Diversity of culturable endophytic fungi vary through time in Manihot esculenta Crantz / A diversidade de fungos endofíticos cultiváveis ​​varia ao longo do tempo em Manihot esculenta Crantz

Abstract Endophytic fungi are a ubiquituos group that colonize all plant species on earth. Studies comparing the location of endophytic fungi within the leaves and the sampling time in Manihot esculenta Crantz (cassava) are limited. In this study, mature leaves of M. esculenta from Panama were collected in order to compare the cultivable diversity of endophytic fungi and to determine their distribution within the leaves. A total of one hundred sixty endophytes belonging to 97 species representing 13 genera and 8 morphospecies determined as mycelia sterilia that containing 63 isolates were isolated. Cladosporium, Nigrospora, Periconia, and mycelia sterilia 1 and 3 were the most predominant isolated endophytes. We detected that endophytes varied across the sampling time, but not amongst locations within leaves. The endophytes composition across sampling and the location of endophytes within leaf was similar, except for Periconia and mycelia sterilia 3 and 7. The data generated in this study contribute to the knowledge on the biodiversity of endophytic fungi in Panama, and establish the bases for future research focused on understanding the function of endophytes in M. esculenta crops.

Resumo Os fungos endofíticos são um grupo ubiquituo que colonizam todas as espécies de plantas na terra. Os estudos que comparam a localização dos fungos endofíticos dentro das folhas de Manihot esculenta Crantz (mandioca) e o tempo de amostragem são muito escassos. Neste estudo, folhas maduras de M. esculenta foram coletadas do Panamá com a finalidade de comparar a diversidade cultivável de endófitos e determinar sua distribuição dentro das folhas. Um total de 170 endófitos foram isolados de 97 espécies que representam 13 gêneros e 8 morfoespécies determinadas como micélios esterilizados contendo 63 isolados. Os fungos Cladosporium, Nigrospora, Periconia e mycelia sterilia 1 e 3 foram os isolados mais predominantes. Também detectamos que os endófitos variaram ao longo do tempo de amostragem, mas não entre os locais dentro das folhas. A composição de endófitos na amostragem e localização de endófitos dentro da folha foi semelhante, exceto para Periconia e mycelia sterilia 3 e 7. Os dados gerados neste estudo contribuem para o conhecimento da biodiversidade de fungos endofíticos no Panamá e estabelecem as bases para pesquisas sobre o entendimento da função de endófitos em culturas de M. esculenta.

Isolation and morphological identification of endophytic fungi from leaves and stems of Aquilaria malaccensis Lamk. originating from Java Island, Indonesia

Aims@#Endophytic fungi are the remarkable category of host-associated fungal community that invades the intercellular regions of host tissues, benefiting their host while obtaining an advantage. Fungal endophytes have lately attracted prominence as a source of active secondary metabolites. This investigation aimed to identify fungal endophytes that reside inside the leaves and stems of Aquilaria malaccensis.@*Methodology and results@#Healthy A. malaccensis stems and leaves samples were collected. Clean leaves and stems were cut to a size of 1 cm, followed by sterilization using 75% ethanol for 1 min, 3% sodium hypochlorite solution for 1 min, and finally, rinsing with sterile water 3 times for 1 min and drying with sterile paper. The sterile samples were put onto Potato Dextrose Agar (PDA) media containing chloramphenicol for 7-14 days until the mycelium grew for morphological identification under a light microscope. Five endophytic fungi were recovered from leaves, while nine endophytic fungi were obtained from stems. Using morphological approaches, nine of the endophytes had observed to produce conidia fungi, whereas the others did not. Neopestalotiopsis sp., Aspergillus sp., Arthrinium sp., Curvularia sp., Podospora sp., Mucor sp. and Verticillium sp. were identified as nine of the fourteen endophytes.@*Conclusion, significance and impact of study@#The number of endophytic fungi discovered in different organs varies. Not all endophytic fungi that grow can create sexual phages. Six genera of endophytic fungi were identified.

Endophytic Bacillus subtilis P10 from Prunus cerasifera as a biocontrol agent against tomato Verticillium wilt / Bacillus subtilis P10 endofítico de Prunus cerasifera como agente de biocontrole contra murcha de Verticillium de tomate

Endophytic bacteria serve key roles in the maintenance of plant health and growth. Few studies to date, however, have explored the antagonistic and plant growth-promoting (PGP) properties of Prunus cerasifera endophytes. To that end, we isolated endophytic bacteria from P. cerasifera tissue samples and used a dual culture plate assay to screen these microbes for antagonistic activity against Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum, and F. moniliforme. Of the 36 strains of isolated bacteria, four (strains P1, P10, P16, and P20) exhibited antagonistic effects against all five model pathogens, and the P10 strain exhibited the strongest antagonistic to five pathogens. This P10 strain was then characterized in-depth via phenotypic assessments, physiological analyses, and 16s rDNA sequencing, revealing it to be a strain of Bacillus subtilis. Application of a P10 cell suspension (1×108 CFU/mL) significantly enhanced the seed germination and seedling growth of tomato in a greenhouse setting. This P10 strain further significantly suppressed tomato Verticillium wilt with much lower disease incidence and disease index scores being observed following P10 treatment relative to untreated plants in pot-based experiments. Tomato plants that had been treated with strain P10 also enhanced defense-related enzymes, peroxidase, superoxide dismutase, and catalase activity upon V. dahliae challenge relative to plants that had not been treated with this endophytic bacterium. The results revealed that the P10 bacterial strain has potential value as a biocontrol agent for use in the prevention of tomato Verticillium wilt.

As bactérias endofíticas desempenham papel fundamental na manutenção da saúde e do crescimento das plantas. Poucos estudos até o momento, no entanto, exploraram as propriedades antagônicas e promotoras de crescimento de plantas (PGP) de endófitos de Prunus cerasifera. Para esse fim, isolamos bactérias endofíticas de amostras de tecido de P. cerasifera e usamos um ensaio de placa de cultura dupla para rastrear esses micróbios quanto à atividade antagonista contra Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum e F. moniliforme. Das 36 cepas de bactérias isoladas, quatro (cepas P1, P10, P16 e P20) exibiram efeitos antagônicos contra todos os cinco patógenos modelo, e a cepa P10 exibiu o antagonista mais forte para cinco patógenos. Essa cepa P10 foi então caracterizada em profundidade por meio de avaliações fenotípicas, análises fisiológicas e sequenciamento de rDNA 16s, revelando ser uma cepa de Bacillus subtilis. A aplicação de uma suspensão de células P10 (1 × 108 UFC / mL) aumentou significativamente a germinação das sementes e o crescimento das mudas de tomate em casa de vegetação. Essa cepa P10 suprimiu ainda mais a murcha de Verticillium do tomate com incidência de doença muito menor e pontuações de índice de doença sendo observadas após o tratamento com P10 em relação a plantas não tratadas em experimentos baseados em vasos. As plantas de tomate que foram tratadas com a cepa P10 também aumentaram as enzimas relacionadas à defesa, peroxidase, superóxido dismutase e atividade da catalase após o desafio de V. dahliae em relação às plantas que não foram tratadas com essa bactéria endofítica. Os resultados revelaram que a cepa bacteriana P10 tem valor potencial como agente de biocontrole para uso na prevenção da murcha de Verticillium em tomate.

Endophytic bacteria Bacillus safensis and Pseudomonas hibiscicola and their ability to increase rice seedling growth

Endophytic bacteria Bacillus safensis RS95 and Pseudomonas hibiscicola RS121 were evaluated for their ability to promote the growth of rice seedlings and produce indole-acetic acid (IAA) and siderophores and to solubilize phosphates. 'Guri' rice seeds were immersed in bacterial endophyte cell suspensions (separated and two-strain mixed), as well as in Escherichia coli DH5α, phosphate-buffered saline (PBS) and water treatments (negative controls). Seeds were sown on agar-water in Petri plates placed vertically at an angle of 65°. The ability of plant growth-promoting endophytic bacteria (PGPEB) to produce IAA and siderophores was determined by Salkowski colorimetric and chrome azurol S (CAS) assays, respectively. Mineral phosphate solubilization activity was calculated by inoculating the endophytes onto medium containing insoluble phosphate. PGPEB showed a positive effect on the growth of rice seedlings, causing a mean growth of shoots and primary-roots of 60 and 67%, respectively. Bacterial strains also showed positive traits for IAA and siderophore production, as well as phosphate-solubilization activity

Biocontrol potential of endophytic fungi in tea (Camellia sinensis (L.) Kuntze) leaves against selected fungal phytopathogens

Aims@#Endophytic fungi are a diverse group of microorganisms that stay asymptomatically in the healthy tissues of the host. Many fungal endophytes are associated with the tea plant (Camellia sinensis) and the pathogens of the tea plant have the potential to grow as endophytes or act as latent pathogens during the initial growth of the plant. The present study aimed at screening tea endophytic fungi with the potential for control of a few critical phytopathogens, Fusarium sp., Lasiodiplodia theobromae, Pestalotiopsis sp. and Sclerotinia sclerotiorum while evaluating the efficiency of growth inhibition of these phytopathogens by the endophytic fungal isolates using in vitro assays.@*Methodology and results@#Five endophytic fungal strains; Colletotrichum gloeosporioides, C. siamense, Daldinia eschscholtzii, Pseudopestalotiopsis chinensis and Phyllosticta capitalensis isolated from leaves of C. sinensis in Sri Lanka were evaluated for growth inhibition against plant pathogens; Fusarium sp., L. theobromae, Pestalotiopsis sp. and S. sclerotiorum using the dual culture assay and volatile compound-mediated inhibition assay. All the fungal endophytes used in this study exhibited antifungal activity against Fusarium sp., Pestalotiopsis sp. and S. sclerotiorum in the dual culture assay on PDA. Daldinia eschscholtzii (67.30%) and C. gloeosporioides (61.54%) showed strong antagonistic activity against S. sclerotiorum, while P. capitalensis (42.30%) demonstrated moderate activity. All the endophytic fungal strains showed moderate antifungal activities against Fusarium sp. The percentage growth inhibitions of Pestalotiopsis sp. by all the endophytic isolates tested were below 25.00%. In the volatile compound-mediated inhibition assay, none of the endophytic isolates showed visible inhibition against the phytopathogens used.@*Conclusion, significance and impact of study@#In this study, the fungal endophytes which showed potential antagonistic activity against the tested phytopathogens can be used to develop commercialized products of fungal biocontrol agents (BCAs) for controlling plant fungal diseases.

Genome analysis and plant growth promoting activity of Pseudonocardia strain DR1-2 from the root of Dendrobium christyanum Rchb.f

Aims@#The objective of this study was to analyze the genome of endophytic actinomycete associated with orchids and evaluate its plant hormone activities, including phytohormone, siderophore, ammonia production, zinc and phosphate solubilization.@*Methodology and results@#Strain DR1-2 isolated from the roots of the Thai orchid, Dendrobium christyanum Rchb.f., was closely related to Pseudonocardia alni DSM 44104T, P. antarctica DSM 44749T and P. carboxydivorans Y8T (99.93-100% similarity) based 16S rRNA gene sequence. This strain exhibited IAA production (294.10 ± 12.17 μg/mL), phosphate solubilization (2.20 ± 0.08 solubilization Index, SI), positive for siderophore production and ammonia production (36.99 ± 2.24 μg/mL). It showed a maximum IAA of 489.73 ± 8.90 μg/mL, when optimized using 0.5% Ltryptophan, pH 6 and incubated at 30 °C for 7 days. The IAA of strain enhanced the root length, shoot length, number of roots and fresh weight of rice seedlings (Oryza sativa L. cv. RD49). The draft genome of strain DR1-2 was 6,077,423 bp in 23 contigs with G+C content of 74.6%. The average nucleotide identity-Blast (ANIb) and average nucleotide identity-MUMmer (ANIm) values of strain DR1-2 and related type strains were 95.81 to 97.25% and the digital DNA-DNA hybridization (dDDH) values were 72.60 to 74.00%, respectively. Genomic analysis of strain DR1-2 revealed that the gene encodes the enzyme involved in the phytohormones biosynthesis and gene clusters involved in the biosynthesis of bioactive metabolites.@*Conclusion, significance and impact of study@#Endophytic actinomycete, Pseudonocardia strain DR1-2 from Thai orchid, D. christyanum Rchb.f., exhibited significant IAA production and affected the growth of the plant, which was the potential source of plant hormones for agricultural applications.

Mycosynthesis of thermostable silver nanoparticles by the endophytic Albifimbria verrucaria with antimicrobial and antiproliferative activities

Aims@#This study was aimed to screen and isolate soil and endophytic fungi with the ability to biosynthesize stable silver nanoparticles (SNPs) with antimicrobial and antiproliferative activities.@*Methodology and results@#A total of 60 fungal isolates isolated from soil and plant samples were screened for their ability to biosynthesize SNPs. Among which, 21 isolates have supported the biosynthesis of SNPs. Furthermore, the endophytic isolate PRR2.1 synthesized highly thermostable SNPs with long shelf life. The PRR2.1 isolate was identified as Albifimbria verrucaria by morphological and molecular means. The synthesis of SNPs was initially monitored by UV-Vis spectroscopy. Further characterization by transmission electron microscopy, X-ray diffraction and dynamic light scattering revealed well-dispersed spherical crystalline in nature SNPs with a mean size of 14 nm and zeta potential of –24.47 mV. Fourier transform infrared spectroscopy showed the presence of biomolecules adsorbed on the surface of biosynthesized SNPs responsible for their synthesis and stability. The mycosynthesized SNPs exhibited stronger antifungal activity against pathogenic strains of Aspergillus niger, A. flavus, A. fumigatus and Candida albicans with respect to its antibacterial activity against Escherichia coli, Staphylococcus aureus, Bacillus cereus and Klebsiella pneumoniae compared to standard antifungal itraconazole and antibiotic cefadroxil with mostly consistent minimum inhibitory concentration of 5.31 μg/mL. The biosynthesized SNPs demonstrated dose-dependent in vitro antiproliferative activity against cancerous HeLa cell line with IC50 value of 2.52 μg/mL and less cytotoxic activity against WI-38 (normal human lung fibroblasts) cell line with CC50 value of 10.2 μg/mL.@*Conclusion, significance and impact of study@#These results show the potential of endophytic fungi biosynthesized SNPs as possible biofriendly, safe and efficient antimicrobial agents with promising antiproliferative activity and low cytotoxicity, which can be furtherly implemented in various biomedical and biotechnological applications.

Secondary metabolites of endophyte fungi Xylaria sp. from Coptis chinensis / 中国中药杂志

Two new polyketides, lasobutone A(1) and lasobutone B(2), along with three known compounds, guignardianone C(3), guignardic acid(4), and 4-hydroxy-17R-methylincisterol(5), were isolated from the endophytic fungi Xylaria sp. by silica gel, MCI, and preparative HPLC, which was separated from the Chinese medicinal material Coptis chinensis and cultivated through solid fermentation with rice. Their structures were elucidated on the basis of spectroscopic methods, such as MS, NMR, IR, UV, and ECD. Compounds 2 and 4 showed inhibitory activities against the nitric oxide(NO) production in the LPS-induced macrophage RAW264.7 with IC_(50) values of 58.7 and 42.5 μmol·L~(-1) respectively, while compound 5 exhibited cytotoxic activities against HT-29 with IC_(50) value of 14.3 μmol·L~(-1).

Ornamental plant Pachystachys lutea as a source of promising endophytes for plant growth and phytoprotective activity

Endophytes are growth-promoting agents capable of synthesizing phytohormones, uptaking nutrients, and controlling pathogens. There is a strong potential to exploit them in the agriculture field like biofertilizers and biocontrol agents. In this work, we aimed to evaluate endophytic fungi isolated from Pachystachys lutea for their potential to solubilize phosphate, synthesise indole acetic acid (IAA), antagonize phytopathogens, and promote plant growth under greenhouse conditions. The phosphate solubilization efficiency was assessed on Pikovskaya’s agar medium. For analysis of IAA production, mycelia plugs of endophytes were cultured in Potato Dextrose Broth medium supplemented with L-tryptophan, with Salkowski Reagent, and the absorbance of the culture was measured. The antagonism evaluation of strain Alternaria sp. PL75 against phytopathogens was performed using the paired-culture technique. The promotion of plant growth provided by Alternaria sp. PL75 was evaluated in tomato plants. All strains evaluated were able to solubilize phosphate; however, the strain Alternaria sp. PL75 was the most effective (4.29). Two strains, Nemania sp. PL27 and Alternaria sp. PL75, produced 1.86 and 1.73 & 956;g mL-1 of IAA, respectively. In the antagonism assay, the endophyte Alternaria sp. PL75 and its fungal extract showed the best results against the pathogen Moniliophthora perniciosa. The greenhouse experiment result showed the endophyte Alternaria sp. PL75 increased the plantlets emergency speed index and the percentage of germination from 60 to 81.63%. It was also observed a statistical significance in the shoot length of the treated plants with the endophyte suspension (55.38 cm) compared to the control (41.67 cm).

Identification and characterization of plant growth-promoting properties of bacterial endophytes from selected Zingiberaceae plants

Aims@#This study aims to isolate, characterize and screen the plant growth-promoting bacteria from Zingiberaceae plants. Plant promoting activities such as indole-3-acetic acid (IAA), phosphate solubilization, zinc solubilization and nitrogen-fixing capabilities are determined, and the IAA production of selected isolates are optimized. @*Methodology and results@#Endophytic bacteria were isolated from the plant samples by surface sterilization on nutrient agar (NA) plates and incubated at 30 °C for 2-3 days. The bacteria were identified based on their phenotypic characteristics and 16S rRNA gene sequence analyses. All isolates were identified as genera Bacillus, Lysinibacillus, Kerstersia, Klebsiella and Brucella. The isolates exhibited phosphate solubilization (1.5 ± 0.75-37.5 ± 8.75 Solubilization Index, SI), zinc solubilization (2.5 ± 0-60 ± 1.5 SI) and IAA production (0.1 ± 0.2-115.7 ± 1.6 µg/mL), while 3 isolates possessed nitrogen-fixing capabilities. Five isolates (PHAS-2, PWS-2, PWR-2, PHBS-2 and SCG-2) were selected for IAA optimization. Isolate PWR-2 produced the maximum IAA at 447.7 ± 0 µg/mL when tryptophan concentration was maintained at 1.0%.@*Conclusion, significance and impact of study@#Genera of bacteria included Bacillus, Lysinibacillus, Kerstersia, Klebsiella and Brucella were successfully isolated from Zingiberaceae plants. All the isolates showed the capability to produce IAA, while some isolates exhibited phosphate solubilization and zinc solubilization, and a few possessed nitrogen-fixing capabilities. The potential IAA production isolates could be applied for the enhancement of agricultural production that will be becoming a more widely accepted practice.

Genetic diversity and antibacterial activity of endophytic fungi from Zanthoxylum nitidum / 中国中药杂志

In order to reveal the distribution and population characteristics of endophytic fungi from Zanthoxylum nitidum and the antibacterial potential,this study performed molecular identification and analyzed the genetic diversity and antibacterial activity of endophytic fungi from Z. nitidum in Guangxi. Through culture and molecular identification,35 strains,belonging to 15 genera,12 families,10 orders,4 classes,and 2 phyla,were isolated from various tissues of Z. nitidum,of which Colletotrichum and Fusarium were the dominant genera,respectively accounting for 20% of total strains. The diversity of endophytic fungi was significantly different among roots,stems,and leaves,as manifested by the significantly higher Shannon index( H') in stems( 1. 678) than in roots( 0. 882 1) and leaves( 0. 515 4). The antimicrobial activity analysis showed that 14. 28% of endophytic fungi inhibited at least one indicator pathogen. Among them,Fusarium sp. ZN-34 and Fusarium sp. ZN-26 separately demonstrated the strongest inhibitory effect on Escherichia coli and Staphylococcus aureus. In general,Fusarium sp. ZN-26 and Phialemoniopsis plurioloculosa ZN-35 were advantageous in suppressing the two bacteria owing to the broad spectrum and strong efficacy. In summary,Z. nitidum in Guangxi boasts rich endophytic fungi with the majority showing strong antibacterial activity,which can be used as candidates for the extraction and separation of basic antibacterial substances and the development of natural antibacterial agents.

Research progress and prospect of endophytes from medicinal plant Atractylodes lancea / 中国中药杂志

The endophytes of medicinal plants play an important role in promoting the quality formation of the host. Therefore, this paper made a review of endophytes of medicinal plant Atractylodes lancea. According to previous studies, A. lancea boasts endophytes, such as fungi, bacteria, and actinomycetes, among which the beneficial microorganisms help the growth and development of A. lancea. There is a close interaction between the volatile oil of A. lancea and endophytes. Different endophytes vary in regulating the composition and content of the volatile oil of A. lancea, which might contribute to the quality formation of A. lancea. However, the information of the endophytic flora of A. lancea obtained by traditional culture and isolation is not enough to reflect the real situation of the endophytes of A. lancea. Little is known about the endophytes of A. lancea from different chemical types and different habitats, which is not conducive to the study of the ecological relationship between A. lancea and endophytes and limits the development and utilization of the endophytes. Therefore, at the end of this paper, the authors put forward suggestions for future research on endophytes in A. lancea, including:(1)mining the core endophyte resources of A. lancea by combining high-throughput sequencing with traditional culture and isolation;(2)exploring the relationship between the diversity of endophytes and chemical types of A. lancea;(3)strengthening the application of endophytes in A. lancea cultivation, in order to facilitate the cultivation efficiency and quality of A. lancea.

Research strategies for endophytes in medicinal plants based on high-throughput sequencing and traditional culture and isolation methods / 中国中药杂志

The research on endophytes of medicinal plants mainly relies on the traditional culture and isolation methods. Because of their functions such as promoting host growth, improving stress resistance, promoting the accumulation of medicinal active ingredients or directly producing medicinal active ingredients, the endophytes of medicinal plants have gradually attracted wide attention. However, it was found that the strains isolated by traditional methods were not the true dominant endophytes of medicinal plants by comparing the results of traditional culture isolation with high-throughput sequencing. The blind and random nature of traditional methods leads to the lack of standards in terms of medium selection, culture time and interaction between species. On the contrary, high-throughput sequencing technology is an emerging molecular biology technology developed in recent decades. Due to its high resolution level and indepen-dent culture, it can be used for thorough analysis of the community structure and diversity of environmental microorganisms. Therefore, we proposed the strategy of using high-throughput sequencing technology to guide the traditional culture and isolation of endophytes from medicinal plants. Firstly, the endophytic structure and diversity of medicinal plants were completely clear by high-throughput sequencing technology, and the dominant endophytes of the host were unequivocal. Then according to the characteristics of each dominant endophytes design or query suitable medium for its growth to culture and isolation. Finally, the function of the isolates was studied. This method can prevent researchers from missing out on the important functional strains of the host, expand the research scope of endophytes of medicinal plants, and facilitate the in-depth excavation and utilization of endophytes of medicinal plants.

Toxicity of ß-(1→3, 1→6)-á´ -glucans produced by Diaporthe sp. endophytes on Metarhizium anisopliae (Metschnikoff) Sorokin assessed by conidia germination speed parameter / Toxicidade de ß- (1 → 3, 1 → 6) -á´ -glucanas produzidas por endófitos Diaporthe sp. sobre Metarhizium anisopliae (Metschnikoff) Sorokin avaliada pelo parâmetro de velocidade de germinação dos conídios

We have previously reported that ß-(1→3,1→6)-ᴅ-glucans produced by endophytes Diaporthe sp. G27-60 and G65-65 (GenBank accession codes JF766998 and JF767007, respectively) are promising anti-proliferation agents against human breast carcinoma (MCF-7) and hepatocellular carcinoma (HepG2-C3A) cells. However, the literature fails to describe the effects of Diaporthe exopolysaccharides (EPS) on eukaryotic healthy cells. The fungus Metarhiziumanisopliae has been employed as model-system to evaluate the toxicity of pharmaceutical and agricultural-interest substances, taking into account, among other parameters, the speed of conidia germination. Current study verified the effect of different concentrations of Diaporthe ß-glucans on the germination speed of M. anisopliae. Conidia were incubated with ß-glucans treatments (50, 200 and 400 µg/mL) at 28ºC, sampled during 24 h and analyzed by light microscopy. At the end of a 24-h incubation, the amount of germinated conidia reached ≈99% for controls and ranged between 97.7 and 98.6% for treatments. Bayesian analysis indicated that Diaporthe glucans had no toxicity on M. anisopliaeand the curve of germination occurred as expected for this fungal strain. Considering the validity of filamentous fungi as model-systems, results are important data on the toxicity of endophytic EPS on healthy cells and may be associated with our previous results obtained for these polymers against tumor cells.

Anteriormente, um estudo mostrou que ß-(1→3,1→6)-ᴅ-glucanas produzidas pelos endófitos Diaporthe sp. G27-60 e G65-65 (códigos de acesso no GenBank JF766998 e JF767007, respectivamente) são agentes promissores com ação antiproliferativa contra células HepG2-C3A (hepatoma humano) e MCF-7 (adenocarcinoma mamário humano). No entanto, os efeitos de exopolissacarídeos (EPS) produzidos por fungos do gênero Diaporthe em células eucarióticas sadias não estão descritos na literatura atual. O fungo Metarhiziumanisopliae tem sido utilizado como sistema-modelo para avaliar a toxicidade de substâncias de interesse farmacêutico e agronômico, considerando, entre outros parâmetros, a velocidade de germinação de conídios. O presente estudo teve como objetivo verificar os efeitos de diferentes concentrações de ß-glucanas produzidas por Diaporthe sp. sobre a velocidade de germinação de M. anisopliae. Os conídios foram incubados com os tratamentos de ß-glucanas (50, 200 e 400 µg/mL) a 28 ºC, com amostras coletadas ao longo de 24 h, e analisados por microscopia de luz. Ao final das 24 h de incubação, o total de conídios germinados nos controles foi de ≈99%, e variou entre 97,7 e 98,6% para os tratamentos. A análise bayesiana indicou que as glucanas de Diaporthe sp. não apresentaram toxicidade sobre M. anisopliae, e a curva de germinação atendeu ao esperado para essa linhagem fúngica. Considerando a validade dos fungos filamentosos como sistemas-modelo, esses resultados representam dados importantes sobre a toxicidade dos EPS de endófitos sobre células sadias e podem ser associados aos resultados anteriormente obtidos para esses polímeros em testes contra células tumorais.

Actividad antimicrobiana de hongos endófitos de las plantas medicinales Mammea americana (Calophyllaceae) y Moringa oleifera (Moringaceae) / Antimicrobial activity of endophytic fungi from the medicinal plants Mammea americana (Calophyllaceae) and Moringa oleifera (Moringaceae)

Introducción. Las enfermedades infecciosas son una causa importante de muertes en el mundo. La resistencia antimicrobiana es un problema global, por lo que es conveniente la investigación de nuevas fuentes de agentes antimicrobianos de origen natural potencialmente efectivos. Objetivo. Evaluar la actividad antimicrobiana de hongos endófitos de Mammea americanay Moringa oleifera en la cepa sensible (ATCC 29213) y en la cepa resistente (USb003) de Staphylococcusaureus, así como en la cepa sensible (ATCC 25922) y la cepa resistente (USb007) de Escherichia coli. Materiales ymétodos. Se aislaron 14 hongos endófitos de las hojas, semillas y tallos de las dos plantas en estudio. Se evaluó su actividad antimicrobiana mediante la formación de halos de sensibilidad por ensayo dual in vitro y pruebas con extractos etanólicos crudos provenientes de los endófitos a los que se les evaluó la concentración mínima inhibitoria (CMI), la concentración bactericida mínima (CBM) y la citotoxicidad. Resultados. Tres extractos etanólicos de Penicillium sp., Cladosporium sp. (001) y Cladosporium sp.(002) presentaron mayores halos de inhibición en cepas sensibles y resistentes de E. coli y S. aureus. La CMI y la CBM halladas fueron estadísticamente significativas (p≤0,05), comparadas con el control de gentamicina. Las pruebas de citotoxicidad (concentración citotóxica, CC50>1.000) demostraron que los hongos endófitos poseen características bactericidas y no ocasionan daño alguno. Conclusión. Se halló una fuente de metabolitos secundarios activos con propiedades antimicrobianas y no tóxicas en los hongos endófitos de M. oleifera y M. americana;estos hallazgos son importantes para continuar con la identificación química de los compuestos y el estudio de sus mecanismos de acción en estas plantas en las que el aislamiento de endófitos ha sido escaso.

Introduction: Infectious diseases represent one of the leading causes of death worldwide. Considering the growing global challenge of antimicrobial resistance, research into new sources of potentially effective antimicrobial agents from natural origins is of great importance for world health. Objective: To evaluate the antimicrobial activity of endophytic fungi from Mammea americana and Moringa oleifera upon Staphylococcus aureus (ATCC 29213), S. aureus(resistant strain USb003), Escherichia coli (ATCC 25922), and E. coli (resistant strain USb007). Materials and methods: We isolated endophytic fungi from the leaves, seeds, and stems of the two plants under study. We evaluated their antimicrobial activity through the formation of sensitivity haloes in dual tests in vitro, as well as in trials using crude ethanolic extracts from the endophytes. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and cytotoxicity o the substances were analyzed. Results: Three ethanolic extracts of Penicillium sp., Cladosporium (001), and Cladosporium (002) exhibited the greatest inhibition halos in sensitive and resistant strains of E. coli and S. aureus. The MIC and CBM found were statistically significant (p≤0.05) compared with the gentamicin control. Furthermore, the cytotoxicity test results of CC50>1,000 demonstrated that the endophytic fungi studied exhibit bactericidal characteristics without causing unintended damage. Conclusion: The endophytic fungi M. oleifera and M. americana represent a source of active secondary metabolites with antimicrobial and non-toxic properties. In light of these findings, further research should proceed with chemical identification of the compounds and the study of their mechanisms of action, especially given the paucity of current scientific knowledge concerning the isolation of endophytes in these plants.

Tolerance of dark septate endophytic fungi (DSE) to 1 agrochemicals in vitro / Tolerancia de hongos endofíticos septados oscuros a agroquímicos in vitro

Abstract Dark septate endophytes (DSE) are a heterogeneous group of fungi, mostly belonging to the Phylum Ascomycota, that are involved in a mutualistic symbiosis with plant roots. The aim of this study is to evaluate the behavior of two strains of DSE isolated from wheat roots of two cropping areas in the province of Buenos Aires, Argentina, against some agrochemicals. Of all the isolates obtained, two strains were identified as Alternaria alternata and Cochliobolus sp. These DSE were found to be tolerant to glyphosate, carbendazim and cypermethrin when evaluated at the recommended agronomic dose (AD), 2 AD and, in some cases, 10 AD. This work contributes to the study of the biology of this group of fungi and their tolerance in the presence of xenobiotics widely used in agriculture.© 2019 Asociaci´on Argentina de Microbiolog´ıa. Published by Elsevier Espa˜na, S.L.U. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/bync-nd/4.0/).

Resumen Los endófitos septados oscuros (DSE) son un grupo heterogéneo de hongos que participan de una simbiosis mutualista con raíces de plantas, perteneciendo principalmente al Phylum Ascomycota. El objetivo de este estudio fue aislar DSE de raíces de trigo proveniente de dos áreas de cultivo de la provincia de Buenos Aires y evaluar el comportamiento de dos cepas de DSE aisladas de raíces de trigo frente a algunos agroquímicos en dos áreas de cultivo de la provincia de Buenos Aires. De todos los aislamientos obtenidos se seleccionaron dos cepas que se identificaron como Alternaria alternata y Cochliobolus sp. Se encontró que estos DSE son tolerantes al glifosato, el carbendazim y la cipermetrina, evaluados a las dosis agronómicas recomendadas (AD), a 2x AD y, en algunos casos, a 10x AD. Este trabajo contribuye al conocimiento de la biología de este grupo de hongos y su tolerancia a xenobióticos ampliamente utilizados en la agricultura.

Adaptation of plants to high-calcium content kart regions: possible involvement of symbiotic microorganisms and underlying mechanisms / Adaptação de plantas a regiões de kart com alto teor de cálcio: possível envolvimento de microrganismos simbióticos e mecanismos subjacentes

Abstract Rhizosphere microorganisms and endophytes can help their hosts absorb nutrients and regulate the levels of plant hormones. Moreover, they can modulate the expressions of host genes, assist hosts in eliminating reactive oxygen species (ROS) and secreting volatile organic compounds. Therefore, rhizosphere microorganisms and endophytes are considered as determinant factors driving processes involved in the growth of host plants. However, the physiological and ecological functions, as well as the molecular mechanism underlying the behavior of rhizosphere microorganisms and endophytes in their role in the adaptive capacity of host plants in the karstic high-calcium environment have not been systematically studied. This review summarizes the physiological and molecular mechanisms of rhizosphere microorganisms and endophytes which help host plants to adapt to various kinds of adverse environments. The adaptive capacities of plants growing in adverse environments, partly, or totally, depends on microorganisms co-existing with the host plants.

Resumo Os microorganismos e endófitos da rizosfera podem ajudar seus hospedeiros a absorver nutrientes e regular os níveis de hormônios vegetais. Além disso, eles podem modular as expressões dos genes hospedeiros, auxiliar os hospedeiros na eliminação de espécies reativas de oxigênio (ROS) e secretar compostos orgânicos voláteis. Portanto, microorganismos e endófitos da rizosfera são considerados determinantes dos processos envolvidos no crescimento de plantas hospedeiras. No entanto, as funções fisiológicas e ecológicas, bem como o mecanismo molecular subjacente ao comportamento dos microrganismos e endofíticos da rizosfera no seu papel na capacidade adaptativa das plantas hospedeiras no ambiente cárstico de alto teor de cálcio, não foram sistematicamente estudados. Esta revisão resume os mecanismos fisiológicos e moleculares de microrganismos e endófitos da rizosfera que ajudam as plantas hospedeiras a se adaptarem a vários tipos de ambientes adversos. As capacidades adaptativas das plantas que crescem em ambientes adversos, em parte ou totalmente, dependem de microrganismos coexistentes com as plantas hospedeiras.

Enzyme Production by Induratia spp. Isolated from Coffee Plants in Brazil

Abstract Endophytic fungi belonging to the genus Muscodor now transferred to Induratia are known producers of bioactive volatile organic compounds (VOCs) with many industrial applications. However, the members of this genus have rarely been reported to produce non-volatile metabolites including enzyme. Enzymes of the endophytes are degraders of the polysaccharides available in the host plants and the knowledge of enzyme production by Induratia spp. may provide insights into their possible biotechnological applications. The aim of this study was to evaluate the activity of amylase, cellulase, lipase, pectinase, phytase, protease, endo β-1,4 glucanase and exo β-1,4 glucanase enzymes produced by fungi of the species Induratia coffeana, Induratia yucatanensis and Induratia sp. isolated from organic coffee plants. All Induratia spp. were able to produce the extracellular enzymes cellulase, pectinase, protease, and phytase. Eight fungi were able to produce lipase and four produced amylase. The specific activity of endo β-1, 4 glucanase and exo β-1,4 glucanase enzymes were detected for 9 and 8 endophytic fungi, respectively. This work demonstrated for the first time, the array of enzymes produced by Induratia spp. isolated from Coffea arabica in organic systems in Brazil.

CO2 Fertilizer Effect on Growth, Polyphenols, and Endophytes in Two Baccharis Species

Abstract In a climate change context, the buildup of CO2 will affect plant communities worldwide. This study evaluated the effects of CO2 enrichment on the development and defense of two Cerrado native species Baccharis dracunculifolia and B. platypoda and their associated endophytic fungi richness. The study took place in Open-Top Chambers, two with ambient CO2 concentration (~400 ppm) and two in an enriched environment (~800 ppm). Baccharis platypoda developed 20% more leaves under enriched CO2 conditions, whereas B. dracunculifolia was 30% taller and showed 27% more leaves than those under ambient conditions. In both species, leaf polyphenol concentration did not differ between treatments. Nevertheless, polyphenol content had a positive correlation with plant height on both species' individuals grown under CO2 enriched conditions. Endophytic fungi richness and colonization rate on both plant species did not differ between ambient and enriched conditions. Our results show the positive effect of CO2 fertilizer in at least one of the measured growth parameters. An important new finding was a synergistic increase in growth and chemical defense in both studied species under enriched CO2 conditions, suggesting higher carbon assimilation and accumulation. This study suggests that the effects on primary productivity and secondary metabolites of Baccharis species will potentially reflect on the diversity and distribution of Cerrado plants and their associated animal communities.