The experiment was concluded at the end of 180 days for canafistula, cutieira and rubber and 210 days for jatoba. At the end of these periods, the root (RDM), shoot (SDM) and total (TDM) the dry matters of the seedlings were determined. Quantification of AMF spores and normalization between samples through SPORES/RDM correction were also performed. The Scott-Knott test at 5% probability was applied. Regarding biomass production, only canafistula had significant difference among the tested substrates. In relation to sporulation, the highest values were observed in cutieira and rubber tree in substrate containing PM. The substrates composed of 40 or 50% soil + 20% sand + 30% or 40 PM for canafistula; 50% soil + 20% sand + 30% PM for cutieira; and for jatoba and rubber tree 60% soil + 20% sand + 20% PM, enabled the best results in terms of biomass production in seedlings and AMF sporulation.

Mycorrhizal association in Euterpe edulis Martius (Palmeira Juçara) in the municipality of Rio Pomba, MG

Este trabalho teve como objetivo avaliar a ocorrencia de associacao micorrizica arbuscular em Euterpe edulis (Palmeira Jucara), na regiao de Rio Pomba/MG. Inicialmente, coletou-se solo da rizosfera de E. edulis em cinco areas distintas. Em seguida, realizouse a extracao de esporos e se estabeleceu o processo de descoloracao e coloracao das raizes. Constatou-se a presenca de estruturas tipicas de associacao micorrizica arbuscular nas raizes em estudo e presenca, em todas as areas, de esporos de fungos micorrizicos arbusculares (FMAs). Estes resultados abrem a possibilidade para estudos mais abrangentes visando a selecao de estirpes fungicas eficientes e possiveis formas de inoculacao desta palmeira em viveiro.

The transcriptional landscape of basidiosporogenesis in mature Pisolithus microcarpus basidiocarp.

BackgroundPisolithus microcarpus (Cooke & Massee) G. Cunn is a gasteromycete that produces closed basidiocarps in symbiosis with eucalypts and acacias. The fungus produces a complex basidiocarp composed of peridioles at different developmental stages and an upper layer of basidiospores free of the hyphae and ready for wind dispersal upon the rupture of the basidiocarp pellis. During basidiosporogenesis, a process that takes place inside the basidiocarp peridioles, a conspicuous reserve of fatty acids is present throughout development. While several previous studies have described basidiosporogenesis inside peridioles, very little is known about gene expression changes that may occur during this part of the fungal life cycle. The objective of this work was to analyze gene transcription during peridiole and basidiospore development, while focusing specifically on cell cycle progression and lipid metabolism.ResultsThroughout different developmental stages of the peridioles we analyzed, 737 genes were regulated between adjacent compartments (>5 fold, FDR-corrected p-value < 0.05) corresponding to 3.49% of the genes present in the P. microcarpus genome. We identified three clusters among the regulated genes which showed differential expression between the peridiole developmental stages and the basidiospores. During peridiole development, transcripts for proteins involved in cellular processes, signaling, and information storage were detected, notably those for coding transcription factors, DNA polymerase subunits, DNA repair proteins, and genes involved in chromatin structure. For both internal embedded basidiospores (hereto referred to as “Internal spores”, IS) and external free basidiospores (hereto referred to as “Free spores”, FS), upregulated transcripts were found to involve primary metabolism, particularly fatty acid metabolism (FA). High expression of transcripts related to β-oxidation and the glyoxylate shunt indicated that fatty acids served as a major carbon source for basidiosporogenesis.ConclusionOur results show that basidiocarp formation in P. microcarpus involves a complex array of genes that are regulated throughout peridiole development. We identified waves of transcripts with coordinated regulation and identified transcription factors which may play a role in this regulation. This is the first work to describe gene expression patterns during basidiocarp formation in an ectomycorrhizal gasteromycete fungus and sheds light on genes that may play important roles in the developmental process.

Respiração basal e induzida por compostos orgânicos de carbono em solos de diferentes agroecossistemas da Zona da Mata de Minas Gerais

The objective of this work was to evaluate soil microorganisms from the agroecosystems of Rio Pomba/MG using basal and induced respiration by organic carbon compounds. Forest soil presented the highest biological quality, a feature revealed by the efficient use of sucrose. Coffee plantation and annually cropped areas were less efficient in the use of sucrose. Nevertheless, the lesser level of basal and cellulose induced respiration of the latter indicated its reduced microbial activity. These results should support studies aiming at the development of sustainable agricultural systems at Zona da Mata de Minas Gerais.

Inoculação com esporos de Pisolithus microcarpus e adição de fosfato natural em mudas de Eucalyptus grandis

The objective of this study was to evaluate the use of spores of Pisolithus microcarpus and rock phosphate (RP) in low-cost inoculation programs of Eucalyptus grandis in nurseries. Although the treatments did not increase plant growth, there was significant difference in dry mass of the root system and partition of carbon in the plant. Inoculation promoted a significant increase in the mycorrhization rates, but the doses of RN used in this study did not affect the establishment of the association. Additional studies are being conducted to optimize the procedure and inoculated with spores.

Inoculation with spores of Pisolithus microcarpus and amendment with rock phosphate in seedlings of Eucalyptus grandis

No solo de ecossistemas florestais de importância economica e para o dia a dia do pequeno produtor, destaca-se a associacao ectomicorrizica (READ; PEREZ-MORENO, 2003). Trata-se de uma associacao simbiotica mutualistica entre as raizes das plantas e fungos especializados do solo que e caracterizada por suas estruturas tipicas: a rede de Hartig e o manto fungico (PETERSON; FARQUHAR, 1994; SMITH; READ, 2008). A rede de Hartig caracteriza-se pelo crescimento de hifas, nos intersticios das celulas das O objetivo deste trabalho foi avaliar a utilizacao dos esporos de Pisolithus microcarpus e de fosfato de natural (FN) em programas de inoculacao de baixo custo de Eucalyptus grandis em viveiros. Embora os tratamentos nao tenham provocado maior crescimento da parte aerea das mudas, verificou-se diferenca significativa na massa seca do sistema radicular e na particao de carbono na planta. A inoculacao promoveu aumento nas taxas de micorrizacao, embora as doses de FN utilizadas neste trabalho nao tenham afetado o estabelecimento da associacao. Estudos adicionais estao sendo conduzidos para otimizar o procedimento de inoculacao com esporos.