Identifying new compounds with potential pharmaceutical and physiological activity in Areca catechu and Areca triandra via a non-targeted metabolomic approach.

INTRODUCTION: The fruits of Areca catechu, also called areca nuts, are widely used as popular masticatory and traditional herbal medicine in Asia. Besides arecoline and related alkaloids, limited information is available about further primary and secondary metabolites and their potential biological activities. OBJECTIVE: Here we aimed to further enhance our knowledge on phytochemical profiles of A. catechu and Areca triandra fruits. We intended to comprehensively identify metabolites in A. catechu and A. triandra fruits. METHODOLOGY: Metabolites were identified by ultra-performance liquid chromatography triple-quadrupole tandem mass spectrometry (UPLC-MS/MS). The occurrence of 12 selected bioactive compounds in 4 different developmental stages of A. catechu and A. triandra was quantified by LC-MS/MS. RESULTS: A total of 791 metabolites was identified. Of these, 115 metabolites could successfully be mapped to 44 Kyoto Encyclopedia of Genes and Genomes metabolic pathways, and 154 metabolites occurred at significantly different levels in A. catechu compared to A. triandra. Several components with known biological activities were identified for the first time in A. catechu and A. triandra. The abundance of many of these new components was similar in A. catechu and A. triandra, but significantly different between the pericarp and the seeds of A. catechu fruits. CONCLUSIONS: Metabolic profiles indicate that fruits of the Areca species compared here have similar primary and secondary metabolites. Our findings provide new insights into A. catechu and A. triandra as valuable sources for traditional medicine and they pave the way for further studies to potentially improve the underlying pharmaceutical and physiological effects.

First report of 16SrI-B subgroup related phytoplasma associated with witches'-broom symptoms in Pericampylus glaucus in China.

Pericampylus glaucus is an important medicinal plant resource containing active components with potential antitumor activity in China (Zhao & Cui, 2009). During July through August 2020, plants displayed disease symptoms including "witches' broom", leaf chlorosis, leaflet and internode shortening that impacted their growth (Fig. 1). These plants were first found in Dingan county of Hainan province, China. Total DNA from 12 plants were extracted using 0.10 g fresh plant leaves based on CTAB method. After amplification using primers specific for phytoplasma 16S rRNA, tuf and secA gene targets, R16mF2R16mR1 (Lee et al, 1993), fTuf1/rTuf1 (Schneider et al., 1997) and secAfor1/secArev3 (Hodgetts et al., 2008), the target bands of the three gene fragments of phytoplasma were detected in the disease sample DNA from six disease plants, and not in the healthy sample DNA from six healthy plants. Nucleotide sequences of the three genes were obtained from the PCR products sequencing and analyzed by DNAMAN 5.0 software. The three gene fragments of the DNA extracted from the disease samples were identical, with length of 1334 bp 16S rRNA (GenBank accession: MT872515), 989 bp tuf (MT755960) and 750 bp secA (MT755961) gene fragments, putatively encoding 329 (tuf) and 249 (secA) amino acids sequence separately. The phytoplasma strain was named as Pericampylus glaucus witches'-broom (PgWB) phytoplasma, PgWB-hnda strain, belonging to 16SrI-B subgroup by iPhyClassifier analysis. Homology and phylogenetic analysis indicated that based on 16S rRNA gene fragments, PgWB-hnda, pepper yellow crinkle phytoplasma PYC-hnhk (MT760793), chinaberry witches'-broom phytoplasma CWB-hnsy1 (KP662119) and CWB-hn (EF990733), periwinkle virescence phytoplasma PeV-hnhk (KP662136), with 100.0 % identity value, arecanut yellow leaf phytoplasma AYL-hnwn (FJ998269) and AYL-hn (FJ694685), with 99.8 % identity value, were clustered into one clade. Based on the analysis of tuf gene sequence fragments, PgWB was closely related to PYC-hnhk (MT755960), CWB-hnsy1 (KP662155), PeV-hnhk (KP662172) with 99.9 % identity value. Based on the analysis of secA gene sequence fragments, PgWB was closely related to CWB-hnsy1 (KP662173) with 99.7 % identity value, PYC-hnhk (MT755961), PeV-hnhk (KP662190) with 99.4 % identity value. To our knowledge, this is the first time that Pericampylus glaucus witches'-broom disease caused by 16SrI-B subgroup phytoplasma strain was found in China. Multilocus sequence analysis showed that PgWB was closely related to the phytoplasma strains causing pepper yellow crinkle, chinaberry witches'-broom, periwinkle virescence and areca palm yellow leaf diseases, all occurred in Hainan Island of China.

First report of phytoplasma belongs to 16SrXXXII group associated with witches'-broom symptoms in Trema tomentosa in China.

Trema tomentosa (Roxb.) Hara belonging to Ulmaceae displayed abnormal symptoms including witches'-broom, internode shortening, leaf chlorosis and leaflet that affected seriously their growth causing financial loss and ecological damage in China. During August through September 2020, these plants with the symptoms were first found and collected in Dingan and Qinghai counties of Hainan province, China. PCR were performed using the primers R16mF2/R16mR1 and secAfor1/secArev3 specific for phytoplasma 16S rRNA and secA gene fragments. The two gene fragments of the DNA extracted from the four disease samples were identical, with length of 1303 bp 16S rRNA and 587 bp secA gene fragments. The phytoplasma strain was named as Trema tomentosa witches'-broom (TtWB) phytoplasma, TtWB-hn strain. Phylogenetic and computer-simulated RFLP analyses based on the nearly full-length 16S rRNA gene sequence indicated that the TtWB phytoplasma strain is more closely related to the 16SrXXXII-A subgroup than to the other subgroups within 16SrXXXII group. It may represent a new subgroup, designed as 16SrXXXII-D subgroup, which is distinct from the other phytoplasma subgroups within the 16SrXXXII group. To our knowledge, this is the first report showing the occurrence of the phytoplasma strain belongs to 16SrXXXII-D subgroup associated with witches'-broom disease in Trema tomentosa in China. Genetic analysis indicated that the TtWB strain was closely related to the phytoplasma strains infecting periwinkle, oil palm, coconut palm in Malyasian, Camptotheca acuminate in Yunnan province of China and Elaeocarpus zollingeri in Japan.

Genetic Transformation and Green Fluorescent Protein Labeling in Ceratocystis paradoxa from Coconut.

Ceratocystis paradoxa, the causal agent of stem-bleeding disease of the coconut palm, causes great losses to the global coconut industry. As the mechanism of pathogenicity of C. paradoxa has not been determined, an exogenous gene marker was introduced into the fungus. In this study, pCT74-sGFP, which contains the green fluorescent protein (GFP) gene, and the hygromycin B resistance gene as a selective marker, was used as an expression vector. Several protoplast release buffers were compared to optimize protoplast preparation. The plasmid pCT74-sGFP was successfully transformed into the genome of C. paradoxa, which was verified using polymerase chain reaction and green fluorescence detection. The transformants did not exhibit any obvious differences from the wild-type isolates in terms of growth and morphological characteristics. Pathogenicity tests showed that the transformation process did not alter the virulence of the X-3314 C. paradoxa strain. This is the first report on the polyethylene glycol-mediated transformation of C. paradoxa carrying a 'reporter' gene GFP that was stably and efficiently expressed in the transformants. These findings provide a basis for future functional genomics studies of C. paradoxa and offer a novel opportunity to track the infection process of C. paradoxa.

[Advances in virulence determinants in Burkholderia cepacia complex--a review].

Most members of the Burkholderia cepacia complex (Bcc) are important human opportunistic pathogens. Although progress has been achieved on the taxonomy and molecular identification of these bacteria, the molecular mechanisms of Bcc pathogenicity remain unclear and little development is made for new therapeutic agents. As Bcc is resistant to many common clinically-relevant antibiotics, revealing its virulence determinants is therefore very important to develop novel antibiotics or alternative anti-infective therapies. In this review, we summarize current advances in principal virulence determinants, limitations and genetic tools for studies of pathogenesis of Bcc. We primarily focus on key pathogenicity factors, including innate resistance to antibiotics, protein secretion system, and quorum-sensing systems.

Molecular Insights into Red Palm Weevil Resistance Mechanisms of Coconut (Cocos nucifera) Leaves.

Red palm weevil (RPW) (Rhynchophorus ferrugineus) threatens most palm species worldwide. This study investigated the molecular responses of coconut (Cocos nucifera) leaves to RPW infestation through metabolomics and transcriptomics analysis. An RPW insect attack model was developed by placing different RPW larval densitiesin coconut plants and measuring the relative chlorophyll content of different leaf positions and physiological indicators of dysfunction after RPW infestation. The metabolomic changes were detected in the leaves of 10, 20, 30, 40, and 50 days after infestation (DAI) using GC-MS. Certain metabolites (glycine, D-pinitol, lauric acid, allylmalonic acid, D-glucaro-1, 4-lactone, protocatechuic acid, alpha, and alpha-trehalose) were found to be possible indicators for distinct stages of infestation using metabolomics analysis. The influence on ABC transporters, glutathione, galactose, and glycolipid metabolism was emphasized by pathway analysis. Differentially expressed genes (DEGs) were identified at 5, 10, 15, and 20 DAI through transcriptomics analysis of infested coconut leaves, with altered expression levels under RPW infestation. The KEGG pathway and GO analysis revealed enrichment in pathways related to metabolism, stress response, and plant-pathogen interactions, shedding light on the intricate mechanisms underlying coconut-RPW interactions. The identified genes may serve as potential markers for tracking RPW infestation progression and could inform strategies for pest control and management.

Combined Analysis of Metabolomics and Biochemical Changes Reveals the Nutritional and Functional Characteristics of Red Palm Weevil Rhynchophus ferrugineus (Coleoptera: Curculionidae) Larvae at Different Developmental Stages.

In this study, the changes in the conventional nutrient and mineral compositions as well as the metabolomics characteristics of the red palm weevil (RPW) Rhynchophus ferrugineus Olivier (Curculionidae: Coleoptera) larvae at early (EL), middle (ML) and old (OL) developmental stages were investigated. Results showed that the EL and ML had the highest content of protein (53.87 g/100 g dw) and fat (67.95 g/100 g), respectively, and three kinds of RPW larvae were all found to be rich in unsaturated fatty acids (52.17-53.12%), potassium (5707.12-15,865.04 mg/kg) and phosphorus (2123.87-7728.31 mg/kg). In addition, their protein contained 17 amino acids with the largest proportion of glutamate. A total of 424 metabolites mainly including lipids and lipid-like molecules, organic acids and their derivatives, organic heterocycle compounds, alkaloids and their derivatives, etc. were identified in the RPW larvae. There was a significant enrichment in the ABC transport, citrate cycle (TCA cycle), aminoacyl-tRNA biosynthesis, and mTOR signaling pathways as the larvae grow according to the analysis results of the metabolic pathways of differential metabolites. The water extract of EL exhibited relatively higher hydroxyl, 2,2-diphenyl-1-pyrroline hydrochloride (DPPH) and 2,2'-azobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging ability with the EC50 values of 1.12 mg/mL, 11.23 mg/mL, and 2.52 mg/mL, respectively. These results contribute to a better understanding of the compositional changes of the RPW larvae during its life cycle and provide a theoretical grounding for its deep processing and high-value utilization.

Four botanical extracts are toxic to the hispine beetle, Brontispa longissima, in laboratory and semi-field trials.

The potential of botanical extracts such as Celosia argenea L. (Caryophyllales: Amaranthaceae), Ricinus communis L. (Malpighiales: Euphorbiaceae), Mikania micrantha Humboldt, Bonpland & Kunth (Astrales: Asteraceae), and Catharanthus roseus (L.) G. Don (Gentianales: Apocynaceae) for the control of Brontispa longissima Gestro was evaluated in a bioassay and semi-field trial. Dose-response bioassay showed no significant difference in oral-toxicity among the extracts of C. argenea, M. micrantha, and C. roseus to larvae and adult of B. longissima. All extracts tested decreased the hatchability of B. longissima eggs. In particular, the extract of M. micrantha showed higher activity than others at the concentration of 5 mg/mL. In an antifeedant bioassay, the extract of C. argenea showed higher activity against the 1(st) larvae than that of other extracts (AF50 0.03 mg/mL), and C. roseus showed higher antifeedant activity to the 2(nd) to 5(th) larvae and adult of B. longissima (AF50 0.34, 0.33, 0.11, 0.43, and 0.20 mg/mL, respectively). The semi-field trial indicated that all extracts used in this study might reduce the pest population. Extracts of C. argenea and M. micrantha showed higher activities than that of C. roseus and R communis, and the decrease in population was 75.56% and 80.00% (without Abbott's correction) after seven days of treatment, respectively, at a concentration of 20 mg/mL. Therefore, these active botanical extracts may possess potential for use in control of B. longissima.

Iron Deficiency Leads to Chlorosis Through Impacting Chlorophyll Synthesis and Nitrogen Metabolism in Areca catechu L.

Deficiency of certain elements can cause leaf chlorosis in Areca catechu L. trees, which causes considerable production loss. The linkage between nutrient deficiency and chlorosis phenomenon and physiological defect in A. catechu remains unclear. Here, we found that low iron supply is a determinant for chlorosis of A. catechu seedling, and excessive iron supply resulted in dark green leaves. We also observed morphological characters of A. catechu seedlings under different iron levels and compared their fresh weight, chlorophyll contents, chloroplast structures and photosynthetic activities. Results showed that iron deficiency directly caused chloroplast degeneration and reduced chlorophyll synthesis in chlorosis leaves, while excessive iron treatment can increase chlorophyll contents, chloroplasts sizes, and inflated starch granules. However, both excessive and deficient of iron decreases fresh weight and photosynthetic rate in A. catechu seedlings. Therefore, we applied transcriptomic and metabolomic approaches to understand the effect of different iron supply to A. catechu seedlings. The genes involved in nitrogen assimilation pathway, such as NR (nitrate reductase) and GOGAT (glutamate synthase), were significantly down-regulated under both iron deficiency and excessive iron. Moreover, the accumulation of organic acids and flavonoids indicated a potential way for A. catechu to endure iron deficiency. On the other hand, the up-regulation of POD-related genes was assumed to be a defense strategy against the excessive iron toxicity. Our data demonstrated that A. catechu is an iron-sensitive species, therefore the precise control of iron level is believed to be the key point for A. catechu cultivation.

Chromosome-scale genome assembly of areca palm (Areca catechu).

Areca palm (Areca catechu L.; family Arecaceae) is an important tropical medicinal crop and is also used for masticatory and religious purposes in Asia. Improvements to areca properties made by traditional breeding tools have been very slow, and further advances in its cultivation and practical use require genomic information, which is still unavailable. Here, we present a chromosome-scale reference genome assembly for areca by combining Illumina and PacBio data with Hi-C mapping technologies, covering the predicted A. catechu genome length (2.59 Gb, variety "Reyan#1") to an estimated 240× read depth. The assembly was 2.51 Gb in length with a scaffold N50 of 1.7Mb. The scaffolds were then further assembled into 16 pseudochromosomes, with an N50 of 172 Mb. Transposable elements comprised 80.37% of the areca genome, and 68.68% of them were long-terminal repeat retrotransposon elements. The areca palm genome was predicted to harbour 31,571 protein-coding genes and overall, 92.92% of genes were functionally annotated, including enriched and expanded families of genes responsible for biosynthesis of flavonoid, anthocyanin, monoterpenoid and their derivatives. Comparative analyses indicated that A. catechu probably diverged from its close relatives Elaeis guineensis and Cocos nucifera approximately 50.3 million years ago (Ma). Two whole genome duplication events in areca palm were found to be shared by palms and monocots, respectively. This genome assembly and associated resources represents an important addition to the palm genomics community and will be a valuable resource that will facilitate areca palm breeding and improve our understanding of areca palm biology and evolution.

Rapid and Efficient Detection of 16SrI Group Areca Palm Yellow Leaf Phytoplasma in China by Loop-Mediated Isothermal Amplification.

Areca palm yellow leaf (AYL) disease caused by the 16SrI group phytoplasma is a serious threat to the development of the Areca palm industry in China. The 16S rRNA gene sequence was utilized to establish a rapid and efficient detection system efficient for the 16SrI-B subgroup AYL phytoplasma in China by loop-mediated isothermal amplification (LAMP). The results showed that two sets of LAMP detection primers, 16SrDNA-2 and 16SrDNA-3, were efficient for 16SrIB subgroup AYL phytoplasma in China, with positive results appearing under reaction conditions of 64oC for 40 min. The lowest detection limit for the two LAMP detection assays was the same at 200 ag/µl, namely approximately 53 copies/µl of the target fragments. Phytoplasma was detected in all AYL disease samples from Baoting, Tunchang, and Wanning counties in Hainan province using the two sets of LAMP primers 16SrDNA-2 and 16SrDNA-3, whereas no phytoplasma was detected in the negative control. The LAMP method established in this study with comparatively high sensitivity and stability, provides reliable results that could be visually detected, making it suitable for application and research in rapid diagnosis of AYL disease, detection of seedlings with the pathogen and breeding of disease-resistant Areca palm varieties.

Preliminary study on biology and feeding capacity of Chelisoches morio (Fabricius) (Dermaptera:Chelisochidae) on Tirathaba rufivena (Walker).

BACKGROUND: Chelisoches morio (Fabricius) (Dermaptera:Chelisochidae) is an important predator of Tirathaba rufivena (Walker) (Lepidoptera:Pyralidae). For better use of the natural enemy, a biological study on C. morio was conducted, particularly its developmental duration, survival, fecundity and sex ratio. And the feeding capacity of C. morio against T. rufivena was also studied under laboratory conditions. RESULTS: The biological study on C. morio was revealed that female adults usually lay eggs in egg masses. The number of eggs per female averaged 140.17 eggs, and the incubation period was 7.50 days. The duration of nymphal development included four instars. The immature period was 83.95 days. The longevity of males and females was 58.60 and 93.55 days, respectively. The results from the study on the feeding capacity of C. morio using T. rufivena as food revealed that C. morio adults could consume 11.08, 7.87, 7.09, 6.82 and 5.89 first-to-fifth-instar T. rufivena larvae, respectively. CONCLUSIONS: C. morio showed a huge amount of predation on this pest at all larval stages, implying a significant potential for the use of C. morio in controlling T. rufivena.

Frozen section and electron microscopy studies of the infection of the red palm weevil, Rhynchophorus ferrugineus (coleoptera:curculionidae) by the entomopathogenic fungus Metarhizium anisopliae.

This study determined the pathogenicity of Metarhizium anisopliae strain SD-3 against invasive red palm weevil (RPW), Rhynchophorus ferrugineus Olivier (coleoptera:curculionidae) larvae in Hainan Province, China. Inoculation of 1 × 108 conidia/mL caused 100 % mortality of R. ferrugineus, indicating that the conidia of strain SD-3 were highly virulent. The process of invasion mechanism was showed by scanning electron microscopy (SEM) and frozen section as follows. Once R. ferrugineus was infected by strain SD-3, M. anisopliae hyphae first invaded the cuticular and body cavity of R. ferrugineus. Secondly, well-developed muscles, fat, tracheaes and digestive tube tissues in the abdomen of R. ferrugineus were then decomposed and absorbed by M. anisopliae hyphae, leading to the total destruction of the larvae. Finally, M. anisopliae hyphae reproduced, resulting in a large number of conidia in the body of RPW. The SEM and frozen section are convenient tools to observe the mode of action of entomopathogenic fungi and to observe how M. anisopliae is able to colonize and infect the host.

Identification and tissue expression profiling of odorant binding protein genes in the red palm weevil, Rhynchophorus ferrugineus.

BACKGROUND: The red palm weevil, Rhynchophorus ferrugineus, is a lethal pest of the palms. The identification of odorant binding protein (OBP) genes will be helpful for clarifing the mechanism of odorant detection of this pest. By sequencing the full length cDNA library of its antenne, 11 OBP genes (RferOBP1-11) were identified. FINDINGS: The result showed RferOBP1-7 and RferOBP8-11 belonged to the minus-C and classic family, respectively qPCR revealed that RferOBP1-10 highly transcribed in the antennae, of which RferOBP1, RferOBP4, RferOBP8 and RferOBP10 were obviously male-biased expression. RferOBP7 and RferOBP11 exhibited highly expression in female head and male thorax. RferOBP2, RferOBP5 and RferOBP6 were highly expressed in the female thorax, leg and abdomen respectively. CONCLUSIONS: The results paved the way towards a future understanding of the olfaction in this species.

Screening of tropical isolates of Metarhizium anisopliae for virulence to the red palm weevil Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae).

The red palm weevil, Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) is a serious pest of the palm tree in tropical regions of the world. One strain of Metarhizium sp. ZJ-1, isolated from Chinese soils, was evaluated for growth characteristics, and screened for its virulence to R. ferrugineus larvae in laboratory conditions. An approximately 685-bp fragment was amplified by ITS (ITS1-5.8S-ITS2) PCR from strain ZJ-1, further phylogenetic analysis revealed that 93 % similarity to Metarhizium anisopliae. Inoculation of 1 × 10(8) conidia/mL caused 100 % mortality of R. ferrugineus, LT50 levels of ZJ-1 were 1.66 days (1 × 10(8) conidia/mL), indicating that the conidia of strain ZJ-1 were highly virulent. These results suggest that M. anisopliae ZJ-1 has potential as an effective and persistent biological control agent for R. ferrugineus.

Effect of temperature on the population growth of Rhynchophorus ferrugineus (Coleoptera: Curculionidae) on sugarcane.

The effect of temperature on the developmental time, survival, and reproduction of Rhynchophorus ferrugineus (Olivier) reared on sugarcane was studied at seven constant temperatures (16, 20, 24, 28, 32, 36, and 40 degrees C). The developmental threshold temperatures and effective accumulated temperatures for the whole generation were 17.41 degrees C and 1,590.72 DD, respectively. One generation had the highest survival rate (26.67%) at 28 degrees C. The egg failed to survive at 16 and 40 degrees C. The population trend index (I = 38.22) and net reproductive rate (R(o) = 38.3) were highest at 28 degrees C. The net reproductive rate (R(o) = 3.36), intrinsic rate of increase (r(m) = 0.0028), and finite capacity of increase (lambda = 1.0028) were lowest at 20 degrees C. The mean generation time (T(o) = 85.82) was shortest at 36 degrees C. The population double time (PDT = 27.08) was shortest at 32 degrees C. Based on these studies, we concluded that the temperatures from 28 to 32 degrees C were the most suitable temperatures for the development of R. ferrugineus.