NMR,HS‐SPME‐GC/MS,and HPLC/MSn Analyses of Phytoconstituents and Aroma Profile of Rosmarinus eriocalyx

In this work, a comprehensive study on the chemical constituents of the aerial parts of Rosmarinus eriocalyx (Lamiaceae), an aromatic shrub traditionally consumed as a food and herbal remedy in Algeria, is presented. The aroma profile was analysed by headspace solid phase microextraction (HS‐SPME) coupled with gas chromatography‐mass spectrometry (GC/MS), whereas the crude extract constituents were analyzed by ¹H‐NMR and by high performance liquid chromatography coupled with mass spectrometry (HPLC/MSⁿ). Thirty‐nine volatile compounds, most of them being monoterpenes, have been identified, with camphor, camphene, and α‐pinene as the most abundant constituents. ¹H‐NMR analysis revealed the presence of phenolic compounds and betulinic acid while HPLC/MSⁿ allowed the identification of glycosilated and aglyconic flavonoids as well as phenylpropanoid derivatives. Some of these constituents, namely as betulinic acid, rosmanol, and cirsimaritin were reported for the first time in R. eriocalyx.     

Proteomic analysis of Arabidopsis thaliana (L.) Heynh responses to a generalist sucking pest (Myzus persicae Sulzer)

Herbivorous insects can cause severe cellular changes to plant foliage following infestations, depending on feeding behaviour. Here, a proteomic study was conducted to investigate the influence of green peach aphid (Myzus persicae Sulzer) as a polyphagous pest on the defence response of Arabidopsis thaliana (L.) Heynh after aphid colony establishment on the host plant (3 days). Analysis of about 574 protein spots on 2‐DE gels revealed 31 differentially expressed protein spots. Twenty out of these 31 differential proteins were selected for analysis by mass spectrometry. In 12 of the 20 analysed spots, we identified seven and nine proteins using MALDI‐TOF‐MS and LC‐ESI‐MS/MS, respectively. Of the analysed spots, 25% contain two proteins. Different metabolic pathways were modulated in Arabidopsis leaves according to aphid feeding: most corresponded to carbohydrate, amino acid and energy metabolism, photosynthesis, defence response and translation. This paper has established a survey of early alterations induced in the proteome of Arabidopsis by M. persicae aphids. It provides valuable insights into the complex responses of plants to biological stress, particularly for herbivorous insects with sucking feeding behaviour.     

Bacterial iturins mediate biocontrol activity of Bacillus sp. against postharvest pear fruit‐rotting fungi

A potential antagonist, Bacillus sp. LYLB4 isolated from pear fruits, was tested for its antifungal activity against postharvest pear pathogens. LYLB4 had a remarkable antifungal effect on Botryosphaeria dothidea. Although it showed a weak inhibition effect on the growth of Rhizopus stolonifer on potato dextrose agar (PDA) plates, LYLB4 almost completely destroyed R. stolonifer during direct contact in potato dextrose broth (PDB). LYLB4 treatment was able to significantly reduce disease incidence (by 68.9% and 100%, respectively) and lesion diameter (by 68.7% and 100%, respectively) of ring rot caused by B. dothidea and soft rot caused by R. stolonifer in pears. LYLB4 also suppressed several other phytopathogens in vitro, suggesting a broad‐spectrum antagonistic activity against fungal pathogens. 16S rRNA and gyrA sequence analysis indicated that LYLB4 is closely related to B. velezensis. Genome mining indicated that LYLB4 had 11 secondary metabolites encoding clusters, but that the surfactin and fengycin gene clusters may not be functional because of a large deletion. Matrix‐assisted laser desorption ionization‐time of flight mass spectra (MALDI‐TOF‐MS) demonstrated that iturins were the major lipopeptides, and that C₁₆/C₁₇ Bacillomycin D synthesis was stimulated when LYLB4 was co‐cultured with B. dothidea compared to the control. Overall, these results demonstrate that the main biocontrol mechanism adopted by LYLB4 could be through the production of toxic metabolites and direct contact with pathogens.     

Utilization of UPLC/Q‐TOF‐MS‐Based Metabolomics and AFLP‐Based Marker‐Assisted Selection to Facilitate/Assist Conventional Breeding of Polygala tenuifolia

As one of the most important traditional Chinese medicine, the quality of Polygala tenuifolia is difficult to control and a new method must be established to facilitate/assist the breeding of P. tenuifolia. In this study, UPLC/Q‐TOF‐MS‐based metabolomics analysis was performed to determine the chemical composition and screen metabolite biomarkers according to agronomic traits. A total of 29 compounds and 18 metabolite biomarkers were found. AFLP‐based marker‐assisted selection (MAS) was used to identify molecular marker bands and screen characteristic bands associated with specific agronomic traits. 184 bands and 76 characteristic AFLP bands were found. The correlation network between compounds and characteristic AFLP bands was built, so we may directly breed certain P. tenuifolia herbs with special agronomic traits (or characteristic AFLP bands), which exhibit specific pharmacological functions depending on the content of the active compounds. The proposed method of metabolomics coupled with MAS could facilitate/assist the breeding of P. tenuifolia.     

Co‐occurrence of bobcats,coyotes, and ocelots in Texas

Interspecific competition among carnivores has been linked to differences in behavior, morphology, and resource use. Insights into these interactions can enhance understanding of local ecological processes that can have impacts on the recovery of endangered species, such as the ocelot (Leopardus pardalis). Ocelots, bobcats (Lynx rufus), and coyotes (Canis latrans) share a small geographic range overlap from South Texas to south‐central Mexico but relationships among the three are poorly understood. From May 2011 to March 2018, we conducted a camera trap study to examine co‐occurrence patterns among ocelots, bobcats, and coyotes on the East Foundation's El Sauz Ranch in South Texas. We used a novel multiseason extension to multispecies occupancy models with ≥2 interacting species to conduct an exploratory analysis to examine interspecific interactions and examine the potential effects of patch‐level and landscape‐level metrics relative to the occurrence of these carnivores. We found strong evidence of seasonal mutual coexistence among all three species and observed a species‐specific seasonal trend in detection. Seasonal coexistence patterns were also explained by increasing distance from a high‐speed roadway. However, these results have important ecological implications for planning ocelot recovery in the rangelands of South Texas. This study suggests a coexistence among ocelots, bobcats, and coyotes under the environmental conditions on the El Sauz Ranch. Further research would provide a better understanding of the ecological mechanisms that facilitate coexistence within this community. As road networks in the region expand over the next few decades, large private working ranches will be needed to provide important habitat for ocelots and other carnivore species.     

Chemical Diversity and Antimicrobial Activity of Volatile Compounds from Zanthoxylum zanthoxyloides Lam. according to Compound Classes,Plant Organs and Senegalese Sample Locations

The chemical diversity of Zanthoxylum zanthoxyloides growing wild in Senegal was studied according to volatile compound classes, plant organs and sample locations. The composition of fruit essential oil was investigated using an original targeted approach based on the combination of gas chromatography (GC) and liquid chromatography (LC) both coupled with mass spectrometry (MS). The volatile composition of Z. zanthoxyloides fruits exhibited relative high amounts of hydrocarbon monoterpenes (24.3 – 55.8%) and non‐terpenic oxygenated compounds (34.5 – 63.1%). The main components were (E)‐β‐ocimene (12.1 – 39%), octyl acetate (11.6 – 21.8%) and decanol (9.7 – 15.4%). The GC and GC/MS profiling of fruit essential oils showed a chemical variability according to geographical locations of plant material. The LC/MS/MS analysis of fruit oils allowed the detection of seven coumarins in trace content. The chemical composition of fruit essential oils was compared with volatile fractions of leaves and barks (root and trunk) from the same plant station. Hexadecanoic acid, germacrene D and decanal were identified as the major constituents of leaves whereas the barks (root and trunk) were dominated by pellitorine (85.8% and 57%, respectively), an atypic linear compound with amide group. The fruit essential oil exhibited interesting antimicrobial activities against Staphylococcus aureus and Candida albicans, particularly the alcohol fraction of the oil.     

Isolation and Identification of Pathogens Causing Marigold (Tagetes erecta L.) Black Spot in Beijing,China

Black spot leads to great marigold losses worldwide. The disease is characterized by black spots on leaves and stems in its early stages, and the whole plant has black rot at the advanced stage. In this report, 6 of 217 Alternaria strains isolated from lesions of marigold plants in Beijing were randomly selected. The morphological characteristics and a pathogenic tree based on two protein‐coding genes (gpd and alt a 1) indicated that Alternaria tagetica is the causal agent of marigold black spot in Beijing. All six Alternaria strains could successfully re‐infect marigold, but they could not infect carrot or zinnia by either spore spray in a greenhouse or planting experiments in the epidemic area. This is the first report of the A. tagetica pathogen being isolated from marigold in Beijing.     

A metabolic signature for long life in the Caenorhabditis elegans Mit mutants

Mit mutations that disrupt function of the mitochondrial electron transport chain can, inexplicably, prolong Caenorhabditis elegans lifespan. In this study we use a metabolomics approach to identify an ensemble of mitochondrial‐derived α‐ketoacids and α‐hydroxyacids that are produced by long‐lived Mit mutants but not by other long‐lived mutants or by short‐lived mitochondrial mutants. We show that accumulation of these compounds is dependent on concerted inhibition of three α‐ketoacid dehydrogenases that share dihydrolipoamide dehydrogenase (DLD) as a common subunit, a protein previously linked in humans with increased risk of Alzheimer's disease. When the expression of DLD in wild‐type animals was reduced using RNA interference we observed an unprecedented effect on lifespan – as RNAi dosage was increased lifespan was significantly shortened, but, at higher doses, it was significantly lengthened, suggesting that DLD plays a unique role in modulating length of life. Our findings provide novel insight into the origin of the Mit phenotype.     

Systematics,genetics, and biogeography of intertidal mites (Acari,Oribatida) from the Andaman Sea and Strait of Malacca

This study demonstrates for the first time the presence of marine‐associated mites in the Andaman Sea and Strait of Malacca and reveals a relatively high diversity of these taxa with six species from two different families: Selenoribatidae and Fortuyniidae. Indopacifica, a new genus of Selenoribatidae, is described from Thailand and Malaysia, with two new species, Indopacifica pantai n. sp. and Indopacifica parva n. sp. The genus is characterized by the unique combination of following characters: lacking lamellar ridges, incomplete dorsosejugal suture, fourteen pairs of notogastral setae, and presence of epimeral foveae. A phylogenetic reconstruction based on 18S ribosomal RNA sequences clearly confirms the distinctness of the new genus Indopacifica and places it close to the genus Rhizophobates. The lack of molecular genetic data of possible relatives impedes a clear assessment, and hence, we emphasize the need for further combined approaches using morphological and molecular genetic sequence data. All species show wide distribution areas within this geographic region suggesting that these taxa are good dispersers despite their minute size and wingless body. Molecular genetic data demonstrate recent gene flow between far distant populations of I. pantai n. sp. from the coasts of Thailand and two islands of Malaysia and hence confirm this assumption. The seasonally changing surface currents within this geographic area may favor hydrochorous dispersal and hence genetic exchange. Nevertheless, morphometric data show a slight trend to morphological divergence among the studied populations, whereas this variation is suggested to be a result of genetic drift but also of habitat differences in one population of Alismobates pseudoreticulatus.     

Matrix‐assisted laser desorption/ionization mass spectrometry imaging: a powerful tool for probing the molecular topology of plant cutin polymer

The cutin polymers of different fruit cuticles (tomato, apple, nectarine) were examined using matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) after in situ release of the lipid monomers by alkaline hydrolysis. The mass spectra were acquired from each coordinate with a lateral spatial resolution of approximately 100 μm. Specific monomers were released at their original location in the tissue, suggesting that post‐hydrolysis diffusion can be neglected. Relative quantification of the species was achieved by introducing an internal standard, and the collection of data was subjected to non‐supervised and supervised statistical treatments. The molecular images obtained showed a specific distribution of ions that could unambiguously be ascribed to cutinized and suberized regions observed at the surface of fruit cuticles, thus demonstrating that the method is able to probe some structural changes that affect hydrophobic cuticle polymers. Subsequent chemical assignment of the differentiating ions was performed, and all of these ions could be matched to cutin and suberin molecular markers. Therefore, this MALDI‐MSI procedure provides a powerful tool for probing the surface heterogeneity of plant lipid polymers. This method should facilitate rapid investigation of the relationships between cuticle phenotypes and the structure of cutin within a large population of mutants.     

Liquid chromatography high‐resolution mass spectrometry for fatty acid profiling

Quantification of fatty acids has been crucial to elucidate lipid biosynthesis pathways in plants. To date, fatty acid identification and quantification has relied mainly on gas chromatography (GC) coupled to flame ionization detection (FID) or mass spectrometry (MS), which requires the derivatization of samples and the use of chemical standards for annotation. Here we present an alternative method based on a simple procedure for the hydrolysis of lipids, so that fatty acids can be quantified by liquid chromatography mass spectrometry (LC‐MS) analysis. Proper peak annotation of the fatty acids in the LC‐MS‐based methods has been achieved by LC‐MS measurements of authentic standard compounds and elemental formula annotation supported by ¹³C isotope‐labeled Arabidopsis. As a proof of concept, we have compared the analysis by LC‐MS and GC‐FID of two previously characterized Arabidopsis thaliana knock‐out mutants for FAD6 and FAD7 desaturase genes. These results are discussed in light of lipidomic profiles obtained from the same samples. In addition, we performed untargeted LC‐MS analysis to determine the fatty acid content of two diatom species. Our results indicate that both LC‐MS and GC‐FID analyses are comparable, but that because of higher sensitivity and selectivity the LC‐MS‐based method allows for a broader coverage and determination of novel fatty acids.