Metabolome and transcriptome related dataset for pheromone biosynthesis in an aggressive forest pest Ips typographus.

Eurasian spruce bark beetle, Ips typographus, is an aggressive pest among spruce vegetation. I. typographus host trees colonization is mediated by aggregation pheromone, consisting of 2-methyl-3-buten-2-ol and cis-verbenol produced in the beetle gut. Other biologically active compounds such as ipsdienol and verbenone have also been detected. 2-Methyl-3-buten-2-ol and ipsdienol are produced de-novo in the mevalonate pathway and cis-verbenol is oxidized from α-pinene sequestrated from the host. The pheromone production is presumably connected with further changes in the primary and secondary metabolisms in the beetle. To evaluate such possibilities, we obtained qualitative metabolomic data from the analysis of beetle guts in different life stages. We used Ultra-high-performance liquid chromatography-electrospray ionization-high resolution tandem mass spectrometry (UHPLC-ESI-HRMS/MS). The data were dereplicated using metabolomic software (XCMS, Camera, and Bio-Conductor) and approximately 3000 features were extracted. The metabolite was identified using GNPS databases and de-novo annotation in Sirius program followed by manual curation. Further, we obtained differential gene expression (DGE) of RNA sequencing data for mevalonate pathway genes and CytochromeP450 (CyP450) genes from the gut tissue of the beetle to delineate their role on life stage-specific pheromone biosynthesis. CyP450 gene families were classified according to subclasses and given individual expression patterns as heat maps. Three mevalonate pathway genes and five CyP450 gene relative expressions were analyzed using quantitative real-time (qRT) PCR, from the gut tissue of different life stage male/female beetles, as extended knowledge of related research article (Ramakrishnan et al., 2022). This data provides essential information on pheromone biosynthesis at the molecular level and supports further research on pheromone biosynthesis and detoxification in conifer bark beetles.

Consumption of gossypol increases fatty acid-amino acid conjugates in the cotton pests Helicoverpa armigera and Heliothis virescens.

Gossypol is a toxic sesquiterpene dimer produced by cotton plants which deters herbivory by insects and vertebrates. Two highly reactive aldehyde groups contribute to gossypol toxicity by cross-linking herbivore proteins. We identified another consequence of consuming gossypol in two insect pests of cotton: increased amounts of fatty acid-amino acid conjugates (FACs). Eight different FACs in the feces of larval Helicoverpa armigera and Heliothis virescens increased when larvae consumed artificial diet containing gossypol, but not a gossypol derivative lacking free aldehyde groups (SB-gossypol). FACs are produced by joining plant-derived fatty acids with amino acids of insect origin in the larval midgut tissue by an unknown conjugase, and translocated into the gut lumen by an unknown transporter. FACs are hydrolyzed back into fatty acids and amino acids by an aminoacylase (L-ACY-1) in the gut lumen. The equilibrium level of FACs in the lumen is determined by a balance between conjugation and hydrolysis, which may differ among species. When heterologously expressed, L-ACY-1 of H. armigera but not H. virescens was inhibited by gossypol; consistent with the excretion of more FACs in the feces by H. armigera. FACs are known to benefit the plant host by inducing anti-herbivore defensive responses, and have been hypothesized to benefit the herbivore by acting as a surfactant and increasing nitrogen uptake efficiency. Thus in addition to its direct toxic effects, gossypol may negatively impact insect nitrogen uptake efficiency and amplify the signal used by the plant to elicit release of volatile compounds that attract parasitoids.

Metabolic Profiling of Rhizobacteria Serratia plymuthica and Bacillus subtilis Revealed Intra- and Interspecific Differences and Elicitation of Plipastatins and Short Peptides Due to Co-cultivation.

Rhizobacteria live in diverse and dynamic communities having a high impact on plant growth and development. Due to the complexity of the microbial communities and the difficult accessibility of the rhizosphere, investigations of interactive processes within this bacterial network are challenging. In order to better understand causal relationships between individual members of the microbial community of plants, we started to investigate the inter- and intraspecific interaction potential of three rhizobacteria, the S. plymuthica isolates 4Rx13 and AS9 and B. subtilis B2g, using high resolution mass spectrometry based metabolic profiling of structured, low-diversity model communities. We found that by metabolic profiling we are able to detect metabolite changes during cultivation of all three isolates. The metabolic profile of S. plymuthica 4Rx13 differs interspecifically to B. subtilis B2g and surprisingly intraspecifically to S. plymuthica AS9. Thereby, the release of different secondary metabolites represents one contributing factor of inter- and intraspecific variations in metabolite profiles. Interspecific co-cultivation of S. plymuthica 4Rx13 and B. subtilis B2g showed consistently distinct metabolic profiles compared to mono-cultivated species. Thereby, putative known and new variants of the plipastatin family are increased in the co-cultivation of S. plymuthica 4Rx13 and B. subtilis B2g. Interestingly, intraspecific co-cultivation of S. plymuthica 4Rx13 and S. plymuthica AS9 revealed a distinct interaction zone and showed distinct metabolic profiles compared to mono-cultures. Thereby, several putative short proline-containing peptides are increased in co-cultivation of S. plymuthica 4Rx13 with S. plymuthica AS9 compared to mono-cultivated strains. Our results demonstrate that the release of metabolites by rhizobacteria alters due to growth and induced by social interactions between single members of the microbial community. These results form a basis to elucidate the functional role of such interaction-triggered compounds in establishment and maintenance of microbial communities and can be applied under natural and more realistic conditions, since rhizobacteria also interact with the plant itself and many other members of plant and soil microbiota.

Response of the wood-decay fungus Schizophyllum commune to co-occurring microorganisms.

Microorganisms are constantly interacting in a given environment by a constant exchange of signaling molecules. In timber, wood-decay fungi will come into contact with other fungi and bacteria. In naturally bleached wood, dark, pigmented lines arising from confrontation of two fungi often hint at such interactions. The metabolites (and pigment) exchange was investigated using the lignicolous basidiomycete Schizophyllum commune, and co-occurring fungi and bacteria inoculated directly on sterilized wood, or on media. In interactions with competitive wood degrading fungi, yeasts or bacteria, different competition strategies and communication types were observed, and stress reactions, as well as competitor-induced enzymes or pigments were analyzed. Melanin, indole, flavonoids and carotenoids were shown to be induced in S. commune interactions. The induced genes included multi-copper oxidases lcc1, lcc2, mco1, mco2, mco3 and mco4, possibly involved in both pigment production and lignin degradation typical for wood bleaching by wood-decay fungi.

Diversity and Distribution of Volatile Secondary Metabolites Throughout Bacillus subtilis Isolates.

Bacillus subtilis releases a broad range of volatile secondary metabolites, which are considered as long- and short distance infochemical signals mediating inter- and intra-specific processes. In addition, they often show antimicrobial or antifungal activities. This review attempts to summarize yet known volatile secondary metabolites produced and emitted by Bacillus subtilis isolates focusing on the structural diversity and distribution patterns. Using in vitro volatile-collection systems, 26 strains of B. subtilis isolated from different habitats were found to produce in total 231 volatile secondary metabolites. These volatile secondary metabolites comprised mainly hydrocarbons, ketones, alcohols, aldehydes, ester, acids, aromatics, sulfur- and nitrogen-containing compounds. Reviewed data revealed to a great extent isolate-specific emission patterns. The production and release of several volatile bioactive compounds was retained in isolates of the species B. subtilis, while volatiles without a described function seemed to be isolate-specifically produced. Detailed analysis, however, also indicated that the original data were strongly influenced by insufficient descriptions of the bacterial isolates, heterogeneous and poorly documented culture conditions as well as sampling techniques and inadequate compound identification. In order to get deeper insight into the nature, diversity, and ecological function of volatile secondary metabolites produced by B. subtilis, it will be necessary to follow well-documented workflows and fulfill state-of-the-art standards to unambiguously identify the volatile metabolites. Future research should consider the dynamic of a bacterial culture leading to differences in cell morphology and cell development. Single cell investigations could help to attribute certain volatile metabolites to defined cell forms and developmental stages.

Untargeted Metabolomics Approach Reveals Differences in Host Plant Chemistry Before and After Infestation With Different Pea Aphid Host Races.

The pea aphid (Acyrthosiphon pisum), a phloem-sucking insect, has undergone a rapid radiation together with the domestication and anthropogenic range expansion of several of its legume host plants. This insect species is a complex of at least 15 genetically different host races that can all develop on the universal host plant Vicia faba. However, each host race is specialized on a particular plant species, such as Medicago sativa, Trifolium pratense, or Pisum sativum, which makes it an attractive model insect to study ecological speciation. Previous work revealed that pea aphid host plants produce a specific phytohormone profile depending on the host plant - host race combination. Native aphid races induce lower defense hormone levels in their host plant than non-native pea aphid races. Whether these changes in hormone levels also lead to changes in other metabolites is still unknown. We used a mass spectrometry-based untargeted metabolomic approach to identify plant chemical compounds that vary among different host plant-host race combinations and might therefore, be involved in pea aphid host race specialization. We found significant differences among the metabolic fingerprints of the four legume species studied prior to aphid infestation, which correlated with aphid performance. After infestation, the metabolic profiles of M. sativa and T. pratense plants infested with their respective native aphid host race were consistently different from profiles after infestation with non-native host races and from uninfested control plants. The metabolic profiles of P. sativum plants infested with their native aphid host race were also different from plants infested with non-native host races, but not different from uninfested control plants. The compounds responsible for these differences were putatively identified as flavonoids, saponins, non-proteinogenic amino acids and peptides among others. As members of these compound classes are known for their activity against insects and aphids in particular, they may be responsible for the differential performance of host races on native vs. non-native host plants. We conclude that the untargeted metabolomic approach is suitable to identify candidate compounds involved in the specificity of pea aphid - host plant interactions.

Publisher Correction: Interspecific formation of the antimicrobial volatile schleiferon.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Interspecific formation of the antimicrobial volatile schleiferon.

Microorganisms release a plethora of volatile secondary metabolites. Up to now, it has been widely accepted that these volatile organic compounds are produced and emitted as a final product by a single organism e.g. a bacterial cell. We questioned this commonly assumed perspective and hypothesized that in diversely colonized microbial communities, bacterial cells can passively interact by emitting precursors which non-enzymatically react to form the active final compound. This hypothesis was inspired by the discovery of the bacterial metabolite schleiferon A. This bactericidal volatile compound is formed by a non-enzymatic reaction between acetoin and 2-phenylethylamine. Both precursors are released by Staphylococcus schleiferi cells. In order to provide evidence for our hypothesis that these precursors could also be released by bacterial cells of different species, we simultaneously but separately cultivated Serratia plymuthica 4Rx13 and Staphylococcus delphini 20771 which held responsible for only one precursor necessary for schleiferon A formation, respectively. By mixing their headspace, we demonstrated that these two species were able to deliver the active principle schleiferon A. Such a joint formation of a volatile secondary metabolite by different bacterial species has not been described yet. This highlights a new aspect of interpreting multispecies interactions in microbial communities as not only direct interactions between species might determine and influence the dynamics of the community. Events outside the cell could lead to the appearance of new compounds which could possess new community shaping properties.

Interspecies interaction of Serratia plymuthica 4Rx13 and Bacillus subtilis B2g alters the emission of sodorifen.

Sodorifen is the major volatile of Serratia plymuthica 4Rx13. It is assumed to be a long-distance communication signal. However, so far the emission patterns of sodorifen had been studied using mono-cultures of S. plymuthica 4Rx13 neglecting that in natura bacteria live in communities. Here, we show that the structured co-cultivation of S. plymuthica 4Rx13 and Bacillus subtilis B2g in a low-diversity model community grown under nutrient-rich conditions led to quantitative changes in sodorifen emission compared to self-paired mono-cultivations. Co-culturing revealed a decreased emission of sodorifen (50%) during exponential growth phase, whereas in the late stationary stage of growth, the amount of headspace sodorifen was increased compared to self-paired mono-cultivation (217% at 500 h of cultivation). Six other compounds that are most probably related to sodorifen or are isomers showed similar emission patterns. These data indicated that S. plymuthica 4Rx13 enhances its communication signal sodorifen as a consequence of interaction with B. subtilis B2g.

Sodorifen Biosynthesis in the Rhizobacterium Serratia plymuthica Involves Methylation and Cyclization of MEP-Derived Farnesyl Pyrophosphate by a SAM-Dependent C-Methyltransferase.

The rhizobacterium Serratia plymuthica 4Rx13 releases a unique polymethylated hydrocarbon (C16H26) with a bicyclo[3.2.1]octadiene skeleton called sodorifen. Sodorifen production depends on a gene cluster carrying a C-methyltransferase and a terpene cyclase along with two enzymes of the 2- C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis. Comparative analysis of wild-type and mutant volatile organic compound profiles revealed a C-methyltransferase-dependent C16 alcohol called pre-sodorifen, the production of which is upregulated in the terpene cyclase mutant. The monocyclic structure of this putative intermediate in sodorifen biosynthesis was identified by NMR spectroscopy. In vitro assays with the heterologously expressed S. plymuthica C-methyltransferase and terpene cyclase demonstrated that these enzymes act sequentially to convert farnesyl pyrophosphate (FPP) into sodorifen via a pre-sodorifen pyrophosphate intermediate, indicating that the S-adenosyl methionine (SAM)-dependent C-methyltransferase from S. plymuthica exhibits unprecedented cyclase activity. In vivo incorporation experiments with 13C-labeled succinate, l-alanine, and l-methionine confirmed a MEP pathway to FPP via the canonical glyceraldehyde-3-phosphate and pyruvate, as well as its SAM-dependent methylation in pre-sodorifen and sodorifen biosynthesis. 13C{1H} NMR spectroscopy facilitated the localization of 13C labels and provided detailed insights into the biosynthetic pathway from FPP via pre-sodorifen pyrophosphate to sodorifen.

Evolutionary stability of antibiotic protection in a defensive symbiosis.

The increasing resistance of human pathogens severely limits the efficacy of antibiotics in medicine, yet many animals, including solitary beewolf wasps, successfully engage in defensive alliances with antibiotic-producing bacteria for millions of years. Here, we report on the in situ production of 49 derivatives belonging to three antibiotic compound classes (45 piericidin derivatives, 3 streptochlorin derivatives, and nigericin) by the symbionts of 25 beewolf host species and subspecies, spanning 68 million years of evolution. Despite a high degree of qualitative stability in the antibiotic mixture, we found consistent quantitative differences between species and across geographic localities, presumably reflecting adaptations to combat local pathogen communities. Antimicrobial bioassays with the three main components and in silico predictions based on the structure and specificity in polyketide synthase domains of the piericidin biosynthesis gene cluster yield insights into the mechanistic basis and ecoevolutionary implications of producing a complex mixture of antimicrobial compounds in a natural setting.

Effects of discrete bioactive microbial volatiles on plants and fungi.

Plants live in association with microorganisms, which are well known as a rich source of specialized metabolites, including volatile compounds. The increasing numbers of described plant microbiomes allowed manifold phylogenetic tree deductions, but less emphasis is presently put on the metabolic capacities of plant-associated microorganisms. With the focus on small volatile metabolites we summarize (i) the knowledge of prominent bacteria of plant microbiomes; (ii) present the state-of-the-art of individual (discrete) microbial organic and inorganic volatiles affecting plants and fungi; and (iii) emphasize the high potential of microbial volatiles in mediating microbe-plant interactions. So far, 94 discrete organic and five inorganic compounds were investigated, most of them trigger alterations of the growth, physiology and defence responses in plants and fungi but little is known about the specific molecular and cellular targets. Large overlaps in emission profiles of the emitters and receivers render specific volatile organic compound-mediated interactions highly unlikely for most bioactive mVOCs identified so far.

Exploring bacterial interspecific interactions for discovery of novel antimicrobial compounds.

Recent studies indicated that the production of secondary metabolites by soil bacteria can be triggered by interspecific interactions. However, little is known to date about interspecific interactions between Gram-positive and Gram-negative bacteria. In this study, we aimed to understand how the interspecific interaction between the Gram-positive Paenibacillus sp. AD87 and the Gram-negative Burkholderia sp. AD24 affects the fitness, gene expression and the production of soluble and volatile secondary metabolites of both bacteria. To obtain better insight into this interaction, transcriptome and metabolome analyses were performed. Our results revealed that the interaction between the two bacteria affected their fitness, gene expression and the production of secondary metabolites. During interaction, the growth of Paenibacillus was not affected, whereas the growth of Burkholderia was inhibited at 48 and 72 h. Transcriptome analysis revealed that the interaction between Burkholderia and Paenibacillus caused significant transcriptional changes in both bacteria as compared to the monocultures. The metabolomic analysis revealed that the interaction increased the production of specific volatile and soluble antimicrobial compounds such as 2,5-bis(1-methylethyl)-pyrazine and an unknown Pederin-like compound. The pyrazine volatile compound produced by Paenibacillus was subjected to bioassays and showed strong inhibitory activity against Burkholderia and a range of plant and human pathogens. Moreover, strong additive antimicrobial effects were observed when soluble extracts from the interacting bacteria were combined with the pure 2,5-bis(1-methylethyl)-pyrazine. The results obtained in this study highlight the importance to explore bacterial interspecific interactions to discover novel secondary metabolites and to perform simultaneously metabolomics of both, soluble and volatile compounds.

Local phytochemical response of Musa acuminata × balbisiana Colla cv. 'Bluggoe' (ABB) to colonization by Sternorrhyncha.

The interaction of two Sternorrhyncha species, the banana aphid (Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae, Aphidinae)), vector of the banana bunchy top virus (BBTV), and the latania scale (Hemiberlesia lataniae Signoret (Hemiptera: Diaspididae, Diaspidinae)) with Musa acuminata × balbisiana Colla (ABB Group) 'Bluggoe' (Musaceae) was investigated by a combination of conventional and spatially resolved analytical techniques, 1H NMR, UHPLC-MS, and matrix-free UV-laser desorption/ionization MS imaging. After infestation, the feeding sites of P. nigronervosa on the pseudostem and the exocarp of banana fruit developed a red tinge, in which tissue-specific accumulations of phenylphenalenones were discovered. Phenylphenalenones were also detected in the black mats of sooty molds growing on the banana aphid exudates and in the dorsal scales of H. lataniae. This suggests that although these secondary metabolites play a role in the reaction of banana plants towards attack by sucking insects, an aphid and an armored scale have established mechanisms to exude these metabolites before they deploy their deleterious effect.

Novel volatiles of skin-borne bacteria inhibit the growth of Gram-positive bacteria and affect quorum-sensing controlled phenotypes of Gram-negative bacteria.

The skin microbiota is import for body protection. Here we present the first comprehensive analysis of the volatile organic compound (VOC) profiles of typical skin-resident corynebacterial and staphylococcal species. The VOC profile of Staphylococcus schleiferi DSMZ 4807 was of particular interest as it is dominated by two compounds, 3-(phenylamino)butan-2-one and 3-(phenylimino)butan-2-one (schleiferon A and B, respectively). Neither of these has previously been reported from natural sources. Schleiferon A and B inhibited the growth of various Gram-positive species and affected two quorum-sensing-dependent phenotypes - prodigiosin accumulation and bioluminescence - of Gram-negative bacteria. Both compounds were found to inhibit the expression of prodigiosin biosynthetic genes and stimulate the expression of prodigiosin regulatory genes pigP and pigS. This study demonstrates that the volatile schleiferons A and B emitted by the skin bacterium S. schleiferi modulate differentially and specifically its interactions with members of diverse bacterial communities. A network of VOC-mediated interspecies interactions and communications must be considered in the establishment of the (skin) microbiome and both compounds are interesting candidates for further investigations to better understand how VOCs emitted by skin bacteria influence and modulate the local microbiota and determine whether they are relevant to antibiotic and anti-virulence therapies.

Bacterial-Plant-Interactions: Approaches to Unravel the Biological Function of Bacterial Volatiles in the Rhizosphere.

Rhizobacteria produce an enormous amount of volatile compounds, however, the function of these metabolites is scarcely understood. Investigations evaluating influences on plants performed in various laboratories using individually developed experimental setups revealed different and often contradictory results, e.g., ranging from a significant plant growth promotion to a dramatic suppression of plant development. In addition to these discrepancies, these test systems neglected properties and complexity of the rhizosphere. Therefore, to pursue further investigations of the role of bacterial volatiles in this underground habitat, the applied methods have to simulate its natural characteristics as much as possible. In this review, we will describe and discuss pros and cons of currently used bioassays, give insights into rhizosphere characteristics, and suggest improvements for test systems that would consider in natura conditions and would allow gaining further knowledge of the potential function and significance of rhizobacterial volatiles in plant life.

Influence of zygomycete-derived D'orenone on IAA signalling in Tricholoma-spruce ectomycorrhiza.

Despite the rising interest in microbial communication, only few studies relate to mycorrhization and the pool of potential morphogenic substances produced by the surrounding soil community. Here, we investigated the effect exerted by the C18 - ketone ß-apo-13-carotenone, D'orenone, on the ectomycorrhizal basidiomycete Tricholoma vaccinum and its symbiosis with the economically important host tree, spruce (Picea abies). D'orenone is an early intermediate in the biosynthesis of morphogens in sexual development of mucoromycetes, the trisporoids. In the ectomycorrhizal fungus T. vaccinum, D'orenone increased the production and/or release of the phytohormone indole-3-acetic acid (IAA) which had been proposed to be involved in the mutual symbiosis. The induced expression of the fungal aldehyde dehydrogenase, Ald5 is associated with IAA synthesis and excretion. In the host tree, D'orenone modulated root architecture by increasing lateral root length and hypertrophy of root cortex cells, likely via changed IAA concentrations and flux. Thus, we report for the first time on carotenoid metabolites from soil fungi affecting both ectomycorrhizal partners. The data imply a complex network of functions for secondary metabolites which act in an inter-kingdom signalling in soil.

Unprecedented Utilization of Pelargonidin and Indole for the Biosynthesis of Plant Indole Alkaloids.

Nudicaulins are a group of indole alkaloid glycosides responsible for the color of yellow petals of Papaver nudicaule (Iceland poppy). The unique aglycone scaffold of these alkaloids attracted our interest as one of the most unusual flavonoid-indole hybrid structures that occur in nature. Stable isotope labeling experiments with sliced petals identified free indole, but not tryptamine or l-tryptophan, as one of the two key biosynthetic precursors of the nudicaulin aglycone. Pelargonidin was identified as the second key precursor, contributing the polyphenolic unit to the nudicaulin molecule. This finding was inferred from the temporary accumulation of pelargonidin glycosides in the petals during flower bud development and a drop at the point in time when nudicaulin levels start to increase. The precursor-directed incorporation of cyanidin into a new 3'-hydroxynudicaulin strongly supports the hypothesis that anthocyanins are involved in the biosynthesis of nudicaulins.

4-Methoxycinnamic acid--An unusual phenylpropanoid involved in phenylphenalenone biosynthesis in Anigozanthos preissii.

In vitro root cultures of Anigozanthos preissii and Wachendorfia thyrsiflora (Haemodoraceae) are suitable biological systems for studying the biosynthesis of phenylphenalenones. Here we report how we used these root cultures to investigate precursor-product relationships between phenylpropanoids and phenylphenalenones whose phenyl rings share identical substitution patterns. Four phenylpropanoic acids, including ferulic acid and the unusual 4-methoxycinnamic acid, were used in (13)C-labeled form as substrates to study their incorporation into phenylphenalenones. In addition to the previously reported 2-hydroxy-9-(4'-hydroxy-3'-methoxyphenyl)-1H-phenalen-1-one (trivial name musanolone F), 2-hydroxy-9-(4'-methoxyphenyl)-1H-phenalen-1-one (proposed trivial name 4'-methoxyanigorufone) was found as a biosynthetic product in A. preissii. The carbon skeleton of 4'-methoxycinnamic acid was biosynthetically incorporated as an intact unit including its 4'-O-methyl substituent at the lateral phenyl ring. 4'-Methoxyanigorufone is reported here for the first time as a natural product.

Antibiotic oxylipins from Alternanthera brasiliana and its endophytic bacteria.

Bioassay-guided fractionation of Alternanthera brasiliana stem extracts resulted in the isolation of an antibiotically active fraction. Five human pathogenic bacteria were used to guide the fractionation process for the isolation of antimicrobial compounds. Finally, 17 linoleate oxylipins were identified by LC-MS/MS and NMR spectroscopy. Five of the isolated compounds present in A. brasiliana tissues were also detected to be synthesized by endophytic bacteria of the genus Bacillus that were isolated from A. brasiliana. It is speculated that the antibiotic oxylipins from A. brasiliana might derive from bacteria and be involved in an ecological relationship between this plant and its endophytes.