Molecular characterization of leaf spot caused by Alternaria alternata on buttonwood (Conocarpus erectus L.) and determination of pathogenicity by a novel disease rating scale.

The buttonwood (Conocarpus erectus L.) is a mangrove shrub belonging to Combretaceae family. It mostly grows on the shorelines of tropical and subtropical regions in the world. It was introduced to Lasbela University of Water, Agriculture & Marine Sciences (LUWMS), Uthal, Baluchistan as an ornamental plant as it grows well under harsh, temperate and saline conditions. During a routine survey, typical leaf spot symptoms were observed on the leaves of buttonwood plants. A disease severity scale for alternaria leaf spot of buttonwood was developed for the first time through this study. Disease severity according to the scale was 38.97%. The microscopic characterizations was accomplished for the identification of Alternaria alternata and Koch's postulates were employed to determine the pathogenicity. For molecular identification, 650 bp internal transcribed spacer (ITS) regions (ITS1, 5.8s and ITS2) were amplified from three representative isolates (LUAWMS1, LUAWMS2 and LUAWMS3) through polymerase chain reaction (PCR). The nucleotide sequences from ITS regions of the isolates were submitted to NCBI with GenBank accession numbers MW585375, MW585376 and MW585377, respectively. The phylogenetic tree of 22 A. alternata isolates was computed and representative isolates exhibited 99.98% genetic similarity with mangroves ecosystem isolates. This study reports the incidence of alternaria leaf spot of buttonwood at LUWMS for the first time. It is suspected that the disease may spread further. Therefore, effective management strategies should be opted to halt the further spread of the disease.

Intraspecific variation on epiphytic bacterial community from Laguncularia racemosa phylloplane.

Mangroves are dynamic and unique ecosystems that provide important ecological services to coastal areas. The phylloplane is one of the greatest microbial habitats, and most of its microorganisms are uncultivated under common laboratory conditions. Bacterial community structure of Laguncularia racemosa phylloplane, a well-adapted mangrove species with salt exudation at foliar levels, was accessed through 16S rRNA amplicon sequencing. Sampling was performed in three different sites across a transect from upland to the seashore in a preserved mangrove forest located in the city of Cananéia, São Paulo State, Brazil. Higher bacterial diversity was observed in intermediary locations between the upland and the seashore, showing that significant intraspecific spatial variation in bacterial communities exists between a single host species with the selection of specific population between an environmental transect.

The remediation of drought stress under VAM inoculation through proline chemical transformation action.

This article discussed the enhanced drought tolerance under arbuscular mycorrhizal (AM) inoculation and normal growth of plants which linked with the activity of photoreceptors. The photoreceptor action in dual symbiosis under drought stress showed not only the high photosynthetic activity but also provide information about the broad range of physiological responses. The pot experiment conducted in a natural environment where drought condition was observed twice a week via regular irrigation with water up to twelve months. Plants analysis showed the high contents of water, hydrogen peroxide, carotenoids, proline, antioxidant enzymes like super dismutase (SOD) and catalase(CAT) in both leaves and roots with a large surface area of leaves over control. The elevated concentration of hydrogen peroxide (0.04 ±â€¯0.0 µmol/g) coupled with singlet oxygen species was the main modified molecular mechanism which was operative in drought condition. The accretion of proline under drought stress in dual symbiosis (32.3 ±â€¯0.3 µg/mL) was related to the highest branching pattern of young leaves and the chemical transformation of reactive oxygen species (ROS) including H2O2 and 3O2 into useful molecules like water and triplet molecular oxygen. The higher contents of carotenoids (5.0 ±â€¯1.2 mg/g) in drought over control (4.8 ±â€¯1.6 mg/g) and AM plant (4.9 ±â€¯1.2 mg/g) was found to be supportive in the conversion of singlet oxygen into triplet one.

Bacterial Succession Decreases Network Complexity During Plant Material Decomposition in Mangroves.

In this study, 16S rRNA gene amplicon sequencing was used to assess bacterial diversity and dynamics throughout different stages of leaves decomposition of three plant species (Rhizophora mangle, Laguncularia racemosa, and Avicennia schaueriana) in three distinct mangroves of São Paulo state, Brazil. The experiments were conducted in microcosms. Phylogenetic diversity (Faiths' PD) index showed differences between samples and suggested that some treatments like R. mangle increased their bacterial diversity through time. Principal coordinate analysis revealed that community's profile varied based on mangroves, followed by plant species and time. A clear succession patterns was observed in this study, i.e., some microorganisms with low abundance in the initial phases gradually became dominant (e.g., Alphaproteobacteria), whereas microbes that were initially predominant became low (e.g., Gammaproteobacteria). Co-occurrence analyses were performed for all times of plant degradation aiming to better understand the relationships between bacterial populations. The c-score index was done to test the randomness of the community assemblage during the stages of decomposition. For all degradation time points, the values of the observed c-score were higher than the values of the simulated c-score. This result indicated that during plant decomposition, the bacterial communities presented less co-occurrence than expected by chance and that these communities were not randomly assembled but instead they are driven by species interactions. Network analyses results showed that in the conditions presented in this experiment, the initial stages of leaf decomposition formed more connected and complex networks than the later stages. These results suggest that resource competition was a determinant in these specific mangroves during plant degradation, mainly in the initial periods.

New Prospective for Enhancement in Bioenergy Resources Through Fungal Engineering.

BACKGROUND: Lignin and cellulose, organic constituents of the plant or plant-based material not commonly used for feeding purpose are referred as Biomass. Patents suggest that this can be used as the best resource of renewable energy. Vesicular Arbuscular Mycorrhizae (VAM) fungi can play an effective role in biomass manufacturing through activated metabolism of the plant under dual symbiosis. During C acclimatization, mycorrhizal inoculated plants existent greater number of leaves with a height of plants as compared to non-mycorrhizal plants. The current article discloses the search of the natural resources for C assimilation into biomass using mycorrhizal symbiosis. METHODS: The pot experiment was conducted in the natural environment for extraction of more bioenergy through biomass of Conocarpus erectus L under VAM (Glomus fasciculatum) inoculation in various environmental conditions with replicates. RESULTS: It observed that these fungal engineered plants showed distinctive prospective to offer, enhanced biomass to energy couple with a strong network for sinking CO2 from the atmosphere via strong roots and large surface area of leaves. There was an increase in biomass (9-17% respectively) of the plant under drought-VAM, VAM inoculation and VAM- enriched CO2 conditions in same period in comparison to control plants through lignin, cellulose and carbohydrate contents. It was followed by enhanced enzyme activities and nutrient ions in dual symbiosis. CONCLUSION: Coupling biomass-originated energy may recover environmental conditions and commercial value for sustainable growth in energy consumption sector. The green energy from fungal engineered plants may replace high demand of fossil fuel as a young biofuel and make the cities more productive in the fabrication of bioenergy too in the form of biomass or biofuel with C impartial atmosphere.

Chemogenomics driven discovery of endogenous polyketide anti-infective compounds from endosymbiotic Emericella variecolor CLB38 and their RNA secondary structure analysis.

In the postgenomic era, a new strategy for chemical dereplication of polyketide anti-infective drugs requires novel genomics and chromatographic strategies. An endosymbiotic fungal strain CLB38 was isolated from the root tissue of Combretum latifolium Blume (Combretaceae) which was collected from the Western Ghats of India. The isolate CLB38 was then identified as Emericella variecolor by its characteristic stellate ascospores culture morphology and molecular analysis of ITS nuclear rDNA and intervening 5.8S rRNA gene sequence. ITS2 RNA secondary structure modeling clearly distinguished fungal endosymbiont E. variecolor CLB38 with other lifestyles in the same monophyletic clade. Ethyl acetate fraction of CLB38 explored a broad spectrum of antimicrobial activity against multidrug resistant pathogens. Biosynthetic PKS type-I gene and chromatographic approach afford two polyketide antimicrobial compounds which identified as evariquinone and isoindolones derivative emerimidine A. MIC of purified compounds against test microorganisms ranged between 3.12 µg/ml and 12.5 µg/ml. This research highlights the utility of E. variecolor CLB38 as an anticipate source for anti-infective polyketide metabolites evariquinone and emerimidine A to combat multidrug resistant microorganisms. Here we demonstrates a chemogenomics strategy via the feasibility of PKS type-I gene and chromatographic approach as a proficient method for the rapid prediction and discovery of new polyketides compounds from fungal endosymbionts.

Intra-specific differentiation of fungal endosymbiont Alternaria longissima CLB44 using RNA secondary structure analysis and their anti-infective potential.

New antimicrobial agents derived from endosymbio-tic fungi with unique and targeted mode of action are crucially rudimentary to combat multidrug-resistant infections. Most of the fungi isolated as endosymbionts show close morphological feature resemblance to plant pathogenic or free-living forms, and it is difficult to differentiate these different lifestyles. A fungal endosymbiont strain CLB44 was isolated from Combretum latifolium Blume (Combretaceae). CLB44 was then identified as Alternaria longissima based on morphological and internal transcribed spacer (ITS) intervening 5.8S rRNA gene sequence analysis. ITS2 RNA secondary structure analysis was carried out using mfold server with temperature 37 °C, and anti-infective potential was determined by MIC and disk diffusion methods. ITS2 RNA secondary structure analysis clearly distinguished endosymbiotic A. longissima CLB44 from free-living and pathogenic A. longissima members in the same monophyletic clade. Secondary metabolites produced effectively inhibited Pseudomonas aeruginosa (25 µg/ml), Escherichia coli (25 µg/ml), methicillin-resistant Staphylococcus aureus (50 µg/ml), Candida albicans (100 µg/ml), and other human pathogens. This study emerges as an innovative finding that explores newly revealed ITS2 RNAs that may be an insight as new markers for refining phylogenetic relations and to distinguish fungal endosymbionts with other free-living or pathogenic forms. A. longissima CLB44, in the emerging field of endosymbionts, will pave the way to a novel avenue in drug discovery to combat multidrug-resistant infections. The sequence data of this fungus is deposited in GenBank under the accession no. KU310611.

Sulfur-containing cytotoxic curvularin macrolides from Penicillium sumatrense MA-92, a fungus obtained from the rhizosphere of the mangrove Lumnitzera racemosa.

Sumalarins A-C (1-3), the new and rare examples of sulfur-containing curvularin derivatives, along with three known analogues (4-6), were isolated and identified from the cytotoxic extract of Penicillium sumatrense MA-92, a fungus obtained from the rhizosphere of the mangrove Lumnitzera racemosa . Their structures were established by detailed interpretation of NMR and MS data, and compound 1 was confirmed by X-ray crystallographic analysis. Compounds 1-3 and 5 showed potent cytotoxicity against some of the tested tumor cell lines. Sulfur substitution at C-11 or a double bond at C-10 significantly increased the cytotoxic activities of the curvularin analogues.

Abundance and genetic diversity of nifH gene sequences in anthropogenically affected Brazilian mangrove sediments.

Although mangroves represent ecosystems of global importance, the genetic diversity and abundance of functional genes that are key to their functioning scarcely have been explored. Here, we present a survey based on the nifH gene across transects of sediments of two mangrove systems located along the coast line of São Paulo state (Brazil) which differed by degree of disturbance, i.e., an oil-spill-affected and an unaffected mangrove. The diazotrophic communities were assessed by denaturing gradient gel electrophoresis (DGGE), quantitative PCR (qPCR), and clone libraries. The nifH gene abundance was similar across the two mangrove sediment systems, as evidenced by qPCR. However, the nifH-based PCR-DGGE profiles revealed clear differences between the mangroves. Moreover, shifts in the nifH gene diversities were noted along the land-sea transect within the previously oiled mangrove. The nifH gene diversity depicted the presence of nitrogen-fixing bacteria affiliated with a wide range of taxa, encompassing members of the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and also a group of anaerobic sulfate-reducing bacteria. We also detected a unique mangrove-specific cluster of sequences denoted Mgv-nifH. Our results indicate that nitrogen-fixing bacterial guilds can be partially endemic to mangroves, and these communities are modulated by oil contamination, which has important implications for conservation strategies.

Denaturing gradient gel electrophoresis and barcoded pyrosequencing reveal unprecedented archaeal diversity in mangrove sediment and rhizosphere samples.

Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages.

Cyanobacterial diversity in the phyllosphere of a mangrove forest.

The cyanobacterial community colonizing phyllosphere in a well-preserved Brazilian mangrove ecosystem was assessed using cultivation-independent molecular approaches. Leaves of trees that occupy this environment (Rhizophora mangle,Avicennia schaueriana and Laguncularia racemosa) were collected along a transect beginning at the margin of the bay and extending upland. The results demonstrated that the phyllosphere of R. mangle and L. racemosa harbor similar assemblages of cyanobacteria at each point along the transect. A. schaueriana, found only in the coastal portions of the transect, was colonized by assemblages with lower richness than the other trees. However, the results indicated that spatial location was a stronger driver of cyanobacterial community composition than plant species. Distinct cyanobacterial communities were observed at each location along the coast-to-upland transect. Clone library analysis allowed identification of 19 genera of cyanobacteria and demonstrated the presence of several uncultivated taxa. A predominance of sequences affiliated with the orders Nostocales and Oscillatoriales was observed, with a remarkable number of sequences similar to genera Symphyonemopsis/Brasilonema (order Nostocales). The results demonstrated that phyllosphere cyanobacteria in this mangrove forest ecosystem are influenced by environmental conditions as the primary driver at the ecosystem scale, with tree species exerting some effect on community structure at the local scale.

Candida wangnamkhiaoensis sp. nov., an anamorphic yeast species in the Hyphopichia clade isolated in Thailand.

Two strains representing a single novel yeast species were isolated from a flower of Calycoopteris floribunda Lame (SK170(T)) and insect frass (ST-122) collected in Thailand. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and the sequence analysis of the D1/D2 domain of the large subunit rRNA gene and the internal transcribed spacer region, the two strains were assigned as a single novel Candida species in the Hyphopichia clade for which the name Candida wangnamkhiaoensis sp. nov. is proposed. The type strain is SK170(T)=BCC 39604(T)=NBRC 106724(T)=CBS 11695(T)).

Investigation of microbes in the rhizosphere of selected trees for the rhizoremediation of hydrocarbon-contaminated soils.

Hydrocarbon-degrading microorganisms (HDMs), associated with the rhizosphere of Conocarpus lancifolius and Ficus infectoria trees grown in bioremediated soil, were isolated under controlled laboratory conditions. The selected trees were used to phytoremediate oil-contaminated soil for three successive growing seasons. At the end of the phytoremediation experiment, 85.7% of measurable total petroleum hydrocarbon was degraded in the rhizosphere soil associated with Conocarpus lancifolius compared to 78.6% in the rhizosphere of Ficus infectoria. The detectable concentrations of some polyaromatic hydrocarbons were less than 0.02 ppm. The HDM isolation process was conducted at 35 degrees C under aerobic conditions. The isolated HDMs were identified using 16S rRNA gene sequencing and fatty acid methyl ester analysis. Differences in the genera of the isolated HDMs and their assessed efficiency in degrading a mixture of hydrocarbon compounds between the two trees were noted.