Genetic diversity of Fusarium pseudocircinatum in the central western region of Mexico: the case of big-leaf mahogany malformation disease.

Fusarium pseudocircinatum is the main causal agent of big-leaf mahogany malformation disease (BLMMD) of mahogany (Swietenia macrophylla) in Mexico. Although, BLMMD is the most important disease for this high-value timber species, there is a lack of information on the genetic variation present in geographically diverse isolates of F. pseudocircinatum. The objective of this study was to determine the genetic diversity of populations of F. pseudocircinatum causing BLMMD in the central western region of Mexico. A total of 611 big-leaf mahogany trees were inspected at eight sites in four states (Colima, Guerrero, Jalisco and Michoacán); of these, 42.7% showed malformation symptoms similar to those of BLMMD. Of 374 Fusarium isolates that were recovered, 277 were identified as F. pseudocircinatum, 56 were F. mexicanum, and 41 were Fusarium spp. An ISSR analysis of the F. pseudocircinatum isolates generated 51 bands of which 38 were polymorphic (76.8%) with a mean of 17 bands per primer. A total of 87 multilocus genotypes (MLGs) were identified. Nei's genetic diversity analysis showed that the isolates had a high genetic diversity average (0.147), with values ranging from 0.070 to 0.365 depending of the geographical location. An analysis of molecular variance revealed that the variation within the populations was low (27.36%), while the variation within MLGs was significant (72.64%), indicating genetic flow. Overall, the genetic variability of F. pseudocircinatum populations was high and the MLGs from Colima (Colima) and Gabriel Zamora (Michoacán) were placed centrally, which possibly is evidence of ancestry and indicates its dispersion routes in the central western region of Mexico.

Genetic diversity of Fusarium mexicanum, causal agent of mango and big-leaf mahogany malformation in Mexico.

In Mexico, Fusarium mexicanum has been reported causing mango malformation disease and big-leaf mahogany malformation disease. Our objective was to determine the genetic diversity of F. mexicanum isolates obtained from malformed big-leaf mahogany and mango trees, using an internal simple sequence repeat (ISSR) analysis. A total of 61 isolates of F. mexicanum, 32 from mango and 29 from big-leaf mahogany, were initially genotyped using fourteen ISSR primers. Data from five primers that produced the highest number of polymorphic bands were selected for further analysis. The primers generated 49 polymorphic bands (85.96%) from a total of 57 fragments ranging in size from 250 to 2800 bp, with an average of 11.4 bands per primer. An analysis of molecular variance (AMOVA) indicated that the variation within populations, isolates grouped by host and geographic origin, was significant (43%), followed by the variation between the big-leaf mahogany versus mango isolates (34%), while among populations the variation was the lowest (22%). The genetic fingerprints suggested that genetic variability of F. mexicanum populations are structured by the host of origin rather than the geographic region.

Three xanthone dimers from the Thai mangrove endophytic fungus Phomopsis sp. xy21.

Three new xanthone dimers, named phomoxanthones C-E (1-3), were obtained from the Thai mangrove fungus Phomopsis sp. xy21, together with four known ones. The structures of these compounds were elucidated by the analysis of HRESIMS and extensive NMR spectroscopic data. The absolute configuration of 1 was established by the analysis of single-crystal X-ray diffraction with Cu Kα radiation. Phomoxanthones C (1) and D (2) possess a highly oxidized hexahydroxanthone skeleton.

Phylogenetic analysis of endophytic bacterial isolates from leaves of the medicinal plant Trichilia elegans A. Juss. (Meliaceae).

Various organisms such as fungi and bacteria can live inside plants, inhabiting the aerial parts (primarily the leaves) without causing damage. These microorganisms, called endophytes, produce an extensive variety of compounds that can be useful for medical and agronomic purposes. Trichilia elegans A. Juss., belonging to the Meliaceae family, shows wide dispersion in South America, and phytochemical analyses from these plants and endophyte isolates have shown biological activity. Accordingly, the aim of this study was to verify the diversity of bacterial endophytes from T. elegans using partial sequencing of 16S rRNA, followed by phylogenetic analysis. Isolation was performed by cutting the leaves, after disinfection with 5% sodium hypochlorite (NaOCl), in 1-2-mm² fragments, which were equally placed on dishes containing TSA and fungicide BENLATE at 75 µg/mL. All dishes were incubated at 28°C in the biochemical oxygen demand system for 5 days and periodically checked. Afterwards, the colonization frequency (%) was determined: (number of fragments colonized by bacteria/total number of fragments) x 100. Three isolations between September 2011 and March 2012 were performed; the growth frequency ranged between 1.6 and 13.6%. Following sequencing of 16S rRNA and phylogenetic analysis, the genera identified were: Staphylococcus, Bacillus, Microbacterium, Pseudomonas, and Pantoea. These results will provide important knowledge on the diversity of endophytic bacteria inhabiting medicinal plants, and a better understanding of the microbiome of T. elegans would reinforce the necessity of endophyte studies with a focus on their future applications in biotechnological areas of agriculture, medicine, and the environment.

An experimental test of the hypothesis of non-homeostatic consumer stoichiometry in a plant litter-microbe system.

Stoichiometric homeostasis of heterotrophs is a common, but not always well-examined premise in ecological stoichiometry. We experimentally evaluated the relationship between substrate (plant litter) and consumer (microorganisms) stoichiometry for a tropical terrestrial decomposer system. Variation in microbial C : P and N : P ratios tracked that of the soluble litter fraction, but not that of bulk leaf litter material. Microbial N and P were not isometrically related, suggesting higher rates of P than N sequestration in microbial biomass. Shifts in microbial stoichiometry were related to changes in microbial community structure. Our results indicate that P in dissolved form is a major driver of terrestrial microbial stoichiometry, similar to aquatic environments. The demonstrated relative plasticity in microbial C : P and N : P and the critical role of P have important implications for theoretical modelling and contribute to a process-based understanding of stoichiometric relationships and the flow of elements across trophic levels in decomposer systems.

New Meliolaceae from the Brazilian Atlantic forest 2: species on host families Annonaceae, Cecropiaceae, Meliaceae, Piperaceae, Rubiaceae, Rutaceae and Tiliaceae.

Continuing the study of black mildews in fragments of the Atlantic forest, three new species and five new records are described herein. Irenopsis luheae-grandiflorae, Meliola vicosensis and Meliola xylopia-sericiae are new species. Cecropia hololeuca, Piper gaudichaudianum and Trichilia lepidota are new hosts for Asteridiella leucosykeae, Asteridiella glabroides and Meliola trichiliae respectively. Asteridiella obesa and Meliola psychotriae var. chiococcae are reported for the first time from Brazil. The new species are described and illustrated based on light and scanning electron microscopy and tables with main characteristics of morphologically similar specimens with species collected in Viçosa are provided. Other species belonging to Meliolaceae collected on hosts belonging to the Annonaceae, Meliaceae and Tiliaceae in Brazil also were studied.

Production of 3-nitropropionic acid by endophytic fungus Phomopsis longicolla isolated from Trichilia elegans A. JUSS ssp. elegans and evaluation of biological activity.

The compound 3-nitropropionic acid is a potent neurotoxic agent in animals and well-known as a potent inhibitor of Mycobacterium tuberculosis. In this research, we were able to extract this compound from the endophytic fungus, Phomopsis longicolla (FJ62759), isolated from Trichilia elegans A. JUSS ssp. elegans. The aim of this study was the isolation of secondary metabolites produced by P. longicolla, the chemical identification of these compounds and evaluation of their antimicrobial and insecticidal activity. To accomplish these goals, the fungus was cultured in BD broth for 25 days without agitation at 28 °C, and then the broth was separated from the mycelium. The supernatant was partitioned with dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and butanol (BuOH) solvents resulting in 3 extracts. However, only the EtOAc extract was used for fractionation and chemical identification because it had the greatest mass. After common chromatographic procedures, the fractions were analyzed by nuclear magnetic resonance to elucidate the chemical components. This procedure resulted in the identification of 3-nitropropionic acid in the D fraction. Evaluation of the insecticidal and antimicrobial activity of this compound has been accomplished, and the results indicate good inhibition of the citrus pathogen Guignardia citricarpa and cocoa pathogen Moniliophthora perniciosa and slight inhibition of the human bacterial pathogens Micrococcus luteus, Salmonella typhi and slight inhibition of phytopathogenic bacteria Xanthomonas axonopodis pv. phaseoli. The evaluation of insecticide activity did not show mortality of the Diatraea saccharalis larvae by the metabolite 3-nitropropionic acid in the D fraction. The results suggest that P. longicolla is a bioactive metabolic producing endophytic fungus with biotechnological properties.

[Diversity and in vitro antitumnor activity of endophytic fungi from mangrove plants Xylocarpus].

A total of 24 biologically pure entophytic fungal strains were isolated from stems, leaves, and seed coats of Xylocarpus plants by repeated purification, and identified with Internal Transcribed Spacer (ITS) rDNA molecular method, which belonging to 14 genera, 11 families, 9 orders and 3 classes. There were differences in genus and species levels among three plant materials from different habitats and species, and it was found that the strains of Phomopsis and Colletotrichum existed in all three plant materials. In vitro assay of antitumor activity by MTT method revealed that the EtOAc extracts of 15 strains exhibited potent antitumor activity. These results suggest that it is of value for further investigation on the above fungal strains.

Fusarium proliferatum, an endophytic fungus from Dysoxylum binectariferum Hook.f, produces rohitukine, a chromane alkaloid possessing anti-cancer activity.

Rohitukine is a chromane alkaloid possessing anti-inflammatory, anti-cancer and immuno-modulatory properties. The compound was first reported from Amoora rohituka (Meliaceae) and later from Dysoxylum binectariferum (Meliaceae) and Schumanniophyton problematicum (Rubiaceae). Flavopiridol, a semi-synthetic derivative of rohitukine is a potent CDK inhibitor and is currently in Phase III clinical trials. In this study, the isolation of an endophytic fungus, Fusarium proliferatum (MTCC 9690) from the inner bark tissue of Dysoxylum binectariferum Hook.f (Meliaceae) is reported. The endophytic fungus produces rohitukine when cultured in shake flasks containing potato dextrose broth. The yield of rohitukine was 186 µg/100 g dry mycelial weight, substantially lower than that produced by the host tissue. The compound from the fungus was authenticated by comparing the LC-HRMS and LC-HRMS/MS spectra with those of the reference standard and that produced by the host plant. Methanolic extract of the fungus was cytotoxic against HCT-116 and MCF-7 human cancer cell lines (IC(50) = 10 µg/ml for both cancer cell lines).

Phylogenetic diversity of endophytic leaf fungus isolates from the medicinal tree Trichilia elegans (Meliaceae).

Various types of organisms, mainly fungi and bacteria, live within vegetal organs and tissues, without causing damage to the plant. These microorganisms, which are called endophytes, can be useful for biological control and plant growth promotion; bioactive compounds from these organisms may have medical and pharmaceutical applications. Trichilia elegans (Meliaceae) is a native tree that grows abundantly in several regions of Brazil. Preparations using the leaves, seeds, bark, and roots of many species of the Meliaceae family have been widely used in traditional medicine, and some members of the Trichilia genus are used in Brazilian popular medicine. We assessed the diversity of endophytic fungi from two wild specimens of T. elegans, collected from a forest remnant, by sequencing ITS1-5.8S-ITS2 of rDNA of the isolates. The fungi were isolated and purified; 97 endophytic fungi were found; they were separated into 17 morpho-groups. Of the 97 endophytic fungi, four genera (Phomopsis, Diaporthe, Dothideomycete, and Cordyceps) with 11 morpho-groups were identified. Phomopsis was the most frequent genus among the identified endophytes. Phylogenetic analysis showed two major clades: Sordariomycetes, which includes three genera, Phomopsis, Diaporthe, and Cordyceps, and the clade Dothideomycetes, which was represented by the order Pleosporales.

Antiangiogenic effect of chamigrane endoperoxides from a Thai mangrove-derived fungus.

As part of our ongoing efforts to investigate natural products with potential for use as cancer treatments, we have recently disclosed the cytotoxicity of unique nor-chamigrane (1) and chamigrane (2, 3) endoperoxides from a Thai mangrove-derived fungus. Reinvestigation of this fungus in a large-scale fermentation led to the isolation of an additional new chamigrane endoperoxide (4) and one known analogue (5). Among these isolated metabolites, compound 3 (merulin C) exhibited potent antiangiogenic activity mainly by suppression of endothelial cell proliferation and migration in a dose-dependent manner, and its effect is mediated by reduction in the phosphorylation of Erk1/2. Merulin C also displayed promising activity in a rat aortic ring sprouting (ex vivo) and a mouse Matrigel (in vivo) assay.

Chaenothecopsis khayensis, a new resinicolous calicioid fungus on African mahogany.

The new species Chaenothecopsis khayensis (Ascomycota, Mycocaliciaceae) is described from Ghana, western Africa, on the resin of Khaya anthotheca and K. ivorensis. The species is distinctive in forming asci without crosiers and in possessing ascospores that are faintly longitudinally striate. Analysis of large subunit rDNA gene sequences positioned this species within a clade corresponding to the Mycocaliciales and identified its closest relative as Sphinctrina leucopoda. Chaenothecopsis khayensis occurs commonly on resin exuding from trees damaged by the larvae of the mahogany shoot borer (Hypsipyla sp.), and we discuss the possible ecological relationship between the fungus and these moths.

A host specialized form of Ceratocystis fimbriata causes seed and seedling blight on native Carapa guianensis (andiroba) in Amazonian rainforests.

Ceratocystis fimbriata Ellis & Halsted recently was recorded causing seed and seedling blight on Carapa guianensis Aubl. (andiroba), a tree species native to the Amazon Rainforest and prized for its valuable timber and medicinal seed oil. C. fimbriata more commonly causes wilt type diseases in woody hosts, especially on non-native host trees. However, on andiroba the disease occurs on seedlings and seeds, affecting the species regeneration. We studied 73 isolates of C. fimbriata on andiroba from three regions of the Amazon Basin to see if they represented natural or introduced populations. Analysis of ITS rDNA sequences and phylogenetic analysis of mating type genes revealed new haplotypes of C. fimbriata from the Latin American Clade that were closely related to other Brazilian populations of the fungus. In mating experiments, andiroba isolates were inter-fertile with tester strains of C. fimbriata from Brazil and elsewhere, confirming that they belong to a single biological species. Using microsatellite markers, 14 genotypes and populations with intermediate levels of genetic variability were found, suggesting that the fungus is indigenous to the Amazon Basin. Inoculation tests indicated that the andiroba isolates are host-specialized on andiroba, supporting the proposition of the special form C. fimbriata f. sp. carapa.

Cytochalasins and an Abietane-Type Diterpenoid with Allelopathic Activities from the Endophytic Fungus Xylaria Species.

Bioactivity-guided isolation of the cultures of the endophytic fugus Xylaria sp. XC-16 residing in a deciduous tree Toona sinensis led to the discovery of four new allelochemicals (1-4), including three cytochalasins, epoxycytochalasin Z17 (1), epoxycytochalasin Z8 (2), and epoxyrosellichalasin (3), and an abietane-type diterpenoid, hydroxyldecandrin G (4), along with four known analogues, 10-phenyl-[12]-cytochalasins Z16 (5) and Z17 (6), cytochalasin K (7), and cytochalasin E (8). The structures of these compounds were elucidated by comprehensive spectroscopic methods, and their absolute configurations were determined by electronic circular dichroism (CD) and X-ray diffraction. All of the chemicals were tested for their allelopathic effects on turnip ( Raphanus sativus) and wheat ( Triticum aestivum). Notably, compounds 3, 4, and 7 strongly inhibited wheat shoot elongation, and compounds 5, 7, and 8 inhibited wheat root elongation, showing comparable IC50 values to the positive control glyphosate. Meanwhile, compound 8 was a potential inhibitor on turnip root elongation, with an IC50 value of 1.57 ± 0.21 µM, which was 50-fold more potent than glyphosate. Nevertheless, compounds 5 and 7 stimulated turnip shoot elongation at lower concentrations.

Multiple-locus variable-number tandem repeat analysis (MLVA) of Leptospira interrogans serovar Pomona from Argentina reveals four new genotypes.

Outbreaks of leptospirosis occur regularly in Argentina, but little is known about their epidemiological relationships. We have analyzed the genetic diversity of a collection of 16 strains of Leptospira interrogans serovar Pomona isolated from animals and humans in Argentina during the past 45 years. Genotyping was performed by multiple-locus variable-number tandem repeat analysis (MLVA) using the loci VNTR4, VNTR7, VNTR9, VNTR10, VNTR19, VNTR23 and VNTR31, as described by Majed et al. [Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto. J Clin Microbiol 2005;43:539-45]. Clustering analysis revealed four new distinct MLVA genotypes, with a dominant one. Strains with this genotype were consistently isolated since 1960 to the present, mainly from cows and pigs, but also from humans, representing 75% of the total strains studied. These strains coexisted temporally and geographically with isolates presenting the other new genotypes. VNTR4 locus, with four different alleles, presented the highest diversity between the VNTR loci analyzed. MLVA patterns obtained will be useful for future diagnostic and epidemiological tracing analysis.

A Novel Disease of Big-Leaf Mahogany Caused by Two Fusarium Species in Mexico.

Big-leaf mahogany (Swietenia macrophylla) is valued for its high-quality wood and use in urban landscapes in Mexico. During surveys of mango-producing areas in the central western region of Mexico, symptoms of malformation, the most important disease of mango in the area, were observed on big-leaf mahogany trees. The objectives of this research were to describe this new disease and determine its cause. Symptoms on big-leaf mahogany at four sites in Michoacán, Mexico resembled those of the vegetative phase of mango malformation, including compact, bunched growth of apical and lateral buds, with greatly shortened internodes and small leaves that curved back toward the supporting stem. Of 163 isolates that were recovered from symptomatic tissues, most were identified as Fusarium pseudocircinatum (n = 121) and F. mexicanum (n = 39) using molecular systematic data; two isolates represented unnamed phylospecies within the F. incarnatum-equiseti species complex (FIESC 20-d and FIESC 37-a) and another was in the F. solani species complex (FSSC 25-m). However, only F. mexicanum and F. pseudocircinatum induced malformation symptoms on 14-day-old seedlings of big-leaf mahogany. The results indicate that F. mexicanum and F. pseudocircinatum, previously reported in Mexico as causal agents of mango malformation disease, also affect big-leaf mahogany. This is the first report of this new disease and the first time that F. mexicanum was shown to affect a host other than mango.

Xanthone-derived polyketides from the Thai mangrove endophytic fungus Phomopsis sp. xy21.

Six new xanthone-derived polyketides, named phomoxanthones F-K (1-6), along with three known ones, were isolated from Phomopsis sp. xy21, which was isolated as an endophytic fungus from the Thai mangrove Xylocarpus granatum. Phomoxanthone F (1) represents the first xanthone-derived polyketide containing a 10a-decarboxylated benzopyranone nucleus that was substituted by a 4-methyldihydrofuran-2(3H)-one moiety at C10a. Phomoxanthones G (2) and H (3) are highly oxidized xanthone-derived polyketides containing a novel 5-methyl-6-oxabicyclo[3.2.1]octane motif. This is the first report of a C6-O-C12 bridge in xanthone-derived polyketides. Additionally, a plausible biogenetic pathway for these xanthone-derived polyketides is proposed.

Phenolic bisabolane sesquiterpenoids from a Thai mangrove endophytic fungus, Aspergillus sp. xy02.

Seven new phenolic bisabolane sesquiterpenoids, (7R,10S)-7,10-epoxysydonic acid (1), (7S,10S)-7,10-epoxysydonic acid (2), (7R,11S)-7,12-epoxysydonic acid (3), (7S,11S)-7,12-epoxysydonic acid (4), 7-deoxy-7,14-didehydro-12-hydroxysydonic acid (5), (Z)-7-deoxy-7,8-didehydro-12-hydroxysydonic acid (6), and (E)-7-deoxy-7,8-didehydro-12-hydroxysydonic acid (7), along with five known analogues (8-12), were obtained from the culture of an endophytic fungus Aspergillus sp. xy02 isolated from the leaves of a Thai mangrove Xylocarpus moluccensis. All structures were assigned on the basis of detailed spectroscopic analyses. The absolute configurations of 1-4, being two pairs of epimers, were established by TDDFT-ECD calculations. Compound 12 showed mild antioxidative activity to scavenge DPPH radical with an IC50 of 72.1 µM, whereas 2, 3, 5, 7, 9, 11, and 12 displayed moderate inhibitory activities against Staphylococcus aureus ATCC 25923 with IC50 values ranging from 31.5 to 41.9 µM.

Arbuscular mycorrhizal fungi activity in the rhizosphere of tree seedlings subjected to residual herbicides / Atividade de fungos micorrízicos arbusculares na rizosfera de mudas de árvores submetidas a herbicidas residuais

Trees occurring on the margins of agricultural areas can mitigate damage from residual herbicides. Rhizospheric microbial activity associated with trees is one of the main remedial capacity indicators. The objective of this study was to evaluate the rhizospheric microbiological activity in tree species subjected to the herbicides atrazine and sulfentrazone via the rhizosphere. The experiment was designed in four blocks and a 6 × 3 factorial scheme. The first factor consisted of six tree species from Brazil and the second of atrazine, sulfentrazone, and water solutions. Four herbicide applications were performed via irrigation. The total dry mass of the plants, mycorrhizal colonization, number of spores, basal respiration of the rhizospheric soil, and survival rate of bioindicator plants after phytoremediation were determined. Trichilia hirta had higher biomass when treated with atrazine and sulfentrazone. Herbicides decreased the microbial activity in Triplaris americana and did not affect the microbiological indicators of Myrsine gardneriana, Schizolobium parahyba, and Toona ciliata. Fewer bioindicator plants survived in soil with Triplaris americana and sulfentrazone. Microbiological indicators were influenced in different ways between species by the presence of herbicides in the rhizosphere.

As árvores que ocorrem nas margens das áreas agrícolas podem mitigar os danos dos herbicidas residuais. A atividade microbiana rizosférica associada às árvores é um dos principais indicadores de capacidade corretiva. O objetivo deste trabalho foi avaliar a atividade microbiológica rizosférica em espécies arbóreas submetidas aos herbicidas atrazina e sulfentrazone via rizosfera. O experimento foi estruturado em quatro blocos e esquema fatorial 6 × 3. O primeiro fator consistiu em seis espécies de árvores do Brasil e o segundo em soluções de atrazine, sulfentrazone e água. Quatro aplicações de herbicidas foram realizadas via irrigação. Foram determinados a massa seca total das plantas, colonização micorrízica, número de esporos, respiração basal do solo rizosférico e taxa de sobrevivência de plantas bioindicadoras após fitorremediação. Trichilia hirta apresentou maior biomassa quando tratada com atrazina e sulfentrazone. Os herbicidas diminuíram a atividade microbiana em Triplaris americana e não afetaram os indicadores microbiológicos de Myrsine gardneriana, Schizolobium parahyba e Toona ciliata. Menos plantas bioindicadoras sobreviveram no solo com Triplaris americana e sulfentrazone. Os indicadores microbiológicos foram influenciados de formas distintas entre as espécies pela presença dos herbicidas na rizosfera.

Antimicrobial activity of endophytic fungi isolated from Swietenia macrophylla leaves.

The endophytic fungi isolated from leaves of Swietenia macrophylla of different ages were examined for antimicrobial activity. The agar plug diffusion assay was used for primary screening, followed by the disc diffusion method. A total of 461 filamentous endophytic fungi were isolated and cultured to examine their antimicrobial properties. In the primary screen, 315 isolates (68.3%) exhibited activity against at least one of the test pathogenic microorganisms. The percentage of isolates exhibiting antimicrobial activity increased with leaf age. Endophytic fungal assemblages, as well as those isolates exhibiting antimicrobial properties appeared to increase with leaf age. The main antimicrobial compounds were produced extracellularly by the endophytic fungi. The results suggest that healthy leaves at older stages of growth can be a potential source for the isolation of endophytic fungi with antimicrobial properties.