Pitchers of Nepenthes khasiana express several digestive-enzyme encoding genes, harbor mostly fungi and probably evolved through changes in the expression of leaf polarity genes.

BACKGROUND: A structural phenomenon seen in certain lineages of angiosperms that has captivated many scholars including Charles Darwin is the evolution of plant carnivory. Evidently, these structural features collectively termed carnivorous syndrome, evolved to aid nutritional acquisition from attracted, captured and digested prey. We now understand why plant carnivory evolved but how carnivorous plants acquired these attributes remains a mystery. In an attempt to understand the evolution of Nepenthes pitcher and to shed more light on its role in prey digestion, we analyzed the transcriptome data of the highly specialized Nepenthes khasiana leaf comprising the leaf base lamina, tendril and the different parts/zones of the pitcher tube viz. digestive zone, waxy zone and lid. RESULTS: In total, we generated around 262 million high-quality Illumina reads. Reads were pooled, normalized and de novo assembled to generate a reference transcriptome of about 412,224 transcripts. We then estimated transcript abundance along the N. khasiana leaf by mapping individual reads from each part/zone to the reference transcriptome. Correlation-based hierarchical clustering analysis of 27,208 commonly expressed genes indicated functional relationship and similar cellular processes underlying the development of the leaf base and the pitcher, thereby implying that the Nepenthes pitcher is indeed a modified leaf. From a list of 2386 differentially expressed genes (DEGs), we identified transcripts encoding key enzymes involved in prey digestion and protection against pathogen attack, some of which are expressed at high levels in the digestive zone. Interestingly, many of these enzyme-encoding genes are also expressed in the unopened N. khasiana pitcher. Transcripts showing homology to both bacteria and fungi were also detected; and in the digestive zone, fungi are more predominant as compared to bacteria. Taking cues from histology and scanning electron microscopy (SEM) photomicrographs, we found altered expressions of key regulatory genes involved in leaf development. Of particular interest, the expression of class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIPIII) and ARGONAUTE (AGO) genes were upregulated in the tendril. CONCLUSIONS: Our findings suggest that N. khasiana pitchers employ a wide range of enzymes for prey digestion and plant defense, harbor microbes and probably evolved through altered expression of leaf polarity genes.

Investigation of an Elevational Gradient Reveals Strong Differences Between Bacterial and Eukaryotic Communities Coinhabiting Nepenthes Phytotelmata.

Elevation is an important determinant of ecological community composition. It integrates several abiotic features and leads to strong, repeatable patterns of community structure, including changes in the abundance and richness of numerous taxa. However, the influence of elevational gradients on microbes is understudied relative to plants and animals. To compare the influence of elevation on multiple taxa simultaneously, we sampled phytotelm communities within a tropical pitcher plant (Nepenthes mindanaoensis) along a gradient from 400 to 1200 m a.s.l. We use a combination of metabarcoding and physical counts to assess diversity and richness of bacteria, micro-eukaryotes, and arthropods, and compare the effect of elevation on community structure to that of regulation by a number of plant factors. Patterns of community structure differed between bacteria and eukaryotes, despite their living together in the same aquatic microhabitats. Elevation influences community composition of eukaryotes to a significantly greater degree than it does bacteria. When examining pitcher characteristics, pitcher dimorphism has an effect on eukaryotes but not bacteria, while variation in pH levels strongly influences both taxa. Consistent with previous ecological studies, arthropod abundance in phytotelmata decreases with elevation, but some patterns of abundance differ between living inquilines and prey.

Transcriptomic de novo analysis of pitaya (Hylocereus polyrhizus) canker disease caused by Neoscytalidium dimidiatum.

BACKGROUND: Canker disease caused by Neoscytalidium dimidiatum is the most serious disease that attacks the pitaya industry. One pathogenic fungus, referred to as ND8, was isolated from the wild-type red-fleshed pitaya (Hylocereus polyrhizus) of Hainan Province. In the early stages of this disease, stems show little spots and a loss of green color. These spots then gradually spread until the stems became rotten due to infection by various strains. Canker disease caused by Neoscytalidium dimidiatum poses a significant threat to pitaya commercial plantations with the growth of stems and the yields, quality of pitaya fruits. However, a lack of transcriptomic and genomic information hinders our understanding of the molecular mechanisms underlying the pitaya defense response. RESULTS: We investigated the host responses of red-fleshed pitaya (H. polyrhizus) cultivars against N. dimidiatum using Illumina RNA-Seq technology. Significant expression profiles of 23 defense-related genes were further analyzed by qRT-PCR. The total read length based on RNA-Seq was 25,010,007; mean length was 744, the N50 was 1206, and the guanine-cytosine content was 44.48%. Our investigation evaluated 33,584 unigenes, of which 6209 (18.49%) and 27,375 (81.51%) were contigs and singlets, respectively. These unigenes shared a similarity of 16.62% with Vitis vinifera, 7.48% with Theobroma cacao, 6.6% with Nelumbo nucifera and 5.35% with Jatropha curcas. The assembled unigenes were annotated into non-redundant (NR, 25161 unigenes), Kyoto Encyclopedia of Genes and Genomes (KEGG, 17895 unigenes), Clusters of Orthologous Groups (COG, 10475 unigenes), InterPro (19,045 unigenes), and Swiss-Prot public protein databases (16,458 unigenes). In addition, 24 differentially expressed genes, which were mainly associated with plant pathology pathways, were analyzed in-depth. CONCLUSIONS: This study provides a basis for further in-depth research on the protein function of the annotated unigene assembly with cDNA sequences.

Role of Curtobacterium herbarum strain CAH5 on aluminum bioaccumulation and enhancement of Lactuca sativa growth under aluminum and drought stresses.

Aluminum (Al) bioaccumulation by a novel Al and drought tolerant Curtobacterium herbarum strain CAH5 isolated from rhizosphere soil of Beta vulgaris grown in acidic Andisols were examined. The rhizobacterial strain also presented important plant growth promoting traits even with Al and drought stresses under in-vitro conditions in broth. In experiments with a 2-6 mM as initial Al concentrations, the percentages of Al removal by bacteria were 89-93% and 78-91% within 72 h incubation under the normal and drought conditions, respectively. Cytogenotoxicity assay revealed that the toxicity of Al was reduced after bioaccumulation process. In the greenhouse study, formulated bio-inoculant CAH5 significantly improves the Lactuca sativa growth under Al and drought stress by reducing oxidative stress, lipid peroxidation and Al accumulation in plant parts. Our results highlighted that strain CAH5 could be used as a promising bioresource for restoration of agricultural soil with presence of phytotoxic Al improving crop production even under drought conditions.

Biosorption of copper by endophytic fungi isolated from Nepenthes ampullaria.

Copper (Cu) tolerance was observed by endophytic fungi isolated from the carnivorous plant Nepenthes ampullaria (collected at an anthropogenically affected site, Kuching city; and a pristine site; Heart of Borneo). The fungal isolates, capable of tolerating Cu up to 1000 ppm (11 isolates in total), were identified through molecular method [internal transcribed spacer 4+5 (ITS4+5); ITS1+NL4; ß-tubulin region using Bt2a + Bt2b], and all of them grouped with Diaporthe, Nigrospora, and Xylaria. A Cu biosorption study was then carried out using live and dead biomass of the 11 fungal isolates. The highest biosorption capacity of using live biomass was achieved by fungal isolates Xylaria sp. NA40 (73·26 ± 1·61 mg Cu per g biomass) and Diaporthe sp. NA41 (72·65 ± 2·23 mg Cu per g biomass), NA27 (59·81 ± 1·15 mg Cu per g biomass) and NA28 (56·85 ± 4·23 mg Cu per g biomass). The fungal isolate Diaporthe sp. NA41 also achieved the highest biosorption capacity of 59·33 ± 0·15 mg g-1 using dead biomass. The living biomass possessed a better biosorption capacity than the dead biomass (P < 0·05) and the roadside fungal strains showed higher Cu biosorption capacities using live biomass compared to the jungle fungal strains (P < 0·05). SIGNIFICANCE AND IMPACT OF THE STUDY: Our study highlights that fungal biosorption capacity is highly dependent on the sampling area (roadside vs jungle) with roadside fungal strains showing significantly higher copper (Cu) biosorption capacities using living biomass compared to fungal strains originating from plants collected in virgin jungle (P < 0·05). It also highlights that different biosorption mechanisms (alive - metabolic dependent and dead biomass - metabolic independent) result in different amounts of Cu being removed from the solutions. The living biomass possessed a better biosorption capacity than the dead biomass (P < 0·05).

Caryophyllales are the main hosts of a unique set of ectomycorrhizal fungi in a Neotropical dry forest.

The ectomycorrhizal symbiosis was long thought to be restricted to temperate forests. However, as tropical forests have been explored, it has become clear that these habitats host unique ectomycorrhizal (ECM) fungi. We have been exploring tropical dry forests (TDF), which are endangered terrestrial ecosystems and hotspots of endemism. Since Fabaceae is the main plant family in this environment, we hypothesized that trees in this lineage would be the main ECM hosts. We sequenced the ITS rDNA region from fungi and both rbcL and trnL cpDNA from plants to identify both symbiotic partners from root tips. The systematic position of each symbiont was confirmed by Bayesian phylogenetic inference. We identified 20 plant species belonging to 10 families that hosted 19 unique ECM fungal species from 5 lineages. Most ECM fungi were associated with Caryophyllales, not with Fabaceae. Achatocarpus and Guapira, the main hosts, are scattered throughout the forest and are not in monodominant patches. The low ECM fungal diversity can be explained by the low density of host plants and their high specificity. Our results indicate that Caryophyllales is an important order of tropical ECM hosts with at least four independent evolutionary lineages that have evolved the ability to form ectomycorrhizae.

Transfer to forest nurseries significantly affects mycorrhizal community composition of Asteropeia mcphersonii wildings.

Mycorrhizal symbiosis is extremely important for tree growth, survival and resistance after transplantation particularly in Madagascar where deforestation is a major concern. The importance of mycorrhizal symbiosis is further increased when soil conditions at the planting site are limiting. To identify technical itineraries capable of improving ecological restoration in Madagascar, we needed to obtain native ectomycorrhizal (ECM) saplings with a wide diversity of ECM fungi. To this end, we transplanted ECM seedlings from the wild (wildlings) to a nursery. Using molecular characterisation of internal transcribed spacer (ITS) rDNA, we tested the effect of transplanting Asteropeia mcphersonii wildlings on ECM communities after 8 months of growth in the nursery. With or without the addition of soil from the site where the seedlings were sampled to the nursery substrate, we observed a dramatic change in the composition of fungal communities with a decrease in the ECM infection rate, a tremendous increase in the abundance of an operational taxonomic unit (OTU) taxonomically close to the order Trechisporales and the disappearance of all OTUs of Boletales. Transplanting to the nursery and/or to nursery conditions was shown to be incompatible with the survival and even less with the development in the nursery of most ECM fungi naturally associated with A. mcphersonii wildings.

Non-halophilic endophytes associated with the euhalophyte Arthrocnemum macrostachyum and their plant growth promoting activity potential.

Numerous microbial taxa establish natural relations with plants, and especially endophytes can be relevant in the development and growth promotion of their host. In this work, we explore the diversity of non-halophilic microorganisms inhabiting the endosphere of the halophyte Arthrocnemum macrostachyum. A total of 1045 isolates were recovered using standard non-saline media, which clustered into 22 operational phylogenetic units (OPUs) including 7 putative new species and 13 OPUs not previously detected as endophytes. The more abundant isolates corresponded to close relatives of Kushneria indalinina/K. marisflavi, Providencia rettgeri, Pseudomonas zhaodongensis and Bacillus safensis, which made up to ∼ 62% of the total isolates. We also isolated OPUs not detected by the culture-independent approach reinforcing the need of culturing to reveal the microbial diversity associated with plants. Additionally, the plant growth promoting activity was evaluated by representative strains of the more abundant OPUs (total = 94 strains) including also some previously isolated halophiles from the same plants. Under both saline and non-saline conditions, some strains principally those affiliated to Paenibacillus borealis, Staphylococcus equorum, Salinicola halophilus and Marinococcus tarijensis, presented growth promoting activity in Arabidopsis thaliana, which was evaluated as an increment of weight and root length.

Antimicrobial activity in cultures of endophytic fungi isolated from Talinum triangulare

Endophytic fungi colonize the interior of plants without causing damage and act in symbiosis with their host. They are also potential sources of compounds with potential applications in industry, agriculture, and medicine, Thus, this study aimed to isolate and identify the endophytic fungi medicinal plant Talinum triangulare and evaluate its potential for the production of antimicrobial substances using the disk diffusion technique and testing in liquid medium front of Staphylococcus aureus ATCC SA 6538, Pseudomonas aeruginosa ATCC PA 0030, and Corynebacterium diphtheria ATCC 27012. Corynebacterium diphtheria was isolated from 3 fungi of the genus Trichoderma and Penicillium, with only the genus Trichoderma fungi showing antimicrobial activity.