The role of Pleistocene dispersal in shaping species richness of sky island wintergreens from the Himalaya-Hengduan Mountains.

In addition to topography and climate, biogeographic dispersal has been considered to influence plant diversity in the Himalaya-Hengduan Mountains (HHM), yet, the mode and tempo of sky island dispersal and its influence on species richness has been little explored. Through phylogenetic analysis of Gaultheria ser. Trichophyllae, a sky island alpine clade within the HHM, we test the hypothesis that dispersal has affected current local species richness. We inferred the dynamics of biogeographic dispersal with correlation tests on direction, distance, occurrence time, and regional species richness. We found that G. ser. Trichophyllae originated at the end of the Miocene and mostly dispersed toward higher longitudes (eastward). In particular, shorter intra-regional eastward dispersals and longer inter-regional westward dispersals were most frequently observed. We detected a prevalence of eastward intra-region dispersals in both glacial periods and interglacials. These dispersals may have been facilitated by the reorganization of paleo-drainages and monsoon intensification through time. We suggest that the timing of dispersal corresponding to glacial periods and the prevalence of intra-region dispersal, rather than dispersal frequency, most influenced the pattern of species richness of G. ser. Trichophyllae. This study facilitates a more comprehensive understanding of biodiversity in the sky islands within the HHM.

2-Furoic acid associated with the infection of nematodes by Dactylellina haptotyla and its biocontrol potential on plant root-knot nematodes.

Dactylellina haptotyla is a typical nematode-trapping fungus that has garnered the attention of many scholars for its highly effective lethal potential for nematodes. Secondary metabolites play an important role in D. haptotyla-nematode interactions, but which metabolites perform which function remains unclear. We report the metabolic functions based on high-quality, chromosome-level genome assembly of wild D. haptotyla YMF1.03409. The results indicate that a large variety of secondary metabolites and their biosynthetic genes were significantly upregulated during the nematode-trapping stage. In parallel, we identified that 2-furoic acid was specifically produced during nematode trapping by D. haptotyla YMF1.03409 and isolated it from fermentation production. 2-Furoic acid demonstrated strong nematicidal activity with an LD50 value of 55.05 µg/mL against Meloidogyne incognita at 48 h. Furthermore, the pot experiment showed that the number of galls of tomato root was significantly reduced in the experimental group treated with 2-furoic acid. The considerable increase in the 2-furoic acid content during the infection process and its virulent nematicidal activity revealed an essential synergistic effect during the process of nematode-trapping fungal infection. IMPORTANCE Dactylellina haptotyla have significant application potential in nematode biocontrol. In this study, we determined the chromosome-level genome sequence of D. haptotyla YMF1.03409 by long-read sequencing technology. Comparative genomic analysis identified a series of pathogenesis-related genes and revealed significant gene family contraction events during the evolution of D. haptotyla YMF1.03409. Combining transcriptomic and metabolomic data as well as in vitro activity test results, a compound with important application potential in nematode biocontrol, 2-furoic acid, was identified. Our result expanded the genetic resource of D. haptotyla and identified a previously unreported nematicidal small molecule, which provides new options for the development of plant biocontrol agents.

A novel fluoropolymer as a protein delivery vector with robust adjuvant effect for cancer immunotherapy.

The inefficient treatment using protein-based nanovaccines is largely attributed to their inadequate immunogenicity. Herein, we developed a novel fluoropolymer (PF) via ring-opening polymerization and constructed a fluoropolymer-based nanovaccine for tumor immunotherapy. Due to the existence of fluoroalkyl chains, PF not only played a crucial role in tumor antigen delivery but also exhibited a remarkable adjuvant effect in enhancing the immunogenicity of nanovaccines. The nanovaccines formed by mixing PF with a model antigen ovalbumin (OVA) enhanced the uptake of antigen proteins by dendritic cells (DCs) and promoted the maturation and antigen presentation of DCs. Compared with free OVA, PF/OVA showed better efficacy in both pre-cancer prevention and tumor treatment. Furthermore, the proportion of CD4+ T and CD8+ T cells was significantly increased in lymph nodes and tumors of mice immunized with PF/OVA. Additionally, there was a great enhancement in the levels of key anti-tumor cytokines (TNF-α and IFN-γ) in the serum of the PF/OVA immunized mice. Our research has shown that fluoropolymer PF applied as a protein vector and adjuvant has great potential for the development of nanovaccines with robust immunogenicity.

AfLaeA, a Global Regulator of Mycelial Growth, Chlamydospore Production, Pathogenicity, Secondary Metabolism, and Energy Metabolism in the Nematode-Trapping Fungus Arthrobotrys flagrans.

Arthrobotrys flagrans (Duddingtonia flagrans) is a typical nematode-trapping fungus which has been used for nematode biocontrol. The global regulator LaeA is widely distributed in filamentous fungi and plays a crucial role in secondary metabolism and development in addition to pathogenicity in fungal pathogens. In this study, the chromosome-level genome of A. flagrans CBS 565.50 was sequenced and homologous sequences of LaeA were identified in A. flagrans. A. flagrans LaeA (AfLaeA) knockout resulted in slower hyphal growth and a smoother hyphal surface. Importantly, deletion of AfLaeA resulted in the absence of chlamydospores and attenuated glycogen and lipid accumulation in hyphae. Similarly, disruption of the AfLaeA gene led to fewer traps and electron-dense bodies, lower protease activity, and a delay in capturing nematodes. The AfLaeA gene had a large effect on the secondary metabolism of A. flagrans, and both the deletion and overexpression of AfLaeA could yield new compounds, whereas some compounds were lost due to the absence of the AfLaeA. Protein-protein interactions between AfLaeA and another eight proteins were detected. Furthermore, transcriptome data analysis showed that 17.77% and 35.51% of the genes were influenced by the AfLaeA gene on days 3 and 7, respectively. AfLaeA gene deletion resulted in the higher expression level of the artA gene cluster, and multiple differentially expressed genes involved in glycogen and lipid synthesis and metabolism showed opposite expression patterns in wild-type and ΔAfLaeA strains. In summary, our results provide novel insights into the functions of AfLaeA in mycelial growth, chlamydospore production, pathogenicity, secondary metabolism, and energy metabolism in A. flagrans. IMPORTANCE The regulation of biological functions, such as the secondary metabolism, development, and pathogenicity of LaeA, has been reported in multiple fungi. But to date, no study on LaeA in nematode-trapping fungi has been reported. Moreover, it has not been investigated whether or not LaeA is involved in energy metabolism and chlamydospore formation has not been investigated. Especially in the formation mechanism of chlamydospores, several transcription factors and signaling pathways are involved in the production of chlamydospores, but the mechanism of chlamydospore formation from an epigenetic perspective has not been revealed. Concurrently, an understanding of protein-protein interactions will provide a broader perspective on the regulatory mechanism of AfLaeA in A. flagrans. This finding is critical for understanding the regulatory role of AfLaeA in the biocontrol fungus A. flagrans and establishes a foundation for developing high-efficiency nematode biocontrol agents.

Appressoria-Small but Incredibly Powerful Structures in Plant-Pathogen Interactions.

Plant-pathogenic fungi are responsible for many of the most severe crop diseases in the world and remain very challenging to control. Improving current protection strategies or designating new measures based on an overall understanding of molecular host-pathogen interaction mechanisms could be helpful for disease management. The attachment and penetration of the plant surface are the most important events among diverse plant-fungi interactions. Fungi evolved as small but incredibly powerful infection structure appressoria to facilitate attachment and penetration. Appressoria are indispensable for many diseases, such as rusts, powdery mildews, and blast diseases, as well as devastating oomycete diseases. Investigation into the formation of plant-pathogen appressoria contributes to improving the understanding of the molecular mechanisms of plant-pathogen interactions. Fungal host attachment is a vital step of fungal pathogenesis. Here, we review recent advances in the molecular mechanisms regulating the formation of appressoria. Additionally, some biocontrol agents were revealed to act on appressorium. The regulation of fungal adhesion during the infective process by acting on appressoria formation is expected to prevent the occurrence of crop disease caused by some pathogenic fungi.

Natural nematicidal metabolites and advances in their biocontrol capacity on plant parasitic nematodes.

Covering: 2010 to 2021Natural nematicidal metabolites are important sources of nematode control. This review covers the isolation and structural determination of nematicidal metabolites from 2010 to 2021. We summarise chemical structures, bioactivity, metabolic regulation and biosynthesis of potential nematocides, and structure-activity relationship and application potentiality of natural metabolites in plant parasitic nematodes' biocontrol. In doing so, we aim to provide a comprehensive overview of the potential roles that natural metabolites can play in anti-nematode strategies.

Identification and Mechanism of Action of the Global Secondary Metabolism Regulator SaraC in Stereum hirsutum.

DNA methylation is an important factor in the regulation of gene expression. In analyzing genomic data of Stereum hirsutum FP-91666, we found a hypothetical bifunctional transcription regulator/O6Meguanine-DNA methyltransferase (named SaraC), which is widely present in both bacteria and fungi, and confirmed that its function in bacteria is mainly for DNA reparation. In this paper, we confirmed that SaraC has the function of DNA binding and demethylation through surface plasma resonance and reaction experiments in vitro. Then, we achieved the overexpression of SaraC (OES) in S. hirsutum, sequenced the methylation and transcription levels of the whole-genome, and further conducted untargeted metabolomics analyses of the OES transformants and the wild type (WT). The results confirmed that the overall-methylation levels of the transformants were significantly downregulated, and various genes related to secondary metabolism were upregulated. Through comparative untargeted metabolomic analyses, it showed that OES SA6 transformant produced a greater number of hybrid polyketides, and we identified 2 novel hybrid polyketides from the fermentation products of SA6. Our results show that overexpression SaraC can effectively stimulate the expression of secondary-metabolism-related genes, which could be a broad-spectrum tool for discovery of metabolites due to its cross-species conservation. IMPORTANCE Fungi are one of the important sources of active compounds. However, in fungi, most of the secondary metabolic biosynthetic gene clusters are weakly expressed or silenced under conventional culture conditions. How to efficiently excavate potential new compounds contained in fungi is becoming a research hot spot in the world. In this study, we found a DNA demethylation protein (SaraC) and confirmed that it is a global secondary metabolism regulator in Stereum hirsutum FP-91666. In the past, SaraC-like proteins were mainly regarded as DNA repair proteins, but our findings proved that it will be a powerful tool for mining secondary metabolites for overexpression of SaraC, which can effectively stimulate the expression of genes related to secondary metabolism.

Identification of Nematicidal Metabolites from Purpureocillium lavendulum.

Purpureocillium lavendulum is a fungus with promising biocontrol applications. Here, transcriptome data acquired during the infection of Caenorhabditis elegans by Purpureocillium lavendulum showed that the transcription of metabolite synthesis genes was significantly up-regulated after 24 and 48 h of the fungus-nematode interaction. Then, the up-regulated transcription level of lipoxygenase was confirmed by RT-qPCR. The ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis of differential metabolites revealed that this interaction resulted in the emergence of new metabolites or enhanced the production of metabolites. The results of the UPLC-MS analysis and the nematicidal assay were used to establish optimal culturing conditions under which 12 metabolites, including 3 hydroxylated C18 fatty acids and 9 steroids, were isolated and identified. Among them, hydroxylated fatty acids showed pronounced nematicidal activity against Meloidogyne incognita, and two degradative sterols showed chemotaxis activity to M. incognita. This study lays a foundation for the function of lipoxygenase and its products during the infection of Purpureocillium lavendulum.

Stereumamides E-H, four new minor quaternary ammonium hybrids from Stereum hirsutum.

In our preview research, four novel quaternary ammonium hybrids were isolated from the Stereum hirsutum FP-91666. To further discover this type of compounds, S. hirsutum was fermented in 30 L YMG broth, and eight hybrid compounds including four new quaternary ammoniums were obtained, which are sesquiterpenes combined with α-amino acids. Their structures were elucidated by extensive spectroscopic analyses, including 1D- and 2D-NMR, and HR-MS experiments.

Activities and metabolomics of Cordyceps gunnii under different culture conditions.

Many active metabolites have been identified from various species of the fungal genus Cordyceps. A predominant species of this genus is Cordyceps gunnii, but there are limited reports on the active ingredients from this species. This study aimed to conduct activity assays and metabolome analysis on extracts of C. gunnii obtained under different culture conditions. Five different solid media were selected to culture the mycelium of C. gunnii and the metabolites were extracted with organic solvents; concurrently, the wild stroma and host complexes of C. gunnii were extracted by ethyl acetate. Extracts were subsequently assayed for various biological activities and were analyzed by untargeted metabolomics. There were significant differences in the activities and metabolites of C. gunnii extracts from different culture conditions and from wild stroma and host complexes. The extracts of stroma and host complexes and mycelia cultured on WGA medium for 21 days exhibited similar effective inhibitory activity against five cell lines. A total of 51 metabolites were annotated and included various structural types. The literatures indicate that most of the identified compounds have a variety of different biological activities. These findings provide the basis for further systematic excavation of C. gunnii and improved utilization of this fungal species.

Recent Advances in the Synthetic Biology of Natural Drugs.

Natural drugs have been transformed and optimized during the long process of evolution. These compounds play a very important role in the protection of human health and treatment of human diseases. Sustainable approaches to the generation of raw materials for pharmaceutical products have been extensively investigated in drug research and development because chemical synthesis is costly and generates pollution. The present review provides an overview of the recent advances in the synthetic biology of natural drugs. Particular attention is paid to the investigations of drugs that may be mass-produced by the pharmaceutical industry after optimization of the corresponding synthetic systems. The present review describes the reconstruction and optimization of biosynthetic pathways for nine drugs, including seven drugs from plant sources and two drugs from microbial sources, suggesting a new strategy for the large-scale preparation of some rare natural plant metabolites and highly bioactive microbial compounds. Some of the suggested synthetic methods remain in a preliminary exploration stage; however, a number of these methods demonstrated considerable application potential. The authors also discuss the advantages and disadvantages of the application of synthetic biology and various expression systems for heterologous expression of natural drugs. Thus, the present review provides a useful perspective for researchers attempting to use synthetic biology to produce natural drugs.

Sesquiterpenoids and their quaternary ammonium hybrids from the mycelium of mushroom Stereum hirsutum by medium optimization.

The fungal genus Stereum (Stereaceae) produces a broad variety of specialised metabolites, including a wide range of terpenes. This probably relates to the presence of an extensive biosynthetic machinery for this group of compounds: genomic analysis of Stereum hirsutum has identified 16 terpene synthase gene clusters, 6 polyketide synthase gene clusters, and 1 polyketide synthase non-ribosomal polypeptide heterodimer gene cluster in S. hirsutum FP-91666. In the present study, the One Strain Many Compounds (OSMAC) approach was employed to discover undescribed metabolites from this strain. Fermentation was carried out in five media and the products of the strain cultivated on different media were analyzed by LC-MS. From cultures grow in WGB medium (30.0 g wheat bran, 20.0 g glucose, 1.5 g KH2PO4, and 1.5 g MgSO4), four previously undescribed metabolites, a sesquiterpene sterostrein X and three mixed terpenes (stereumamides I-K) were isolated, together with seven known compounds (drimene-2,11-diol, stereumamide E, stereumamide D, stereumamide B, stereumamide A, stereumamide C, and sterostrein Q). The drimane-type sesquiterpene drimene-2,11-diol was found in S. hirsutum FP-91666 for the first time. All structures were elucidated by spectroscopic data analysis. The absolute configurations of stereumamides I, J and K were assigned by comparing their experimental and calculated electronic circular dichroism (ECD) spectra. An anti-Mycobacterium tuberculosis experiment showed that stereumamides I-K and sterostrein Q had weak antibacterial activity against this pathogen.

Advances in the Study of Metabolomics and Metabolites in Some Species Interactions.

In the natural environment, interactions between species are a common natural phenomena. The mechanisms of interaction between different species are mainly studied using genomic, transcriptomic, proteomic, and metabolomic techniques. Metabolomics is a crucial part of system biology and is based on precision instrument analysis. In the last decade, the emerging field of metabolomics has received extensive attention. Metabolomics not only provides a qualitative and quantitative method for studying the mechanisms of interactions between different species, but also helps clarify the mechanisms of defense between the host and pathogen, and to explore new metabolites with various biological activities. This review focuses on the methods and progress of interspecies metabolomics. Additionally, the prospects and challenges of interspecies metabolomics are discussed.

External Validation of and Factors Associated with the Overuse Index: a Nationwide Population-Based Study from Taiwan.

BACKGROUND: The Overuse Index (OI), previously called the Johns Hopkins Overuse Index, is developed and validated as a composite measure of systematic overuse/low-value care using United States claims data. However, no information is available concerning whether the external validation of the OI is sustained, especially for international application. Moreover, little is known about which supply and demand factors are associated with the OI. OBJECTIVE: We used nationwide population-based data from Taiwan to externally validate the OI and to examine the association of regional healthcare resources and socioeconomic factors with the OI. DESIGN AND PARTICIPANTS: We analyzed 1,994,636 beneficiaries randomly selected from all people enrolled in the Taiwan National Health Insurance in 2013. MAIN MEASURES: The OI was calculated for 2013 to 2015 for each of 50 medical regions. Spearman correlation analysis was applied to examine the association of the OI with total medical costs per capita and mortality rate. Generalized estimating equation linear regression analysis was conducted to examine the association of regional healthcare resources (number of hospital beds per 1000 population, number of physicians per 1000 population, and proportion of primary care physicians [PCPs]) and socioeconomic factors (proportion of low-income people and proportion of population aged 20 and older without a high school diploma) with the OI. RESULTS: Higher scores of the OI were associated with higher total medical costs per capita (ρ = 0.48, P < 0.001) and not associated with total mortality (ρ = - 0.01, P = 0.882). Higher proportions of PCPs and higher proportions of low-income people were associated with lower scores of the OI (ß = - 0.022, P = 0.016 and ß = - 0.224, P < 0.001, respectively). CONCLUSIONS: Our study supported the external validation of the OI by demonstrating a similar association within a universal healthcare system, and it showed the association of a higher proportion of PCPs and a higher proportion of low-income people with less overuse/low-value care.

Secondary Metabolites and Their Bioactivities Produced by Paecilomyces.

Paecilomyces, a common saprobic filamentous fungus, not only plays an important role in biological control, but also has applications in medicine, food, and environmental protection. In this paper, 223 secondary metabolites and their bioactivities from 13 known species and various unidentified strains of Paecilomyces are reviewed. Their structures can be described as polyketide, terpenoid, peptide, alkaloid, quinone, pyrone, sterol, and fatty acid. They have been demonstrated varying biological activities, including antimicrobial, antitumor, insecticidal, antiplasmodial, antimalarial, nematicidal, herbicidal, and enzyme-inhibiting. This review provides a comprehensive overview of secondary metabolites and their biological activities from strains of Paecilomyces.

The general public's ability to operate automated external defibrillator: A controlled simulation study.

BACKGROUND: Automated external defibrillators (AEDs) enable laypeople to provide early defibrillations to patients undergoing cardiac arrest, but scant information is available on the general public's ability to use AEDs. This study assessed the ability of laypeople to operate AEDs, the effect of a 15-minute training, and whether skills differed by age. METHODS: From May 1 to December 31, 2018, a prospective simulation study was conducted with 94 laypeople aged 18-65 years (32 aged 18-24 years, 34 aged 25-54 years, and 28 aged 55-65 years) with no prior AED training. The participants' AED skills were assessed individually pre-training, post-training, and at a three-month follow-up using a simulated cardiac arrest scenario. The critical actions and time intervals were evaluated during the AED operating process. RESULTS: Only 14 (14.9%) participants (eight aged 18-24 years, four aged 25-54 years, and two aged 55-65 years) successfully delivered defibrillations before training. AED operation errors were more likely to occur among the participants aged 55-65 years than among other age groups. After training, the proportion of successful defibrillations increased significantly (18-24 years old: 25.0% vs. 71.9%, P<0.01; 25-54 years old: 11.8% vs. 70.6%, P<0.01; 55-65 years old: 7.1% vs. 67.9%, P<0.01). After three months, 26.1% of the participants aged 55-65 years successfully delivered defibrillations, which was significantly lower than that of participants aged 18-24 years (54.8%) and 25-54 years (64.3%) (P=0.02). There were no differences in time measures among three age groups in each test. CONCLUSIONS: The majority of untrained laypeople cannot effectively operate AEDs. More frequent training and refresher courses are crucial to improve AED skills.

Vib-PT, an Aromatic Prenyltransferase Involved in the Biosynthesis of Vibralactone from Stereum vibrans.

Vibralactone, a hybrid compound derived from phenols and a prenyl group, is a strong pancreatic lipase inhibitor with a rare fused bicyclic ß-lactone skeleton. Recently, a researcher reported a vibralactone derivative (compound C1) that caused inhibition of pancreatic lipase with a half-maximal inhibitory concentration of 14 nM determined by structure-based optimization, suggesting a potential candidate as a new antiobesity treatment. In the present study, we sought to identify the main gene encoding prenyltransferase in Stereum vibrans, which is responsible for the prenylation of phenol leading to vibralactone synthesis. Two RNA silencing transformants of the identified gene (vib-PT) were obtained through Agrobacterium tumefaciens-mediated transformation. Compared to wild-type strains, the transformants showed a decrease in vib-PT expression ranging from 11.0 to 56.0% at 5, 10, and 15 days in reverse transcription-quantitative PCR analysis, along with a reduction in primary vibralactone production of 37 to 64% at 15 and 21 days, respectively, as determined using ultra-high-performance liquid chromatography-mass spectrometry analysis. A soluble and enzymatically active fusion Vib-PT protein was obtained by expressing vib-PT in Escherichia coli, and the enzyme's optimal reaction conditions and catalytic efficiency (Km /kcat) were determined. In vitro experiments established that Vib-PT catalyzed the C-prenylation at C-3 of 4-hydroxy-benzaldehyde and the O-prenylation at the 4-hydroxy of 4-hydroxy-benzenemethanol in the presence of dimethylallyl diphosphate. Moreover, Vib-PT shows promiscuity toward aromatic compounds and prenyl donors.IMPORTANCE Vibralactone is a lead compound with a novel skeleton structure that shows strong inhibitory activity against pancreatic lipase. Vibralactone is not encoded by the genome directly but rather is synthesized from phenol, followed by prenylation and other enzyme reactions. Here, we used an RNA silencing approach to identify and characterize a prenyltransferase in a basidiomycete species that is responsible for the synthesis of vibralactone. The identified gene, vib-PT, was expressed in Escherichia coli to obtain a soluble and enzymatically active fusion Vib-PT protein. In vitro characterization of the enzyme demonstrated the catalytic mechanism of prenylation and broad substrate range for different aromatic acceptors and prenyl donors. These characteristics highlight the possibility of Vib-PT to generate prenylated derivatives of aromatics and other compounds as improved bioactive agents or potential prodrugs.

Griseaketides A-D, New Aromatic Polyketides from the Pathogenic Fungus Magnaporthe grisea.

Magnaporthe grisea is the causal agent of rice blast disease, which is the most serious disease of cultivated rice. Aromatic polyketides are its typical metabolites and are involved in the infection process. In the search for novel lead compounds, chemical investigation of the fungus M. grisea M639 has led to the isolation of four new aromatic polyketides (salicylaldehyde skeleton bearing an unsaturated side chain), griseaketides A-D (1-4), as well as 15 known compounds (5-19). The structures of the new compounds were elucidated on the basis of extensive spectroscopic analyses, including HR-MS, 2D NMR. Compound 12 showed prominent activity that killed 94.5% of C. elegans at 400 ppm and 66.9% at 200 ppm over 24 h. This is the first report describing the nematicidal activity of this type aromatic polyketide.

Antibacterial diketopiperazines from an endophytic fungus Bionectria sp. Y1085.

Two new diketopiperazines (1, 2), one new polyprenol (3), together with 19 known compounds (4-22) were obtained from the EtOAc extract of Bionectria sp. Y1085, an endophytic fungus isolated from the plant Huperzia serrata. Their structures were elucidated by extensive NMR and MS analysis. Bionectin D (1) is a rare diketopiperazine with a single methylthio substitution at the α-carbon of cyclized amino acid residue. The antibacterial activity of compounds was assayed against Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium ATCC 6539, and some metabolites (1, 2, 10, 11, and 14) exhibited evident antibacterial activity.

Non-Volatile Metabolites from Trichoderma spp.

The genus Trichoderma is comprised of many common fungi species that are distributed worldwide across many ecosystems. Trichoderma species are well-known producers of secondary metabolites with a variety of biological activities. Their potential use as biocontrol agents has been known for many years. Several reviews about metabolites from Trichoderma have been published. These reviews are based on their structural type, biological activity, or fungal origin. In this review, we summarize the secondary metabolites per Trichoderma species and elaborate on approximately 390 non-volatile compounds from 20 known species and various unidentified species.