Molecular and morphological diversity, qualitative chemical profile and antioxidant activity of filamentous fungi of the digestive tract of Phylloicus sp. (Trichoptera: Calamoceratidae) / Diversidade molecular e morfológica, perfil químico qualitativo e atividade antioxidante de fungos filamentosos do trato digestivo de Phylloicus sp. (Trichoptera: Calamoceratidae)

This study aimed to identify by molecular analysis, morphology, chemistry and antioxidant extracts of filamentous fungi isolated from the digestive tract of Phylloicus sp, an aquatic insect that lives on leaf packages in tropical streams and participates together with fungi of the decomposition of plant substrates in aquatic habitats. Insect larvae of Phylloicus sp. were collected in streams in the state of Tocantins, Brazil. Fungi were isolated from the digestive tract of larvae after disinfection and dissection, then described and purified for identification purposes and testing for antioxidant activity. Molecular identity was performed of ITS1 and ITS4, TUB e TEF sequencing. Fungal extracts were produced in 70% ethanol solution and later lyophilized. For analysis of chemical groups of extracts, thin layer chromatography (TLC) was performed in two mobile phases and different developers. Morphology was performed by optical microscopy stained with Toluidine Blue and measurement performed using the ImageJ program. Antioxidant activity performed in TLC and by quantitative method for DPPH and hydrogen peroxide (H2O2) radicals. Four fungi were identified: Endomelanconiopsis endophytica, Myxospora musae, Neopestalotiopsis cubana and Fusarium pseudocircinatum. The TLC showed several spots with acetone/chloroform mobile phase and UV 254 nm developers and I2 vapor. Fungal extracts demonstrate antioxidant action to reduce the DPPH free radical and especially for H2O2 above 50%, E. endophytica 91.6%, M. musae 87.8%, N. cubana 89.5% and 92.3% for F. pseudocircinatum. This study demonstrated that the molecular technique by PCR was satisfactory for identifying fungi, and extracts with numerous chemical groups and potent reducing agents. Thus future work, should be carried out evaluating these four species for industrial use.

Diversidade molecular, morfológica, perfil químico qualitativo e atividade antioxidante de fungos filamentosos do trato digestivo de Phylloicus sp. (Trichoptera: Calamoceratidae). Este estudo visou identificar por análises molecular, morfológica, química e por extrato antioxidante, fungos filamentosos isolados do trato digestivo de Phylloicus sp, um inseto aquático que vive entre folhagens em riachos tropicais, e participam junto com os fungos da decomposição de substratos vegetais em habitats aquáticos. Larvas de insetos de Phylloicus sp. foram coletadas em riachos no Estado do Tocantins, Brasil. Os fungos foram isolados a partir do trato digestivo das larvas após desinfecção e dissecção, depois descritos e purificados para fins de identificação e testes para atividade antioxidante. A identidade molecular foi realizada do sequenciamento ITS1 e ITS4, TUB e TEF. Os extratos fúngicos foram produzidos em solução etanólica 70% e posterior liofilização. Para análise de grupos químicos dos extratos, foi realizada cromatografia em camada delgada (CCD) em duas fases móveis e diferentes reveladores. A morfologia foi realizada por microscopia óptica corada com Azul de Toluidina e a mensuração realizada no programa ImageJ. A atividade antioxidante realizada em CCD e por método quantitativo para os radicais DPPH e peróxido de hidrogênio (H2O2). Foram identificados quatro fungos: Endomelanconiopsis endophytica, Myxospora musae, Neopestalotiopsis cubana e Fusarium pseudocircinatum. Na CCD foi evidenciado várias manchas com fase móvel acetona/clorofórmio e reveladores UV 254 nm e vapor de I2. Os extratos fúngicos demonstram ação antioxidante para redução do radical livre DPPH e em especial para H2O2 acima de 50%, E. endophytica 91.6%, M. musae 87.8%, N. cubana 89.5% e 92.3% para F. pseudocircinatum. Este estudo demonstrou que a técnica molecular por PCR foi satisfatória para identificação dos fungos, e os extratos com inúmeros grupos químicos e potentes agentes redutores. Com isso, trabalhos futuros deverão ser realizados avaliando essas quatro espécies para uso industrial.

Diversity of cellulolytic and xylanolytic fungi associated with the digestive tract of aquatic insect larvae in streams of the Amazon Forest and Cerrado in Brazil.

The study of the relationship between fungi and insects brings important contributions to the knowledge of fungal biodiversity and to the understanding of mutualistic ecological interactions. This study reports the occurrence of a community of filamentous fungi in the digestive tract (DT) of mining insect larvae belonging to genus Stenochironomus in streams of two Brazilian biomes. Fungi were obtained from the digestive tract of larvae found on trunks and leaves of low-order streams in the Amazon Forest and Cerrado in the north of Brazil. The fungal community was screened for xylanolytic and cellulolytic activities. The diversity of fungal species in the DT of larvae is possibly related to the diversity of diets of species of that genus and the diversity of substrates in the ecosystems. The diversity and richness of fungal species were influenced by ecological differences between locations more than by the types of substrates in which they were collected (trunk and leaf). Most fungi in the DT of Stenochironomus larvae sampled in leaves exhibited cellulolytic enzyme activity. Such results stress that the mycobiomes of the DT of Stenochiromonus larvae produce enzymes that contribute to the process of breaking down plant remains in their hosts.

Diversity and enzymatic capabilities of fungi associated with the digestive tract of larval stages of a shredder insect in Cerrado and Amazon Forest, Brazil.

Tropical biomes such as Brazilian Cerrado and Amazon Forest have a great diversity of fungi and insects. Interactions between these organisms can be beneficial to both partners. In streams, these interactions contribute to litter decomposition. Studying the digestive tract (DT) of shredder insects as a habitat for fungal microorganisms is an opportunity to obtain fungal strains with biotechnological potential, which may help to understand the symbiotic relationships between these organisms in tropical forests. This study investigated the fungal community in the DT of larvae of Triplectides (Trichoptera: Leptoceridae) collected in low-order streams in the Cerrado and Amazon Forest biomes in Brazil. Forty-nine fungal isolates were obtained and identified among 32 species and 12 genera. The genus Roussoella was only found in the DT of insects in Amazon Forest streams, while 7 genera only occurred in the DT of insects in Cerrado streams. The genus Penicillium (40%) was the most frequent. In the Cerrado, 78% were producers of CMCase, more than two-fold that in the Amazon Forest (35%). And 62% were producers of xylanase, in the Cerrado and 71% in the Amazon Forest. In this context, the fungal community in the DT of Triplectides larvae may play an important role in the insect diet by breaking down lignocellulosic material.

Molecular and morphological diversity, qualitative chemical profile and antioxidant activity of filamentous fungi of the digestive tract of Phylloicus sp. (Trichoptera: Calamoceratidae).

This study aimed to identify by molecular analysis, morphology, chemistry and antioxidant extracts of filamentous fungi isolated from the digestive tract of Phylloicus sp, an aquatic insect that lives on leaf packages in tropical streams and participates together with fungi of the decomposition of plant substrates in aquatic habitats. Insect larvae of Phylloicus sp. were collected in streams in the state of Tocantins, Brazil. Fungi were isolated from the digestive tract of larvae after disinfection and dissection, then described and purified for identification purposes and testing for antioxidant activity. Molecular identity was performed of ITS1 and ITS4, TUB e TEF sequencing. Fungal extracts were produced in 70% ethanol solution and later lyophilized. For analysis of chemical groups of extracts, thin layer chromatography (TLC) was performed in two mobile phases and different developers. Morphology was performed by optical microscopy stained with Toluidine Blue and measurement performed using the ImageJ program. Antioxidant activity performed in TLC and by quantitative method for DPPH and hydrogen peroxide (H2O2) radicals. Four fungi were identified: Endomelanconiopsis endophytica, Myxospora musae, Neopestalotiopsis cubana and Fusarium pseudocircinatum. The TLC showed several spots with acetone/chloroform mobile phase and UV 254 nm developers and I2 vapor. Fungal extracts demonstrate antioxidant action to reduce the DPPH free radical and especially for H2O2 above 50%, E. endophytica 91.6%, M. musae 87.8%, N. cubana 89.5% and 92.3% for F. pseudocircinatum. This study demonstrated that the molecular technique by PCR was satisfactory for identifying fungi, and extracts with numerous chemical groups and potent reducing agents. Thus future work, should be carried out evaluating these four species for industrial use.

Diversity and enzymatic capabilities of fungi associated with the digestive tract of larval stages of a shredder insect in Cerrado and Amazon Forest, Brazil / Diversidade e capacidades enzimáticas de fungos associados ao trato digestivo da fase larval de um inseto triturador em Cerrado e Floresta Amazônica, Brasil

Tropical biomes such as Brazilian Cerrado and Amazon Forest have a great diversity of fungi and insects. Interactions between these organisms can be beneficial to both partners. In streams, these interactions contribute to litter decomposition. Studying the digestive tract (DT) of shredder insects as a habitat for fungal microorganisms is an opportunity to obtain fungal strains with biotechnological potential, which may help to understand the symbiotic relationships between these organisms in tropical forests. This study investigated the fungal community in the DT of larvae of Triplectides (Trichoptera: Leptoceridae) collected in low-order streams in the Cerrado and Amazon Forest biomes in Brazil. Forty-nine fungal isolates were obtained and identified among 32 species and 12 genera. The genus Roussoella was only found in the DT of insects in Amazon Forest streams, while 7 genera only occurred in the DT of insects in Cerrado streams. The genus Penicillium (40%) was the most frequent. In the Cerrado, 78% were producers of CMCase, more than two-fold that in the Amazon Forest (35%). And 62% were producers of xylanase, in the Cerrado and 71% in the Amazon Forest. In this context, the fungal community in the DT of Triplectides larvae may play an important role in the insect diet by breaking down lignocellulosic material.(AU)

Biomas tropicais como o Cerrado brasileiro e a Floresta Amazônica apresentam uma grande diversidade de fungos e insetos. As interações entre esses organismos podem ser benéficas para ambos os parceiros. Em riachos, essas interações contribuem para a decomposição da serapilheira. O estudo do trato digestório (TD) de insetos como um habitat para microrganismos fúngicos é uma oportunidade para obtenção de linhagens fúngicas com potencial biotecnológico, podendo trazer luz para o entendimento das relações simbióticas entre esses organismos em florestas tropicais. Esse estudo investigou a comunidade fúngica do TD de larvas de Triplectides (Trichoptera: Leptoceridae) coletados em riachos de baixa ordem nos biomas Cerrado e Floresta Amazônica no Brasil. Foram obtidos 49 isolados fúngicos e identificados entre 32 espécies de 12 gêneros. O gênero Roussoella foi encontrado apenas no DT de insetos em riachos da Floresta Amazônica, enquanto sete gêneros ocorreram apenas no DT de insetos em riachos do Cerrado. O gênero Penicillium (40%) foi o mais frequente. No Cerrado, 78% foram produtoras de CMCase, mais que o dobro da Floresta Amazônica (35%). E 62% foram produtoras de xilanase, no Cerrado, e 71% na Floresta Amazônica. Nesse contexto, a comunidade fúngica do TD de larvas Triplectides pode desempenhar um papel importante na dieta de insetos por quebrar o material lignocelulósico.(AU)

Diversity of cellulolytic and xylanolytic fungi associated with the digestive tract of aquatic insect larvae in streams of the Amazon Forest and Cerrado in Brazil / Diversidade de fungos celulolíticos e xilanolíticos associados ao trato digestivo de larvas de insetos aquáticos em riachos de Floresta Amazônica e Cerrado no Brasil

The study of the relationship between fungi and insects brings important contributions to the knowledge of fungal biodiversity and to the understanding of mutualistic ecological interactions. This study reports the occurrence of a community of filamentous fungi in the digestive tract (DT) of mining insect larvae belonging to genus Stenochironomus in streams of two Brazilian biomes. Fungi were obtained from the digestive tract of larvae found on trunks and leaves of low-order streams in the Amazon Forest and Cerrado in the north of Brazil. The fungal community was screened for xylanolytic and cellulolytic activities. The diversity of fungal species in the DT of larvae is possibly related to the diversity of diets of species of that genus and the diversity of substrates in the ecosystems. The diversity and richness of fungal species were influenced by ecological differences between locations more than by the types of substrates in which they were collected (trunk and leaf). Most fungi in the DT of Stenochironomus larvae sampled in leaves exhibited cellulolytic enzyme activity. Such results stress that the mycobiomes of the DT of Stenochiromonus larvae produce enzymes that contribute to the process of breaking down plant remains in their hosts.

O estudo da relação entre fungos e insetos traz importantes contribuições para o conhecimento da biodiversidade fúngica e para o entendimento das interações ecológicas mutualísticas. Este estudo relata a ocorrência de uma comunidade de fungos filamentosos no trato digestivo (TD) de larvas minadoras de insetos do gênero Stenochironomus em riachos de dois biomas brasileiros. Os fungos foram obtidos do trato digestivo de larvas encontradas em troncos e folhas de riachos de baixa ordem na Floresta Amazônica e Cerrado no norte do Brasil. A comunidade fúngica foi triada para atividades xilanolíticas e celulolíticas. A diversidade de espécies fúngicas no TD de larvas possivelmente está relacionada à diversidade de dietas das espécies desse gênero e à diversidade de substratos nos ecossistemas. A diversidade e riqueza de espécies fúngicas foram influenciadas mais pelas diferenças ecológicas entre os locais do que pelos tipos de substratos em que foram coletados (tronco e folha). A maioria dos fungos no TD de larvas de Stenochironomus amostradas em folhas exibiu atividade enzimática celulolítica. Tais resultados reforçam que os micobiomas do DT de larvas de Stenochiromonus produzem enzimas que contribuem para o processo de decomposição de restos vegetais em seus hospedeiros.

Lipolytic Enzymes with Hydrolytic and Esterification Activities Produced by Filamentous Fungi Isolated from Decomposition Leaves in an Aquatic Environment.

Microbial lipases are prominent biocatalysts able to catalyze a wide variety of reactions in aqueous and nonaqueous media. In this work, filamentous fungi isolated from leaves decomposed in an aquatic environment were screened for lipase production with hydrolytic activity and esterification. Agar plates with Tween 20 and Rhodamine B were used for selection, while submerged cultures with olive oil were subsequently used to select 38 filamentous fungi. Trichoderma harzianum, Fusarium solani, Trichoderma harzianum F5, and Penicillium sp. F36 were grown in six different culture media. F. solani presented the highest lipase production (2.37 U/mL) with esterification activity of 0.07 U/mL using medium composed of (g.L-1) KH2PO4 1.00, MgSO4 H2O 1.123, and CuSO4 0.06. Supplementation of this culture medium with organic nitrogen sources increased lipase production by 461.3% using tryptone and by 419.4% using yeast extract. Among the vegetable oils from the Amazon region, degummed cotton oil induced lipase production up to 8.14 U/mL. The lipase produced by F. solani F61 has great potential to application in conventional processes and biodiesel production by transesterification of vegetable oils, as well as food industries in the production of fatty acid esters by hydrolysis and esterification.

Feeding preference of the shredder Phylloicus sp. for plant leaves of Chrysophyllum oliviforme or Miconia chartacea after conditioning in streams from different biomes / Preferência alimentar do fragmentador Phylloicus sp. por folhas de Chrysophyllum oliviforme e Miconia chartacea após condicionadas em riachos de dois biomas

Abstract Macroinvertebrate shredders consume preferably leaves conditioned by fungi and bacteria which offer greater palatability to them. Plant species in Cerrado present high concentration of chemical elements such as lignin and cellulose, phenols and tanins thus making them less attractive for shredders consumption and limiting the palatability. This study aimed to evaluate the feeding preference of a macroinvertebrate shredder of the genus Phylloicus for plant material from two different biomes (Cerrado and Mata Atlântica), after conditioning in a stream of Mata Atlântica and observing their physical and chemical characteristics. Senescent leaves were collected, monthly from the litterfall of riparian vegetation in a 500 m stretch of a stream in each biome from August 2014 to January 201. The most abundant species in each stream was selected for the experiment. The experimental design consisted in with two treatments. The first (T1) comprised leaf discs from Chrysophyllum oliviforme (Cerrado species) together with leaf discs of Miconia chartacea (Atlantic Forest species) which were conditioned in the Atlantic Forest stream. The second treatment (T2) involved leaf discs of Miconia chartacea conditioned in Mata Altlântica and Cerrado streams. Both tests had showed significant differences between the two treatments (T1 and T2). For T1, there was consumption of M. chartacea leaf discs by Phylloicus sp., but there was no consumption of C. oliviforme discs. For T2, there was preference for M. chartaceae leaves conditioned in a stream of Mata Atlântica than in Cerrado stream. The results showed that Phylloicus sp., had presented preference for food detritus of the Mata Antlântica biome and rejection to the one from Cerrado biome.

Resumo Macroinvertebrados fragmentadores consomem folhas preferencialmente condicionadas por fungos e bactérias que lhes oferece uma maior palatabilidade. Nas espécies do cerrado esse condicionamento está também associado às altas concentrações de elementos químicos limitantes à palatabilidade como alto teor de lignina e celulose, que tornam as folhas menos atrativas para os fragmentadores. O trabalho teve como objetivo avaliar a preferência alimentar de macroinvertebrados fragmentador ( Phylloicus sp.) por material vegetal de dois diferentes biomas (Cerrado e Mata Atlântica), após condicionamento em riacho de Mata Atlântica, observando suas características físicas e químicas. Foram coletadas folhas senescentes do aporte vegetal (AV) de espécies nativas da vegetação ripária nesses dois biomas, com periodicidade mensal em um trecho de 500 m de um córrego em cada bioma. O experimento foi delineado com dois tratamentos. O primeiro (T1) compreendeu discos de folhas do Cerrado (Chrysophyllum oliviforme) mais discos de folhas da Mata Atlântica (Miconia chartacea) que foram condicionadas em córrego de Mata Atlântica. O segundo tratamento (T2) envolveu discos de folhas da Mata Atlântica condicionadas em córrego da Mata Atlântica mais discos de folhas da Mata Atlântica condicionadas em córrego do Cerrado. Os dois testes apontaram diferenças significativas entre os dois tratamentos (T1 e T2). Para T1 houve consumo de discos de folha de M. chartacea por Phylloicus sp, mas não houve consumo dos discos de C. oliviforme , de Cerrado. Para T2, houve o consumo, porém a preferência pelas folhas de M. chartaceae condicionadas no córrego da Mata Atlantica foi consideravelmete maior. Os resultados apontam que Phylloicus sp, apresentou preferência alimentar pelo detrito de bioma de Mata Atlântica e rejeição pelo detrito do bioma Cerrado.

Feeding preference of the shredder Phylloicus sp. for plant leaves of Chrysophyllum oliviforme or Miconia chartacea after conditioning in streams from different biomes.

Macroinvertebrate shredders consume preferably leaves conditioned by fungi and bacteria which offer greater palatability to them. Plant species in Cerrado present high concentration of chemical elements such as lignin and cellulose, phenols and tanins thus making them less attractive for shredders consumption and limiting the palatability. This study aimed to evaluate the feeding preference of a macroinvertebrate shredder of the genus Phylloicus for plant material from two different biomes (Cerrado and Mata Atlântica), after conditioning in a stream of Mata Atlântica and observing their physical and chemical characteristics. Senescent leaves were collected, monthly from the litterfall of riparian vegetation in a 500 m stretch of a stream in each biome from August 2014 to January 201. The most abundant species in each stream was selected for the experiment. The experimental design consisted in with two treatments. The first (T1) comprised leaf discs from Chrysophyllum oliviforme (Cerrado species) together with leaf discs of Miconia chartacea (Atlantic Forest species) which were conditioned in the Atlantic Forest stream. The second treatment (T2) involved leaf discs of Miconia chartacea conditioned in Mata Altlântica and Cerrado streams. Both tests had showed significant differences between the two treatments (T1 and T2). For T1, there was consumption of M. chartacea leaf discs by Phylloicus sp., but there was no consumption of C. oliviforme discs. For T2, there was preference for M. chartaceae leaves conditioned in a stream of Mata Atlântica than in Cerrado stream. The results showed that Phylloicus sp., had presented preference for food detritus of the Mata Antlântica biome and rejection to the one from Cerrado biome.

Feeding preference of the shredder Phylloicus sp. for plant leaves of Chrysophyllum oliviforme or Miconia chartacea after conditioning in streams from different biomes / Preferência alimentar do fragmentador Phylloicus sp. por folhas de Chrysophyllum oliviforme e Miconia chartacea após condicionadas em riachos de dois biomas

Macroinvertebrate shredders consume preferably leaves conditioned by fungi and bacteria which offer greater palatability to them. Plant species in Cerrado present high concentration of chemical elements such as lignin and cellulose, phenols and tanins thus making them less attractive for shredders consumption and limiting the palatability. This study aimed to evaluate the feeding preference of a macroinvertebrate shredder of the genus Phylloicus for plant material from two different biomes (Cerrado and Mata Atlântica), after conditioning in a stream of Mata Atlântica and observing their physical and chemical characteristics. Senescent leaves were collected, monthly from the litterfall of riparian vegetation in a 500 m stretch of a stream in each biome from August 2014 to January 201. The most abundant species in each stream was selected for the experiment. The experimental design consisted in with two treatments. The first (T1) comprised leaf discs from Chrysophyllum oliviforme (Cerrado species) together with leaf discs of Miconia chartacea (Atlantic Forest species) which were conditioned in the Atlantic Forest stream. The second treatment (T2) involved leaf discs of Miconia chartacea conditioned in Mata Altlântica and Cerrado streams. Both tests had showed significant differences between the two treatments (T1 and T2). For T1, there was consumption of M. chartacea leaf discs by Phylloicus sp., but there was no consumption of C. oliviforme discs. For T2, there was preference for M. chartaceae leaves conditioned in a stream of Mata Atlântica than in Cerrado stream. The results showed that Phylloicus sp., had presented preference for food detritus of the Mata Antlântica biome and rejection to the one from Cerrado biome.(AU)

Macroinvertebrados fragmentadores consomem folhas preferencialmente condicionadas por fungos e bactérias que lhes oferece uma maior palatabilidade. Nas espécies do cerrado esse condicionamento está também associado às altas concentrações de elementos químicos limitantes à palatabilidade como alto teor de lignina e celulose, que tornam as folhas menos atrativas para os fragmentadores. O trabalho teve como objetivo avaliar a preferência alimentar de macroinvertebrados fragmentador ( Phylloicus sp.) por material vegetal de dois diferentes biomas (Cerrado e Mata Atlântica), após condicionamento em riacho de Mata Atlântica, observando suas características físicas e químicas. Foram coletadas folhas senescentes do aporte vegetal (AV) de espécies nativas da vegetação ripária nesses dois biomas, com periodicidade mensal em um trecho de 500 m de um córrego em cada bioma. O experimento foi delineado com dois tratamentos. O primeiro (T1) compreendeu discos de folhas do Cerrado (Chrysophyllum oliviforme) mais discos de folhas da Mata Atlântica (Miconia chartacea) que foram condicionadas em córrego de Mata Atlântica. O segundo tratamento (T2) envolveu discos de folhas da Mata Atlântica condicionadas em córrego da Mata Atlântica mais discos de folhas da Mata Atlântica condicionadas em córrego do Cerrado. Os dois testes apontaram diferenças significativas entre os dois tratamentos (T1 e T2). Para T1 houve consumo de discos de folha de M. chartacea por Phylloicus sp, mas não houve consumo dos discos de C. oliviforme , de Cerrado. Para T2, houve o consumo, porém a preferência pelas folhas de M. chartaceae condicionadas no córrego da Mata Atlantica foi consideravelmete maior. Os resultados apontam que Phylloicus sp, apresentou preferência alimentar pelo detrito de bioma de Mata Atlântica e rejeição pelo detrito do bioma Cerrado.(AU)

Integrated control of penicillium digitatum by the predacious yeast Saccharomycopsis crataegensis and sodium bicarbonate on oranges

Our investigation of integrated biological control (IBC) started with an assay testing activity of the predacious yeast Saccharomycopsis crataegensis UFMG-DC19.2 against Penicillium digitatum LCP 4354, a very aggressive fungus that causes postharvest decay in oranges. Under unfavourable environmental conditions, the yeast showed a high potential for control (39.9 percent disease severity reduction) of this fungus. This result was decisive for the next step, in which S. crataegensis was tested in association with sodium bicarbonate salt, a generally regarded as safe (GRAS) substance. The yeast was able to survive at different concentrations of the salt (1 percent, 2 percent and 5 percent), and continued to grow for a week at the wound site, remaining viable at high population for 14 days on the fruit surface. The yeast alone reduced the severity of decay by 41.7 percent and sodium bicarbonate alone reduced severity of decay by 19.8 percent, whereas the application of both led to a delay in the development of symptoms from 2 to 10 days. Ingredients of the formulations were not aggressive to fruits since no lesions were produced in control experiments.

Integrated control of Penicillium digitatum by the predacious yeast Saccharomycopsis crataegensis and sodium bicarbonate on oranges.

Our investigation of integrated biological control (IBC) started with an assay testing activity of the predacious yeast Saccharomycopsis crataegensis UFMG-DC19.2 against Penicillium digitatum LCP 4354, a very aggressive fungus that causes postharvest decay in oranges. Under unfavourable environmental conditions, the yeast showed a high potential for control (39.9% disease severity reduction) of this fungus. This result was decisive for the next step, in which S. crataegensis was tested in association with sodium bicarbonate salt, a generally regarded as safe (GRAS) substance. The yeast was able to survive at different concentrations of the salt (1%, 2% and 5%), and continued to grow for a week at the wound site, remaining viable at high population for 14 days on the fruit surface. The yeast alone reduced the severity of decay by 41.7% and sodium bicarbonate alone reduced severity of decay by 19.8%, whereas the application of both led to a delay in the development of symptoms from 2 to 10 days. Ingredients of the formulations were not aggressive to fruits since no lesions were produced in control experiments.

Trehalose accumulation by tropical yeast strains submitted to stress conditions.

Trehalose, a non-reducing disaccharide that accumulates in Saccharomyces cerevisiae, has been implicated in survival under various stress conditions by acting as membrane protectant, as a supplementary compatible solute or as a reserve carbohydrate which may be mobilized during stress. However, most of these studies have been done with strains isolated from European or Asian habitats of temperate climate. In this study, yeasts living in tropical environments, isolated from different microhabitats in Southeastern Brazil, were used to evaluate whether trehalose contributes to survival under osmotic, ethanol and heat stress. The survival under severe stress was compared to a well-characterized laboratorial wild-type strain (D273-10B). Most of the Saccharomyces cerevisiae strains isolated from Drosophila in Tropical Rain Forest were able to accumulate trehalose after a preconditioning treatment at 40 degrees C for 1 h. The amount of intracellular trehalose levels was better correlated with survival during a challenging heat shock at 50.5 degrees C for 8 min. Saccharomyces cerevisiae and Candida guilliermondii were observed to be thermotolerant as well as osmotolerant. No clear correlation between intracellular trehalose levels and survival could be derived during ethanol stress. In some cases, the amount of trehalose accumulated before the ethanol stress seemed to play an important role for the survival of these strains.

Screening of yeasts from Brazilian Amazon rain forest for extracellular proteinases production.

Eighty seven yeast strains representing 34 species isolated from Parahancornia amapa fruit and associated Drosophila flies collected in the Brazilian Amazon rain forest, were screened for proteinase production. Proteolytic activity was tested through casein hydrolysis in solid medium supplemented with 0.5% casein and glucose. Among 23 strains, 18 from genus Candida and 5 from Pichia were caseinolytic and produced proteinases in yeast carbon base liquid medium supplemented with casein 0.01%. The proteolytic activity was tested on pH ranging from 2.0 to 9.0 in correspondence to the pH of the cultures media in which the yeasts were grown. Six highly proteolytic strains: Candida parapsilosis AP153A, C. krusei AP176, C. sorbosa DR215, C. sorbosa AP259, C. valida AP209A and C. sorboxylosa AP287 were selected and the pH optima of production and the proteolytic activity were determined. In general the secretion of proteinase was maximum throughout the exponential and the stationary phases. Greater production occurred in acidic culture and high activity was observed at pH 3.0, 4.0 and 5.0.

The incidence of killer activity and extracellular proteases in tropical yeast communities.

The presence of killer and proteolytic yeasts was studied among 944 isolates representing 105 species from tropical yeast communities. We found 13 killer toxin producing species, with Pichia kluyveri being the most frequent. Other killer yeast isolates were Candida apis, Candida bombicola, Candida fructus, Candida krusei, Candida sorbosa, Hanseniaspora uvarum, Issatchenkia occidentalis, Kloeckera apis, Kluyveromyces marxianus, Pichia membranaefaciens, Pichia ohmeri-like, and Sporobolomyces roseus. The communities from which killer yeasts were isolated had strains sensitive to them, and there were interspecific and intraspecific differences in the spectra of their killer activities. Pichia kluyveri had the broadest spectra of activity against sensitive isolates, and it apparently produced different toxins. The coexistence of sensitive and killer yeasts using the same substrate suggests that there is spatial separation in microhabitats or temporal separation in different stages of successions. Basidiomycetous yeasts were more frequently proteolytic than ascomycetous yeasts. Extracellular proteases could be important for the yeasts to have access to more nitrogen nutrients and obtain a better balance with available carbon sources.

Yeast succession in the Amazon fruit Parahancornia amapa as resource partitioning among Drosophila spp.

The succession of yeasts colonizing the fallen ripe amapa fruit, from Parahancornia amapa, was examined. The occupation of the substrate depended on both the competitive interactions of yeast species, such as the production of killer toxins, and the selective dispersion by the drosophilid guild of the amapa fruit. The yeast community associated with this Amazon fruit differed from those isolated from other fruits in the same forest. The physiological profile of these yeasts was mostly restricted to the assimilation of a few simple carbon sources, mainly L-sorbose, D-glycerol, DL-lactate, cellobiose, and salicin. Common fruit-associated yeasts of the genera Kloeckera and Hanseniaspora, Candida guilliermondii, and Candida krusei colonized fruits during the first three days after the fruit fell. These yeasts were dispersed and served as food for the invader Drosophila malerkotliana. The resident flies of the Drosophila willistoni group fed selectively on patches of yeasts colonizing fruits 3 to 10 days after the fruit fell. The killer toxin-producing yeasts Pichia kluyveri var. kluyveri and Candida fructus were probably involved in the exclusion of some species during the intermediate stages of fruit deterioration. An increase in pH, inhibiting toxin activity and the depletion of simple sugars, may have promoted an increase in yeast diversity in the later stages of decomposition. The yeast succession provided a patchy environment for the drosophilids sharing this ephemeral substrate.

Yeast communities as descriptors of habitat use by the Drosophila fasciola subgroup (repleta group) in Atlantic rain forests.

Yeast communities associated with four species of the Drosophila fasciola subgroup (repleta group) in tropical rain forests were surveyed in an abandoned orchard, and rain forest sites of Rio de Janeiro and Ilha Grande, State of Rio de Janeiro, Brazil. Adult flies of Drosophila carolinae, Drosophila coroica, Drosophila fascioloides and Drosophila onca frequently carried Candida colliculosa, Geotrichum sp, Kloeckera apiculata and a Pichia membranaefaciens-like species. The most frequent yeasts in the crop of flies included Candida collicullosa, C. krusei, Pichia kluyveri and a P. membranaefaciens-like species. The physiological abilities and species composition of these yeast communities differed from those of other forest-inhabiting Drosophila. The narrow feeding niches of the fasciola subgroup suggested the use of only part of the substrates available to the flies as food in the forest environment, as noted previously for cactophilic Drosophila serido (mulleri subgroup of the repleta group) in a sand dune ecosystem. The cactophilic yeasts that were isolated have not been previously found in forests. The fasciola subgroup probably used epiphytic cactus substrates as breeding and feeding sites in the forest. The physiological profile of yeasts associated with the fasciola flies was broader than that of yeasts associated with the cactophilic Drosophila serido, suggesting that the fasciola subgroup represents an older lineage from which the South American repleta species evolved.

Yeast communities of the cactus Pilosocereus arrabidae and associated insects in the Sandy coastal plains of southeastern Brazil.

The yeast communities from necrotic tissues, decaying flowers and fruits, and from larval feeding sites of the moth Sigelgaita sp. in the cactus Pilosocereus arrabidae were surveyed in three restinga ecosystems in Southeastern Brazil. Insects associated with these substrates were sampled to verify the vectoring of yeasts. The cactus Pilosocereus arrabidae was shown to have four different yeast communities associated with it. Necrotic stems had a diverse yeast community with the prevalent species Pichia barkeri, Candida sonorensis, Pichia cactophila, Geotrichum sp., Myxozyma mucilagina and Sporopachydermia sp. A, representing about 80% of the total isolates. Pichia sp. A and a Candida domercqii-like species represented more than 90% of the yeast isolates from decaying flowers. Fruits had a heterogeneous yeast community with typical fruit yeasts of the genus Kloeckera, basidiomicetous anamorphs of the genus Cryptococcus, the black yeast Aureobasidium pullulans, Pichia sp. A, a Candida domercqii-like species, and some cactophilic yeasts, especially Clavispora opuntiae. The feeding site of Sigelgaita sp. larvae had Clavispora opuntiae as the prevalent species. Insect vectors are suggested as one the most important factors influencing the composition of these yeast communities.

Yeast communities of the cactus Pilosocereus arrabidae as resources for larval and adult stages of Drosophila serido.

The feeding behavior of Drosophila serido on the yeast communities of necrotic stem tissue of Pilosocereus arrabidae were studied in a sand dune ecosystem of Rio de Janeiro, Brazil. The prevalence of cactophilic yeasts including Pichia barkeri, Candida sonorensis and Geotrichum sp. in the crops and external surfaces of D. serido reflected its association with the cactus habitat. The effective number of yeasts vectored on the surface of flies was higher than that in the crops. Also overlap between the yeasts from stems and from crops was partial suggesting selective feeding by the flies in the substrates visited. The females had a higher effective number of yeast species and a lower similarity than males with the yeast community of P. arrabidae. This was probably related to the search for oviposition sites by females. The presence of Pichia thermotolerans-like and Pichia amethionina var pachycereana in the flies, but not in P. arrabidae stems, indicated that D. serido was not limited to this cactus species. The larvae and adults lived in different patches with the adults feeding in patches with higher yeast species richness. The larvae had a narrower feeding niche and higher overlap with P. arrabidae, and preferred P. barkeri and Pichia cactophila as food. Adult flies fed on patches with the most frequent yeasts except for P. cactophila. Pichia caribaea was found in higher frequency in the adult crops than in the stems. Our data suggested that there was food selection and diet partitioning between adult and larval stages of D. serido.

Yeasts and coliform bacteria of water accumulated in bromeliads of mangrove and sand dune ecosystems of southeast Brazil.

Yeasts and coliform bacteria were isolated from water that accumulated in the central cups and adjacent leaf axilae of two bromeliads, Neoregelia cruenta of a coastal sand dune and Quesnelia quesneliana of a mangrove ecosystem near the city of Rio de Janeiro, Brazil. The mean total coliform counts were above 10,000 per 100 mL for waters of both plants, but the mean fecal coliform counts were only 74 per 100 mL for Q. quesneliana and mostly undetected in water from N. cruenta. Of 90 fecal coliform isolates, 51 were typical of Escherichia coli in colony morphology and indol, methyl red, Volges-Proskauer, and citrate (IMViC) tests. Seven representatives of the typical E. coli cultures were identified as this species, but the identifications of nine other coliform bacteria were mostly dubious. The yeast community of N. cruenta was typical of plant surfaces with basidiomycetous yeasts anamorphs, and the black yeast Aureobasidium pullulans was prevalent. Quesnelia quesneliana had a substantial proportion of ascomycetous yeasts and their anamorphs, including a probable new biotype of Saccharomyces unisporus. Our results suggested that the microbial communities in bromeliad waters are typically autochtonous and not contaminants.