Micropropagation of Seed-Derived Clonal Lines of the Endangered Agave marmorata Roezl and Their Compatibility with Endophytes.

A. marmorata is the raw material used for tepextate mescal production but is classified as an endangered species. In the present study, we obtain and multiply clonal lines of Agave marmorata Roezl by selecting seedlings derived from seeds. Ten seedlings from two lots of 400 germinated seeds were selected for axillary bud proliferation induced by BAP 5 mg/L in vitamin-free Murashige and Skoog's medium. Differences in shoot numbers, heights and senescent tissue formation were observed. Notably, the AM32 line formed 84 shoots and presented low senescent tissue after 60 d of culture. We also selected the AM31 and AM33 clonal lines. Four-month shoots were extracted with 80% methanol in water to determine the total content of saponins, flavonoids, and phenolic acids and compare the three clonal lines. Some bioactive molecules were identified using HPLC techniques and MALDI-TOF mass spectrometry none showed significant differences in content. Additionally, plants derived from the clonal lines were inoculated with four endophytic bacteria. Among these, Achromobacter xylosoxidans supported plant growth of AM32. A notable effect of plant death was observed after inoculation with Enterobacter cloacae, an endophyte of A. tequilana. Additionally, Pseudomonas aeruginosa, an endophyte from A. marmorata, reduced biomass. Our results demonstrate the incompatibility of A. marmorata to E. cloacae and specialization between the host plant and its endophytes. The compatibility of the plant-endophyte could be exploited to boost the establishment and stability of mutualisms to benefit plant development, stress tolerance and pathogen resistance. The differences in multiplication capacity, stable tissue formation, and endophyte biotization responses may indicate genetic variability. Clonal selection and micropropagation from seed-derived plants could contribute to conserving the endangered A. marmorata plant for reforestation in their natural habitats, thus, assuring mass propagation for sustainable industrial production of mescal, bioactive compounds, and prebiotics.

Biomolecules resveratrol + coenzyme Q10 recover the cell state of human mesenchymal stem cells after 1-methyl-4-phenylpyridinium-induced damage and improve proliferation and neural differentiation.

Neurodegenerative disorders are a critical affection with a high incidence around the world. Currently, there are no effective treatments to solve this problem. However, the application of mesenchymal stem cells (MSCs) and antioxidants in neurodegenerative diseases has shown to be a promising tool due to their multiple therapeutic effects. This work aimed to evaluate the effects of a combination of resveratrol (RSV) and coenzyme Q10 (CoQ10) on the proliferation and differentiation of MSC and the protector effects in induced damage. To characterize the MSCs, we performed flow cytometry, protocols of cellular differentiation, and immunocytochemistry analysis. The impact of RSV + CoQ10 in proliferation was evaluated by supplementing 2.5 and 10 µM of RSV + CoQ10 in a cellular kinetic for 14 days. Cell viability and lactate dehydrogenase levels (LDH) were also analyzed. The protective effect of RSV + CoQ10 was assessed by supplementing the treatment to damaged MSCs by 1-methyl-4-phenylpyridinium (MPP+); cellular viability, LDH, and reactive oxygen species (ROS) were evaluated.. MSCs expressed the surface markers CD44, CD73, CD90, and CD105 and showed multipotential ability. The combination of RSV + CoQ10 increased the proliferation potential and cell viability and decreased LDH levels. In addition, it reverted the effect of MPP+-induced damage in MSCs to enhance cell viability and decrease LDH and ROS. Finally, RSV + CoQ10 promoted the differentiation of neural progenitors. The combination of RSV + CoQ10 represents a potential treatment to improve MSCs capacities and protect against neurodegenerative damage.

Probiotic Endophytes for More Sustainable Banana Production.

Climatic factors and pathogenic fungi threaten global banana production. Moreover, bananas are being cultivated using excessive amendments of nitrogen and pesticides, which shift the microbial diversity in plants and soil. Advances in high-throughput sequencing (HTS) technologies and culture-dependent methods have provided valuable information about microbial diversity and functionality of plant-associated endophytic communities. Under stressful (biotic or abiotic) conditions, plants can recruit sets of microorganisms to alleviate specific potentially detrimental effects, a phenomenon known as "cry for help". This mechanism is likely initiated in banana plants infected by Fusarium wilt pathogen. Recently, reports demonstrated the synergistic and cumulative effects of synthetic microbial communities (SynComs) on naturally occurring plant microbiomes. Indeed, probiotic SynComs have been shown to increase plant resilience against biotic and abiotic stresses and promote growth. This review focuses on endophytic bacterial diversity and keystone taxa of banana plants. We also discuss the prospects of creating SynComs composed of endophytic bacteria that could enhance the production and sustainability of Cavendish bananas (Musa acuminata AAA), the fourth most important crop for maintaining global food security.

Introduction to Special Issue: Plant Microbiome Augmentation and Stimulation-New Strategies to Grow Crops with Reduced Agrochemicals.

Since the early work of Justus von Liebig on nutrient absorption in plants in the 1800s [...].

Enterobacter cloacae, an Endophyte That Establishes a Nutrient-Transfer Symbiosis With Banana Plants and Protects Against the Black Sigatoka Pathogen.

Banana (Musa spp.) is an important crop worldwide, but black Sigatoka disease caused by the fungus Pseudocercospora fijiensis threatens fruit production. In this work, we examined the potential of the endophytes of banana plants Enterobacter cloacae and Klebsiella pneumoniae, as antagonists of P. fijiensis and support plant growth in nutrient limited soils by N-transfer. The two bacterial isolates were identified by MALDI-TOF mass spectrometry and corroborated by 16S rRNA sequence analysis. Both bacteria were positive for beneficial traits such as N-fixation, indole acetic acid production, phosphate solubilization, negative for 1-aminocyclopropane 1-carboxylic acid deaminase and were antagonistic to P. fijiensis. To measure the effects on plant growth, the two plant bacteria and an E. coli strain (as non-endophyte), were inoculated weekly for 60 days as active cells (AC) and heat-killed cells (HKC) into plant microcosms without nutrients and compared to a water only treatment, and a mineral nutrients solution (MMN) treatment. Bacterial treatments increased growth parameters and prevented accelerated senescence, which was observed for water and mineral nutrients solution (MMN) treatments used as controls. Plants died after the first 20 days of being irrigated with water; irrigation with MMN enabled plants to develop some new leaves, but plants lost weight (-30%) during the same period. Plants treated with bacteria showed good growth, but E. cloacae AC treated plants had significantly greater biomass than the E. cloacae HKC. After 60 days, plants inoculated with E. cloacae AC showed intracellular bacteria within root cells, suggesting that a stable symbiosis was established. To evaluate the transference of organic N from bacteria into the plants, the 3 bacteria were grown with 15NH4Cl or Na15NO3 as the nitrogen source. The 15N transferred from bacteria to plant tissues was measured by pheophytin isotopomer abundance. The relative abundance of the isotopomers m/z 872.57, 873.57, 874.57, 875.57, 876.57 unequivocally demonstrated that plants acquired 15N atoms directly from bacterial cells, using them as a source of N, to support plant growth in restricted nutrient soils. E. cloacae might be a new alternative to promote growth and health of banana crops.

Role of Lipid Composition, Physicochemical Interactions, and Membrane Mechanics in the Molecular Actions of Microbial Cyclic Lipopeptides.

Several experimental and theoretical studies have extensively investigated the effects of a large diversity of antimicrobial peptides (AMPs) on model lipid bilayers and living cells. Many of these peptides disturb cells by forming pores in the plasma membrane that eventually lead to the cell death. The complexity of these peptide-lipid interactions is mainly related to electrostatic, hydrophobic and topological issues of these counterparts. Diverse studies have shed some light on how AMPs act on lipid bilayers composed by different phospholipids, and how mechanical properties of membranes could affect the antimicrobial effects of such compounds. On the other hand, cyclic lipopeptides (cLPs), an important class of microbial secondary metabolites, have received comparatively less attention. Due to their amphipathic structures, cLPs exhibit interesting biological activities including interactions with biofilms, anti-bacterial, anti-fungal, antiviral, and anti-tumoral properties, which deserve more investigation. Understanding how physicochemical properties of lipid bilayers contribute and determining the antagonistic activity of these secondary metabolites over a broad spectrum of microbial pathogens could establish a framework to design and select effective strategies of biological control. This implies unravelling-at the biophysical level-the complex interactions established between cLPs and lipid bilayers. This review presents, in a systematic manner, the diversity of lipidated antibiotics produced by different microorganisms, with a critical analysis of the perturbing actions that have been reported in the literature for this specific set of membrane-active lipopeptides during their interactions with model membranes and in vivo. With an overview on the mechanical properties of lipid bilayers that can be experimentally determined, we also discuss which parameters are relevant in the understanding of those perturbation effects. Finally, we expose in brief, how this knowledge can help to design novel strategies to use these biosurfactants in the agronomic and pharmaceutical industries.

Cultivable endophytic bacteria from leaf bases of Agave tequilana and their role as plant growth promoters

Agave tequilana Weber var. 'Azul' is grown for the production of tequila, inulin and syrup. Diverse bacteria inhabit plant tissues and play a crucial role for plant health and growth. In this study culturable endophytic bacteria were extracted from leaf bases of 100 healthy Agave tequilana plants. In plant tissue bacteria occurred at mean population densities of 3 million CFU/g of fresh plant tissue. Three hundred endophytic strains were isolated and 16s rDNA sequences grouped the bacteria into eight different taxa that shared high homology with other known sequences. Bacterial endophytes were identified as Acinectobacter sp., A. baumanii, A. bereziniae, Cronobacter sakazakii, Enterobacter hormaechei, Bacillus sp. Klebsiella oxytoca, Pseudomonas sp., Enterococcus casseliflavus, Leuconostoc mesenteroides subsp. mesenteroides and Gluconobacter oxydans. Isolates were confirmed to be plant growth promoting bacteria (PGPB) by their capacities for nitrogen fixation, auxin production, phosphate solubilization, or antagonism against Fusarium oxysporum AC132. E. casseliflavus JM47 and K. oxytoca JM26 secreted the highest concentrations of IAA. The endophyte Acinectobacter sp. JM58 exhibited the maximum values for nitrogen fixation and phosphate solubilization index (PSI). Inhibition of fungi was found in Pseudomonas sp. JM9p and K. oxytoca JM26. Bacterial endophytes show promise for use as bio-inoculants for agave cultivation. Use of endophytes to enhance cultivation of agave may be particularly important for plants produced by micropropagation techniques, where native endophytes may have been lost.

Cultivable endophytic bacteria from leaf bases of Agave tequilana and their role as plant growth promoters

Agave tequilana Weber var. 'Azul' is grown for the production of tequila, inulin and syrup. Diverse bacteria inhabit plant tissues and play a crucial role for plant health and growth. In this study culturable endophytic bacteria were extracted from leaf bases of 100 healthy Agave tequilana plants. In plant tissue bacteria occurred at mean population densities of 3 million CFU/g of fresh plant tissue. Three hundred endophytic strains were isolated and 16s rDNA sequences grouped the bacteria into eight different taxa that shared high homology with other known sequences. Bacterial endophytes were identified as Acinectobacter sp., A. baumanii, A. bereziniae, Cronobacter sakazakii, Enterobacter hormaechei, Bacillus sp. Klebsiella oxytoca, Pseudomonas sp., Enterococcus casseliflavus, Leuconostoc mesenteroides subsp. mesenteroides and Gluconobacter oxydans. Isolates were confirmed to be plant growth promoting bacteria (PGPB) by their capacities for nitrogen fixation, auxin production, phosphate solubilization, or antagonism against Fusarium oxysporum AC132. E. casseliflavus JM47 and K. oxytoca JM26 secreted the highest concentrations of IAA. The endophyte Acinectobacter sp. JM58 exhibited the maximum values for nitrogen fixation and phosphate solubilization index (PSI). Inhibition of fungi was found in Pseudomonas sp. JM9p and K. oxytoca JM26. Bacterial endophytes show promise for use as bio-inoculants for agave cultivation. Use of endophytes to enhance cultivation of agave may be particularly important for plants produced by micropropagation techniques, where native endophytes may have been lost.

Nitrogen acquisition in Agave tequilana from degradation of endophytic bacteria.

Plants form symbiotic associations with endophytic bacteria within tissues of leaves, stems, and roots. It is unclear whether or how plants obtain nitrogen from these endophytic bacteria. Here we present evidence showing nitrogen flow from endophytic bacteria to plants in a process that appears to involve oxidative degradation of bacteria. In our experiments we employed Agave tequilana and its seed-transmitted endophyte Bacillus tequilensis to elucidate organic nitrogen transfer from (15)N-labeled bacteria to plants. Bacillus tequilensis cells grown in a minimal medium with (15)NH4Cl as the nitrogen source were watered onto plants growing in sand. We traced incorporation of (15)N into tryptophan, deoxynucleosides and pheophytin derived from chlorophyll a. Probes for hydrogen peroxide show its presence during degradation of bacteria in plant tissues, supporting involvement of reactive oxygen in the degradation process. In another experiment to assess nitrogen absorbed as a result of endophytic colonization of plants we demonstrated that endophytic bacteria potentially transfer more nitrogen to plants and stimulate greater biomass in plants than heat-killed bacteria that do not colonize plants but instead degrade in the soil. Findings presented here support the hypothesis that some plants under nutrient limitation may degrade and obtain nitrogen from endophytic microbes.

Chemical management in fungicide sensitivity of Mycosphaerella fijiensis collected from banana fields in México.

The chemical management of the black leaf streak disease in banana caused by Mycosphaerella fijiensis (Morelet) requires numerous applications of fungicides per year. However this has led to fungicide resistance in the field. The present study evaluated the activities of six fungicides against the mycelial growth by determination of EC50 values of strains collected from fields with different fungicide management programs: Rustic management (RM) without applications and Intensive management (IM) more than 25 fungicide application/year. Results showed a decreased sensitivity to all fungicides in isolates collected from IM. Means of EC50 values in mg L(-1) for RM and IM were: 13.25 ± 18.24 and 51.58 ± 46.14 for azoxystrobin, 81.40 ± 56.50 and 1.8575 ± 2.11 for carbendazim, 1.225 ± 0.945 and 10.01 ± 8.55 for propiconazole, 220 ± 67.66 vs. 368 ± 62.76 for vinclozolin, 9.862 ± 3.24 and 54.5 ± 21.08 for fludioxonil, 49.2125 ± 34.11 and 112.25 ± 51.20 for mancozeb. A molecular analysis for ß-tubulin revealed a mutation at codon 198 in these strains having an EC50 greater than 10 mg L(-1) for carbendazim. Our data indicate a consistency between fungicide resistance and intensive chemical management in banana fields, however indicative values for resistance were also found in strains collected from rustic fields, suggesting that proximity among fields may be causing a fungus interchange, where rustic fields are breeding grounds for development of resistant strains. Urgent actions are required in order to avoid fungicide resistance in Mexican populations of M. fijiensis due to fungicide management practices.

Singlet molecular oxygen generation by light-activated DHN-melanin of the fungal pathogen Mycosphaerella fijiensis in black Sigatoka disease of bananas.

In pathogenic fungi, melanin contributes to virulence, allowing tissue invasion and inactivation of the plant defence system, but has never been implicated as a factor for host cell death, or as a light-activated phytotoxin. Our research shows that melanin synthesized by the fungal banana pathogen Mycosphaerella fijiensis acts as a virulence factor through the photogeneration of singlet molecular oxygen O2 (1Δg). Using analytical tools, including elemental analysis, ultraviolet/infrared absorption spectrophometry and MALDI-TOF mass spectrometry analysis, we characterized both pigment content in mycelia and secreted to the culture media as 1,8-dihydroxynaphthalene (DHN)-melanin type compound. This is sole melanin-type in M. fijiensis. Isolated melanins irradiated with a Nd:YAG laser at 532 nm produced monomol light emission at 1270 nm, confirming generation of O2 (1Δg), a highly reactive oxygen specie (ROS) that causes cellular death by reacting with all cellular macromolecules. Intermediary polyketides accumulated in culture media by using tricyclazole and pyroquilon (two inhibitors of DHN-melanin synthesis) were identified by ESI-HPLC-MS/MS. Additionally, irradiation at 532 nm of that mixture of compounds and whole melanized mycelium also generated O2 (1Δg). A pigmented-strain generated more O2 (1Δg) than a strain with low melanin content. Banana leaves of cultivar Cavendish, naturally infected with different stages of black Sigatoka disease, were collected from field. Direct staining of the naturally infected leaf tissues showed the presence of melanin that was positively correlated to the disease stage. We also found hydrogen peroxide (H2O2) but we cannot distinguish the source. Our results suggest that O2 (1Δg) photogenerated by DHN-melanin may be involved in the destructive effects of Mycosphaerella fijiensis on banana leaf tissues. Further studies are needed to fully evaluate contributions of melanin-mediated ROS to microbial pathogenesis.

Cultivable endophytic bacteria from leaf bases of Agave tequilana and their role as plant growth promoters.

Agave tequilana Weber var. 'Azul' is grown for the production of tequila, inulin and syrup. Diverse bacteria inhabit plant tissues and play a crucial role for plant health and growth. In this study culturable endophytic bacteria were extracted from leaf bases of 100 healthy Agave tequilana plants. In plant tissue bacteria occurred at mean population densities of 3 million CFU/g of fresh plant tissue. Three hundred endophytic strains were isolated and 16s rDNA sequences grouped the bacteria into eight different taxa that shared high homology with other known sequences. Bacterial endophytes were identified as Acinectobacter sp., A. baumanii, A. bereziniae, Cronobacter sakazakii, Enterobacter hormaechei, Bacillus sp. Klebsiella oxytoca, Pseudomonas sp., Enterococcus casseliflavus, Leuconostoc mesenteroides subsp. mesenteroides and Gluconobacter oxydans. Isolates were confirmed to be plant growth promoting bacteria (PGPB) by their capacities for nitrogen fixation, auxin production, phosphate solubilization, or antagonism against Fusarium oxysporum AC132. E. casseliflavus JM47 and K. oxytoca JM26 secreted the highest concentrations of IAA. The endophyte Acinectobacter sp. JM58 exhibited the maximum values for nitrogen fixation and phosphate solubilization index (PSI). Inhibition of fungi was found in Pseudomonas sp. JM9p and K. oxytoca JM26. Bacterial endophytes show promise for use as bio-inoculants for agave cultivation. Use of endophytes to enhance cultivation of agave may be particularly important for plants produced by micropropagation techniques, where native endophytes may have been lost.

Chemical management in fungicide sensivity of Mycosphaerella fijiensis collected from banana fields in México

The chemical management of the black leaf streak disease in banana caused by Mycosphaerella fijiensis (Morelet) requires numerous applications of fungicides per year. However this has led to fungicide resistance in the field. The present study evaluated the activities of six fungicides against the mycelial growth by determination of EC50 values of strains collected from fields with different fungicide management programs: Rustic management (RM) without applications and Intensive management (IM) more than 25 fungicide application/year. Results showed a decreased sensitivity to all fungicides in isolates collected from IM. Means of EC50 values in mg L-1 for RM and IM were: 13.25 ± 18.24 and 51.58 ± 46.14 for azoxystrobin, 81.40 ± 56.50 and 1.8575 ± 2.11 for carbendazim, 1.225 ± 0.945 and 10.01 ± 8.55 for propiconazole, 220 ± 67.66 vs. 368 ± 62.76 for vinclozolin, 9.862 ± 3.24 and 54.5 ± 21.08 for fludioxonil, 49.2125 ± 34.11 and 112.25 ± 51.20 for mancozeb. A molecular analysis for β-tubulin revealed a mutation at codon 198 in these strains having an EC50 greater than 10 mg L-1 for carbendazim. Our data indicate a consistency between fungicide resistance and intensive chemical management in banana fields, however indicative values for resistance were also found in strains collected from rustic fields, suggesting that proximity among fields may be causing a fungus interchange, where rustic fields are breeding grounds for development of resistant strains. Urgent actions are required in order to avoid fungicide resistance in Mexican populations of M. fijiensis due to fungicide management practices.

Chemical management in fungicide sensivity of Mycosphaerella fijiensis collected from banana fields in México

The chemical management of the black leaf streak disease in banana caused by Mycosphaerella fijiensis (Morelet) requires numerous applications of fungicides per year. However this has led to fungicide resistance in the field. The present study evaluated the activities of six fungicides against the mycelial growth by determination of EC50 values of strains collected from fields with different fungicide management programs: Rustic management (RM) without applications and Intensive management (IM) more than 25 fungicide application/year. Results showed a decreased sensitivity to all fungicides in isolates collected from IM. Means of EC50 values in mg L-1 for RM and IM were: 13.25 ± 18.24 and 51.58 ± 46.14 for azoxystrobin, 81.40 ± 56.50 and 1.8575 ± 2.11 for carbendazim, 1.225 ± 0.945 and 10.01 ± 8.55 for propiconazole, 220 ± 67.66 vs. 368 ± 62.76 for vinclozolin, 9.862 ± 3.24 and 54.5 ± 21.08 for fludioxonil, 49.2125 ± 34.11 and 112.25 ± 51.20 for mancozeb. A molecular analysis for β-tubulin revealed a mutation at codon 198 in these strains having an EC50 greater than 10 mg L-1 for carbendazim. Our data indicate a consistency between fungicide resistance and intensive chemical management in banana fields, however indicative values for resistance were also found in strains collected from rustic fields, suggesting that proximity among fields may be causing a fungus interchange, where rustic fields are breeding grounds for development of resistant strains. Urgent actions are required in order to avoid fungicide resistance in Mexican populations of M. fijiensis due to fungicide management practices.(AU)