Leaf markers for root colonization by arbuscular mycorrhizal fungi in plants

Abstract

The invention relates to a method of determining an association of a first plant with an arbuscular mycorrhizal fungus (AMF), said method comprising comparing the amount of a blumenol in an aerial part of said first plant to the amount of said blumenol in an aerial part of a second plant, wherein said second plant belongs to the same species as said first plant, and wherein an increased amount is indicative of increased association in said first plant as compared to said second plant, and a decreased amount is indicative of decreased association.

Claims

1. A method of determining a mycorrhizal association of a first plant with an arbuscular mycorrhizal fungus (AMF), said method comprising: (a) determining the amount of a blumenol marker for mycorrhizal association in an aerial part of said first plant, wherein said blumenol marker is 11-hydroxyblumenol-C-9-O-Glc and said first plant is a vascular plant selected from a Nicotiana plant, a Triticum plant, a Hordeum plant, and an Oryza plant, optionally wherein said first plant has been brought into contact with an AMF prior to said determining, and wherein said determining comprises taking a sample from said aerial part and measuring the amount of said blumenol marker at a time point between about two weeks and about ten weeks after a said contact with an AMF (b) selecting a second plant from one of (i) a second plant wherein said second plant belongs to the same species as said first plant and is free of any association with an AMF, (ii) a second plant wherein said second plant belongs to the same species as said first plant and has an association with an AMF, (iii) a second plant wherein said first plant and said second plant are the same individual at different points in time, optionally wherein said second plant is the same individual at an earlier point in time than said first plant, and (iv) a second plant wherein the mycorrhization status of said second plant is not known; (c) comparing said amount of said blumenol marker in said aerial part of said first plant to the amount of said blumenol marker determined in an aerial part of said second plant; and (d) determining said association of said first plant with an AMF after said comparing step, selected from at least one of (i) selecting said first plant for it having a higher amount of said blumenol marker in said aerial part of said first plant as compared to said second plant when said second plant is free of any association with an AMF, and, thereby, having an increased AMF association as compared to said second plant, (ii) selecting said first plant for it having a higher amount of said blumenol marker in said aerial part of said first plant as compared to said second plant when said second plant has an association with an AMF or when said second plant is the same individual at a different point in time, or when said mycorrhization status of said second plant is not known, and, thereby, having a higher degree of AMF association as compared to said second plant, (iii) selecting said first plant for it having a lower amount of said blumenol marker in said aerial part of said first plant as compared to said second plant when said second plant has an association with an AMF, and, thereby, having a lower degree of AMF association as compared to said second plant, and (iv) selecting said first plant for it having an about equal amount of said blumenol marker in said aerial part of said first plant as compared to said second plant when said second plant is free of any association with an AMF, and, thereby, not having an AMF association.

2. The method of claim 1, wherein said amount of said blumenol marker in said aerial part of said first plant is determined in a sample from said aerial part of said first plant wherein said amount is optionally determined for one, two, or a plurality of first plants, and for one, two, or a plurality of samples from each said first plant, and said amount of said blumenol marker in said aerial part of said second plant, is selected from at least one of: (i) the amount of said blumenol marker determined in a sample from an aerial part of said second plant wherein said second plant is known to be free of any mycorrhizal association with an AMF, (ii) the amount of said blumenol marker determined in a sample from an aerial part of said second plant wherein said second plant is known to have a mycorrhizal association with an AMF, (iii) the amount of said blumenol marker determined in a sample from an aerial part of said second plant wherein said first plant and said second plant are the same individual at different points in time, (iv) the amount of said blumenol marker determined in a sample from an aerial part of said second plant wherein the mycorrhization status of said second plant is not known, (v) a reference value taken from a knowledge base, wherein said value is the amount of said blumenol marker determined in an aerial part of a plant of the same species as said first plant wherein the mycorrhization status of said second plant is known, (vi) a reference value taken from a knowledge base wherein said value is a baseline value for a plant of the same species, and (vii) a predefined value for a said amount of said blumenol marker indicative of a given mycorrhization status for a plant of the same species; and wherein said amount of said blumenol marker determined in said sample from said aerial part of said first plant is indicative of the degree of said mycorrhizal association.

3. The method of claim 1, wherein said determining on the basis of said amount of said blumenol marker for said mycorrhizal association is selected from at least one of qualitative determining and quantitative determining.

4. The method of claim 1, for determining whether said first plant has AMF receptivity, comprising: (a) contacting said first plant with an AMF known to be capable of colonization wherein said contacting is prior to said determining the amount of said blumenol marker in a sample from said aerial part of said contacted first plant; (b) selecting a point in time between about two weeks and about ten weeks after said contacting and determining the amount of said blumenol marker in said sample from said aerial part of said contacted first plant at said point in time; (c) comparing said amount of said blumenol marker determined in said sample from said contacted first plant in step (b) and a reference amount of said blumenol marker in said aerial part of said second plant wherein said reference amount is selected from one of (i) a reference amount wherein said first plant and said second plant are the same individual at different points in time, and said reference amount is the amount of said blumenol determined in a sample from an aerial part of said individual prior to said first point of time in step (b), optionally wherein said reference amount is determined prior to said contacting in step (a); (ii) a reference amount of said blumenol marker determined in a sample from an aerial part of said second plant wherein said second plant is free of any association with an AMF; and (iii) a reference amount wherein said amount is a reference value taken from a knowledge base and said value is a baseline value for a plant of the same species, (d) obtaining a difference between said amount determined in step (b) and said reference amount of step (c); wherein a higher amount of said blumenol marker in said sample from said contacted first plant determined in step (b) as compared to said reference amount of step (c) is indicative of said AMF receptivity of said first plant, and said difference of step (d) is a measure of said receptivity; and (e) selecting each said contacted first plant for having said higher amount of said blumenol marker in said sample determined in step (b) as compared to said reference amount of step (c) and therefore said first plant has AMF receptivity.

5. The method of claim 4, further wherein step (a) comprises providing a plurality of said first plants and contacting each said plant of said plurality of said first plants with an AMF, thereby providing a contacted plurality of said first plants, wherein each said contacted first plant of said contacted plurality of said first plants belongs to the same species, and steps (b) to (e) comprise determining AMF receptivity for each said contacted first plant of said contacted plurality of said first plants, and selecting each said contacted first plant for having AMF receptivity.

6. The method of claim 1, for determining whether an AMF has colonization capability for said first plant, and quantifying said capability, comprising: (a) selecting said first plant wherein said first plant is known to be receptive for colonization, selecting an AMF, and contacting said first plant with said AMF prior to determining the amount of said blumenol marker in a sample from said aerial part of said contacted first plant; (b) selecting a point in time between about two weeks and about ten weeks after said contacting and determining the amount of said blumenol marker in said sample from an aerial part of said contacted first plant at said point in time; (c) comparing said amount of said blumenol marker in said sample from said contacted first plant determined in step (b) and a reference amount of said blumenol marker in an aerial part of said second plant selected from one of (i) a reference amount wherein said first plant and said second plant are the same individual at different points in time, and said reference amount is the amount of said blumenol marker in said aerial part of said second plant taken from said individual prior to said point in time of step (b), optionally wherein said reference amount is determined prior to said contacting in step (a), (ii) a reference amount of said blumenol marker determined in a sample from an aerial part of said second plant wherein said second plant is free of any association with an AMF, and (iii) a reference amount wherein said amount is a value taken from a knowledge base and said value is a baseline value; (d) obtaining a difference between said amount determined in step (b) and said reference amount of step (c), wherein a higher amount of said blumenol marker in said sample from said contacted first plant determined in step (b) as compared to said reference amount of step (c) is indicative of said AMF having said colonization capability for said first plant and said difference of step (d) is a measure of said colonization capability; and (e) selecting each of said contacted first plant for having said higher amount of said blumenol marker in said sample determined in step (b) as compared to said reference amount of step (c), thereby selecting said AMF having colonization capability for said first plant.

7. The method of claim 6, wherein step (a) comprises selecting a plurality of AMFs comprising at least two different AMF species, and contacting at least one said first plant known to be receptive to colonization, and selecting each said AMF for having said colonization capability.

8. A method of producing a mycorrhizal association of a plant with an AMF, said method comprising: (a) providing at least one first plant wherein said first plant is a vascular plant selected from a Nicotiana plant, a Triticum plant, a Hordeum plant, and an Oryza plant, and contacting said first plant with an AMF; (b) selecting a point in time between about two weeks and about ten weeks after said contacting in step (a) and determining the amount of a blumenol marker for mycorrhizal association in a sample from an aerial part of said contacted first plant at said point in time, wherein said blumenol marker is 11-hydroxyblumenol-C-9-O-Glc, wherein said determining comprises taking a sample from said aerial part and measuring the amount of said blumenol marker; (c) obtaining a reference value selected from one of (i) a reference value determined for a second plant wherein the second plant is the same individual as said contacted first plant of step (a) at a different point in time, wherein said reference value is the amount of said blumenol marker determined in a sample from an aerial part of said individual taken from said individual prior to said point in time of step (b), optionally wherein said reference amount is determined for said individual prior to said contacting in step (a), (ii) a reference value determined from a second plant of the same species, wherein said second plant is free of any association with an AMF and said reference value is the amount of said blumenol marker determined in a sample from an aerial part of said second plant, and (iii) a reference value taken from a knowledge base, wherein said reference value is a baseline value for a plant of the same species; (d) comparing said amount of said blumenol marker in said contacted first plant determined in step (b) and said reference value of step (c) to obtain a difference between said amount in said contacted first plant and said reference value; and (e) selecting each said contacted first plant for it having an amount of said blumenol marker determined in step (b) that is increased as compared to said reference value of step (c) and, thereby, having a mycorrhizal association, thereby said selected first plant thereby produces said mycorrhizal association.

9. The method of claim 1, comprising determining said amount of said blumenol marker in each said aerial part of each said first plant and second plant using a mass spectrometer configured for the quantitative analysis of a blumenol as defined in claim 1.

10. The method of claim 8, wherein step (a) comprises contacting said first plant with a plurality of AMFs comprising at least two different AMF species, step (b) comprises determining the amount of said blumenol marker in a sample from an aerial part of said contacted first plant at said selected point in time, and step (e) comprises selecting each said contacted first plant for having an increased amount of said blumenol marker determined in step (b) compared to said reference value of step (c), wherein each said selected said first plant thereby produces a mycorrhizal association of said plant with at least one AMF of said plurality of AMFs.

11. The method of claim 8, wherein step (a) comprises contacting a plurality of said first plants with an AMF, step (b) comprises determining the amount of said blumenol marker in a sample from an aerial part of each said contacted first plant at said selected point in time, and step (e) comprises selecting each said contacted first plant for having an increased amount of said blumenol marker determined in step (b) compared to said reference value of step (c), wherein each said selecting thereby produces a mycorrhizal association of said selected plant with said AMF.

12. The method of claim 4, wherein said first plant and said second plant are the same individual at different points in time according to step (c)(i) and said method comprises monitoring mycorrhization status by determining said mycorrhizal association in said individual at two or more time points, wherein the amount of said blumenol marker in said sample from said individual at each time point is indicative of the degree of mycorrhizal association of said individual at each said time point.

Description

(1) The figures show:

(2) FIG. 1 Combined targeted and untargeted metabolomics identified blumenol derivatives as AMF-indicative in-planta fingerprints in the roots and leaves of Nicotiana attenuata plants. A Experimental set-up. EV and irCCaMK plants were co-cultured and inoculated with or without Rhizophagus irregularis. Six weeks after inoculation (wpi), root samples were harvested for metabolite profiling. B Covariance network visualizing m/z features from UHPLC-qTOF-MS untargeted analysis (n=8). Known compounds, including nicotine, phenylalanine and various phenolics, and unknowns (Unk.) are annotated by dashed ellipses. C Normalized Z-scored m/z features were clustered using STEM Clustering; 5 of 8 significant clusters are shown in different grey levels and mapped onto the covariance network. The intensity variation (mean+SE) of 2 selected features (Compounds 1 and 2) are shown in bar plots (n.d, not detected). D Representative chromatograms of Compounds 1 and 2 in roots and leaves of plants with and without AMF inoculation, as analyzed by targeted UHPLC-triple quadrupole-MS metabolomics.

(3) FIG. 2 Compounds 1 and 2 are leaf markers of root AMF colonization in N. attenuata. A Time lapse accumulations of Compounds 1 and 2 in leaves of EV plants with (EV+) or without (EV) AMF inoculation and of irCCaMK plants with AMF inoculation (irCCaMK+)(meansSE, n6). B Leaf abundances of Compounds 1 and 2 (5 wpi) of plants inoculated with different inoculum concentrations (means+SE, n=8); different letters indicate significant differences (p<0.05, one-way ANOVA followed by Fisher's LSD). C Compounds 1 and 2 in leaf samples of EV and irCCaMK plants inoculated with (+) or without () AMF inoculum isolated from the plant's native habitat (6 wpi); different letters indicate significant differences (p<0.05, one-way ANOVA followed by Tukey's HSD, n=10). D Field experiment (Great Basin Desert, Utah, USA): Compounds 1 and 2 in leaf samples of EV (n=20) and irCCaMK (n=19) plants sampled 8 weeks after planting. (Student's t-test: ***, p<0.001). E Representative images of WGA-488 stained roots of plants shown in B (bar=100 m). F Leaf Compounds 1 and 2 relative to the percentage of root colonization by hyphae, arbuscules, vesicles and total root length colonization of the same plants (linear regression model). G Compounds 1 and 2 in 17 different tissues of plants with (+AMF, n=3, left aligned bars) or without (AMF, n=1, right aligned bars) AMF inoculation harvested at 6 wpi.

(4) FIG. 3 The AMF-indicative accumulations of Compounds 1 and 2 in shoots originate from roots. A Hierarchical clustering analysis of transcript abundance from RNA-seq of methylerythritol 4-phosphate (MEP) and (apo)carotenoid biosynthetic genes. B Compounds 1, 2 and 6 (not AMF-specific) in AMF-inoculated i-irPDS and EV plants. On each plant a single stem leaf (leaf 0) was elicited with 100 M DEX-containing paste for 3 weeks; treated and adjacent, untreated control leaves (leaf 1 and leaf +1) were harvested. Representative leaves are shown (bleaching indicates PDS silencing); (means+SE, n=9). The same leaf positions in i-irPDS and EV plants were compared by Student's t-tests. C Contents of Compounds 1, 2 and 6 in the roots and shoots of seedlings whose roots were dipped for 1 d into an aqueous solution with or without AMF-indicative blumenols. D Model of the blumenol distribution in plants with (right panel) and without (left panel) AMF colonization. The model illustrates constitutive blumenols (e.g., Compound 6 in N. attenuata) and AMF-indicative ones (e.g., Compounds 1 and 2 in N. attenuata) and their inferred transport.

(5) FIG. 4 AMF-indicative changes in blumenols in aerial plant parts are valuable research tools providing accurate assessments of functional AMF associations in high-throughput screenings of multiple plant and AMF species. A Root colonization analysis between two N. attenuata accessions (UT/AZ). H: hyphae; A: arbuscules; V: vesicles; T: total colonization (n=8; Student's t-test, *, p<0.05, **, p<0.01, ***, p<0.001). B Representative images of trypan blue stained roots (6 wpi; bar=100 m). C Compound 2 in roots and leaves of UT and AZ plants with and without AMF-inoculation (means+SE, n=8). D Heatmap of the normalized abundance of foliar Compound 2 of plants from a UT-AZ RIL population (728 plants) across a 7,200 m.sup.2 field plot. E QTL mapping analysis of the data from D. QTL locus on linkage group 3 contains NaNOPE1, an AMF-associated gene, in addition to others. LOD, logarithm of the odds ratio. F Blumenol contents of different crop plants with and without AMF inoculation. Different plant and AMF species were used as indicated (means+SE; n.d, not detected).

(6) FIG. 5 AMF-indicative changes in blumenols in aerial plant parts are valuable research tools providing accurate assessments of functional AMF associations of multiple plant and AMF species (continued from FIG. 4F). Blumenol contents of different crop plants with and without AMF inoculation. Different plant and AMF species were used, as indicated; means+SE, n.d, not detected.

(7) FIG. 6 Comparison of 11-hydroxyblumenol C glucoside (Compound 1) signals in wild-type Nipponbare (NB) and ccamk-2 mutant plants without (mock) or with inoculation with crude or pure-culture (plate) Rhizophagus irregularis inoculum.

(8) FIG. 7 Comparison of 11-carboxyblumenol C glucoside (Compound 2) signals in wild-type Nipponbare (NB) and ccamk-2 mutant plants without (mock) or with inoculation with crude or pure-culture (plate) Rhizophagus irregularis inoculum.

(9) FIG. 8 Compounds 1 and 2 are leaf markers of root AMF colonization in O. sativa. Leaf abundances of AMF marker compounds were quantified in rice wild-type Nipponbare (NB), ccamk-1 and ccamk-2 mutant plants without (mock) or with inoculation with crude or pure-culture (plate) Rhizophagus irregularis inoculum.

(10) The examples illustrate the invention:

EXAMPLE 1

Materials and Methods

(11) Plant Growth and AMF Inoculation

(12) For our experiments with Nicotiana attenuata (Torr. ex S. Wats.), we used plants from the 31.sup.st inbred generation of the inbred UT line, irCCaMK (A-09-1212-1; Groten et al. (2015) Silencing a key gene of the common symbiosis pathway in Nicotiana attenuata specifically impairs arbuscular mycorrhizal infection without influencing the root-associated microbiome or plant growth. Plant, Cell & Environment 38, 2398-2416) plants that are stably silenced in CCaMK via RNAi, the i-irPDS plants (A-11-92-4A-11-325-4; Schfer et al. (2013) Real time genetic manipulation: A new tool for ecological field studies. The Plant Journal 76, 506-518) harboring the LhGR/pOp6 system for chemically-inducible RNAi-mediated gene silencing of phytoene desaturase (PDS) and the respective empty vector (EV) transformed plants (A-04-266-3; Bubner et al. (2006) Occurrence of tetraploidy in Nicotiana attenuata plants after Agrobacterium-mediated transformation is genotype specific but independent of polysomaty of explant tissue. Plant Cell Reports 25, 668-675) as control. Details about the transformation and screening of the irCCaMK plants are described by Groten, et al. ((2015) Silencing a key gene of the common symbiosis pathway in Nicotiana attenuata specifically impairs arbuscular mycorrhizal infection without influencing the root-associated microbiome or plant growth. Plant, Cell & Environment 38, 2398-2416 and for the i-irPDS plants by Schfer et al. ((2013) Real time genetic manipulation: A new tool for ecological field studies. The Plant Journal 76, 506-518). Seeds were germinated on Gamborg B5 as described by Krgel et al. ((2002) Agrobacterium-mediated transformation of Nicotiana attenuata, a model ecological expression system. Chemoecology 12, 177-183). The used advance intercross recombinant inbred line (RIL) population was developed by crossing two N. attenuata inbred lines originating from accessions collected in Arizona (AZ) and Utah (UT), USA (Glawe et al. (2003) Ecological costs and benefits correlated with trypsin protease inhibitor production in Nicotiana attenuata. Ecology 84, 79-90, Zhou et al. (2017) Tissue-specific emission of (E)-alpha-bergamotene helps resolve the dilemma when pollinators are also herbivores. Current Biology 27, 1336-1341). Additionally, we used Solanum lycopersicum Moneymaker, Hordeum vulgare Elbany and Triticum aestivum Chinese Spring plants.

(13) For glasshouse experiments, plants were treated according to Groten et al. ((2015) Silencing a key gene of the common symbiosis pathway in Nicotiana attenuata specifically impairs arbuscular mycorrhizal infection without influencing the root-associated microbiome or plant growth. Plant, Cell & Environment 38, 2398-2416). In brief, they were transferred into dead (autoclaved twice at 121 C. for 30 min; non-inoculated controls) or living inoculum (R. irregularis, Biomyc Vital, www.biomyc.de, inoculated plants) diluted 1:10 with expanded clay (size: 2-4 mm). Pots were covered with a thin layer of sand. Plants were watered with distilled water for 7 d and subsequently fertilized every second day either with a full strength hydroponic solution (for 1 L: 0.1292 g CaSO.sub.42H.sub.2O, 0.1232 g MgSO.sub.47H.sub.2O, 0.0479 g K.sub.2HPO.sub.4, 0.0306 g KH.sub.2PO.sub.4, 2 mL KNO.sub.3 (1 M), 0.5 mL micronutrients, 0.5 mL Fe diethylene triamine pentaacetic acid) or with a low P hydroponics solution containing only 1/10 of the regular P-concentration (0.05 mM). Plants were grown separately in 1 L pots, if not stated otherwise. In the paired design (FIG. 1), irCCaMK plants were grown together with EV plants in 2 L pots and the watering regime was changed to % of the regular P-concentration after plants started to elongate. Glasshouse experiments with natural inoculum (FIG. 2C) were conducted in a mesocosm system (4 boxes, each 2 pairs of EV and irCCaMK plants). Plants were maintained under standard glasshouse conditions (16 h light, 24-28 C., and 8 h dark, 20-24 C. and 45-55% humidity) with supplemental light supplied by high-pressure sodium lamps (Son-T-Agro).

(14) The field experiments were conducted as described by Schuman et al. ((2012) Herbivory-induced volatiles function as defenses increasing fitness of the native plant Nicotiana attenuata in nature. Elife 1, e00007). Seedlings were transferred to Jiffy pots and planted into a field plot at the Lytle Ranch Preserve in the Great Basin Desert (Utah, USA: N 37.1412, W 114.0275). Field season 2016 (FIG. 2D): field experiments were conducted under the US Department of Agriculture Animal and Plant Health Inspection Service (APHIS) import permission numbers 10-004-105m (irCCaMK) and 07-341-101n (EV) and the APHIS release permission number 16-013-102r. EV and irCCaMK plants were planted in communities of six plants, either of the same genotype or with both genotypes in equal number.

(15) Samples Prepared at Other Laboratory Facilities:

(16) Medicago truncatula (FIGS. 4 and 5) and Brachypodium distachyon (FIGS. 4 and 5) samples were prepared at the laboratory of Prof. Maria Harrison from the Boyce Thompson Institute for Plant Research (Ithaca, NY., USA).

(17) Solanum lycopersicum Moneymaker (FIG. 5) and Solanum tuberosum (FIG. 4) samples were prepared at the laboratory of Prof. Philipp Franken by Dr. Michael Bitterlich from the Leibniz-Institute of Vegetable and Ornamental Crops (IGZ, Grobeeren/Erfurt, Germany).

(18) Inducible PDS Silencing

(19) For the temporal and spatial restriction of PDS gene silencing, we treated the petiole of the second oldest stem leaf of AMF-inoculated and non AMF-inoculated i-irPDS and EV plants with a 100 M dexamethasone-containing lanolin paste (1% v/v DMSO). The lanolin paste was prepared and applied as described by Schfer et al. ((2013) Real time genetic manipulation: A new tool for ecological field studies. The Plant Journal 76, 506-518). The treatment started 3 weeks after potting and was conducted for 3 weeks. The lanoline paste was refreshed twice per week. On each plant the treated leaf and the adjacent, untreated leaves were harvested for analysis.

(20) Sample Collection

(21) During harvests, roots were washed and briefly dried with a paper towel. Subsequently, they were cut into 1 cm pieces and mixed. Plant tissues were shock-frozen in liquid nitrogen immediately after collection, ground to a fine powder and stored at 20 C. (short-term storage)/80 C. (long-term storage) until extraction. From the root samples, an aliquot was stored in root storage solution (25% ethanol and 15% acetic acid in water) at 4 C. for microscopic analysis. S. lycopersicum and S. tuberosum samples from IGZ were provided as dry material.

(22) For stem sap collection, branches of N. attenuata plants were cut into 1.5 cm long pieces and placed into small 0.5 mL reaction tubes with a small hole in the tip, which were placed in a larger 1.5 mL reaction tube. The tubes were centrifuged for 15 min at 10 000g. The stem sap from the larger reaction tubes were collected and stored at 20 C.

(23) Stress Treatments

(24) Herbivory treatments were conducted by placing Manduca sexta neonates, originating from an in-house colony, on the plants. After feeding for 2 weeks, rosette leaves were harvested. As controls, we harvested leaves from untreated plants.

(25) For bacteria and virus infection, plants were inoculated plants with Agrobacterium tumefaciens carrying the Tobacco rattle virus. The inoculation was conducted by infiltrating leaves with a bacteria solution using a syringe. The treatment was conducted as described for virus-induced gene silencing described by Ratcliff et al. ((2001) Technical Advance. Tobacco rattle virus as a vector for analysis of gene function by silencing. The Plant Journal 25, 237-45) and by Saedler and Baldwin ((2004) Virus-induced gene silencing of jasmonate-induced direct defences, nicotine and trypsin proteinase-inhibitors in Nicotiana attenuata. Journal of Experimental Botany 55, 151-157). After incubation for 3 weeks, stem leaves of the treated plants and untreated control plants were harvested.

(26) The fungal infection was done with Botrytis cinerea. On each plant, three leaves were treated by applying 6 droplets each containing 10 L of B. cinerea spore suspension (10.sup.6 spores/mL in Potato Extract Glucose Broth, Carl Roth GmbH) to the leaf surface. As control, plants were treated with broth without spores in the same way. Samples were collected after 4 days incubation.

(27) Drought stress was induced by stopping the watering for 4 d. Subsequently, stem leaves of the drought-stressed plants and the continuously watered control plants were harvested. In contrast to the other samples of the stress experiment, leaves were dried before analysis to compensate for weight differences caused by changes in the water content.

(28) Sample PreparationExtraction and Purification

(29) For extraction, samples were aliquoted into reaction tubes, containing two steel balls. Weights were recorded for later normalization. Per 100 mg plant tissues, approximately 1 mL 80% MeOH was added to the samples before being shaken in a GenoGrinder 2000 (SPEX SamplePrep) for 60 s at 1150 strokes min.sup.1. After centrifugation, the supernatant was collected and analyzed. For triple-quadrupole MS quantification, the extraction buffer was spiked with 10 ng stable isotope-labeled abscisic acid (D.sub.6-ABA, HPC Standards GmbH) as an internal standard.

(30) Stem sap was diluted 1:1 with MeOH spiked with D.sub.6-ABA as an internal standard. After centrifugation, the supernatant was collected and analyzed.

(31) The purification of N. attenuata leaf extracts for high resolution MS was conducted by solid-phase-extraction (SPE) using the Chromabond HR-XC 45 m benzensulfonic acid cation exchange columns (Machery-Nagel) to removed abundant constituents, such as nicotine and phenolamides. After purification the samples were evaporated to dryness and reconstituted in 80% methanol.

(32) Compound identification was conducted by NMR with purified fractions of root and leaf extracts. Compounds 1, 3 and 4 were extracted from root tissues of N. attenuata and purified by HPLC (Agilent-HPLC 1100 series; Grom-Sil 120 ODS-4 HE, C18, 2508 mm, 5 m; equipped with a Gilson 206 Abimed fraction collector). Compounds 2 and 7 were extracted from a mixture of leaf tissues from different plant species (M. truncatula, Z. mays, S. lypersicum and N. attenuata). The first purification step was conducted by SPE using the Chromabond HR-XC 45 m benzensulfonic acid cation exchange columns (Machery-Nagel) to remove hydrophilic and cationic constituents. Additional purification steps were conducted via HPLC (Agilent-HPLC 1100 series; Phenomenex Luna C18(2), 25010 mm, 5 m; equipped with a Foxy Jr. sample collector) and UHPLC (Dionex UltiMate 3000; Thermo Acclaim RSLC 120 C18, 1502.1 mm, 2.2 m; using the auto-sampler for fraction collection).

(33) Untargeted MS Based Analyses

(34) For high resolution mass spectrometry (MS), indiscriminate tandem mass spectrometry (idMS/MS), tandem MS (MS2) and pseudo-MS3 we developed a chromatographic method using a mixture of solvent A: water (Milli-Q, Merck, emdmillipore.com) with 0.1% acetonitrile and 0.05% formic acid and solvent B: acetonitrile with 0.05% formic acid. Ultra high performance liquid chromatography (UHPLC) was performed using a Dionex UltiMate 3000 rapid separation LC system (Thermo Fisher, thermofisher.com), combined with a Thermo Acclaim RSLC 120 C18, 1502.1 mm, 2.2 m column. The solvent composition changed from a high % A in a linear gradient to a high % B followed by column equilibration steps and the return to the starting conditions. The flow rate was set to 0.3 mL/min. MS detection was performed using a microTOF-Q II MS system (Bruker Daltonics, bruker.com), equipped with an electrospray ionization (ESI) source operating in positive ion mode. ESI conditions for the micrOTOF-Q II system were end plate offset 500 V, capillary voltage 4500 V, capillary exit 130 V, dry temperature 180 C. and a dry gas flow of 10 L min-1. Mass calibration was performed using sodium formiate (250 mL isopropanol, 1 mL formic acid, 5 mL 1 M NaOH in 500 mL water). Data files were calibrated using the Bruker high-precision calibration algorithm. Instrument control, data acquisition and reprocessing were performed using HyStar 3.1 (Bruker Daltonics).

(35) idMS/MS was conducted in order to gain structural information on the overall detectable metabolic profile. For this, samples were first analyzed by UHPLC-ESI/qTOF-MS using the single MS mode (producing low fragmentations resulting from in-source fragmentation) by scanning from m/z 50 to 1400 at a rate of 5000 scans/s. MS/MS analyses were conducted using nitrogen as collision gas and involving independent measurements at the following 4 different collision-induced dissociation (CID) voltages: 20, 30, 40 and 50 eV. The quadrupole was operated throughout the measurement with the largest mass isolation window, from m/z 50 to 1400. Mass fragments were scanned between m/z 50 to 1400 at a rate of 5000 scans/s. For the idMS/MS assembly, we used a previously designed precursor-to-product assignment pipeline (Li et al. (2015) Navigating natural variation in herbivory-induced secondary metabolism in coyote tobacco populations using MS/MS structural analysis. Proceedings of the National Academy of Sciences 112, E4147-E4155, Li et al. (2016) Illuminating a plant's tissue-specific metabolic diversity using computational metabolomics and information theory. Proceedings of the National Academy of Sciences 113, E7610-E7618) using the output results for processing with the R packages XCMS and CAMERA.

(36) Additional MS/MS experiments were performed on the molecular ion at various CID voltages. For the fragmentation of the proposed aglycones via pseudo-MS.sup.3, we applied a 60 eV in-source-CID transfer energy which produced spectra reflecting the loss of all sugar moieties.

(37) Structure Elucidation by NMR

(38) Purified fractions were dried completely and reconstituted in MeOH-d.sub.3 before analysis by nuclear magnetic resonance spectroscopy (NMR) on an Avance AV700 MD NMR spectrometer (Bruker-Biospin, Karlsruhe, Germany) at 298 K using a 1.7 mm TCl CryoProbe. Chemical shift values () are given relative to the residual solvent peaks at .sub.H 3.31 and .sub.C 49.05, respectively. Carbon shifts were determined indirectly from .sup.1H-.sup.13C HSQC and HMBC spectra.

(39) Targeted Metabolite Analysis

(40) For chromatographic separations, a UHPLC (Dionex UltiMate 3000) was used to provide a maximum of separation with short run times. This reduced the disturbance by other extract components (matrix effects), increased the specificity of the method, and met the requirements of a HTP analysis. The auto-sampler was cooled to 10 C. As a stationary phase, we used a reversed phase column (Agilent ZORBAX Eclipse XDB C18, 503.0 mm, 1.8 m) suitable for the separation of moderately polar compounds. Column temperature was set to 42 C. As mobile phases, we used: A, 0.05% HCOOH, 0.1% ACN in H.sub.2O and B, MeOH, the composition of which was optimized for an efficient separation of blumenol-type compounds within a short run time. We included in the method a cleaning segment at 100% B and an equilibration segment allowing for reproducible results across large samples sets. The gradient program was as follows: 0-1 min, 10% B; 1-1.2 min, 10-35% B; 1.2-5 min, 35-50% B; 5-5.5 min, 50-100% B; 5.5-6.5 min, 100% B; 6.5-6.6 min, 100-10% B and 6.6-7.6 min, 10% B. The flow rate was set to 500 L min Analysis was performed on a Bruker Elite EvoQ triple quadrupole MS equipped with a HESI (heated electrospray ionization) ion source. Source parameters were as follows: spray voltage (+), 4500V; spray voltage (), 4500V; cone temperature, 350 C.; cone gas flow, 35; heated probe temperature, 300 C.; probe gas flow, 55 and nebulizer gas flow, 60. Samples were analyzed in multi-reaction-monitoring (MRM) mode (Table 4).

(41) TABLE-US-00004 TABLE 4 MRM-settings used for targeted blumenol analysis Q1 Nr. Compound Name RT [m/z] .sup.a, b Q3 [m/z] .sup.c, d (CE [V]) 1 11-hydroxyblumenol C-Glc .sup.f, g 2.82 +389.22 227.16 (2.5), 209.15 (7.5), 191.14 (12.5), 163.10 (15), 149.10 (17.5) 2 11-carboxyblumenol C-Glc .sup.f, g 3.22 +403.22 241.16 (2.5), 223.15 (7.5), 177.10 (15), 195.14 (12.5) .sup.+241.16 .sup.e 223.15 (5), 177.10 (15), 195.14 (10) 3 11-hydroxyblumenol C-Glc.Glc .sup.f, g 2.5 +551.27 389.22 (2.5), 227.16 (7.5), 209.15 (10), 191.14 (15), 149.10 (20) 4 Blumenol C - Glc-Glc .sup.f, g 3.47 +535.27 373.22 (2.5), 211.00 (10), 193.10 (17.5), 135.00 (22.5), 109.00 (22.5) 5 Blumenol C - Glc .sup.f, h 4.18 +373.22 211.20 (6), 193.16 (9), 175.10 (15), 135.12 (16), 109.10 (20) 7 Blumenol B - Glc .sup.f, g 2.5 +389.22 227.16 (5), 209.15 (7.5), 191.14 (12.5), 153.10 (17.5), 149.10 (17.5) 8 Blumenol C - Glc-GlcU .sup.i 3.25 +549.27 373.22 (2.5), 211.00 (10), 193.10 (17.5), 135.00 (22.5), & 3.38 109.00 (22.5) 9 11-hydroxylumenol C - Glc-Rha .sup.i 2.8 +535.27 389.22 (2.5), 227.16 (7.5), 209.15 (10), 191.14 (15), 149.10 (20) 10 Blumenol C - Glc-Rha .sup.i 4.1 +519.27 373.22 (2.5), 211.00 (10), 193.10 (17.5), 135.00 (22.5), 109.00 (22.5) 11 Hydroxyblumenol C-Hex-Pen .sup.i 2.5 +521.27 389.22 (2.5), 227.16 (7.5), 209.15 (10), 191.14 (15), 149.10 (20) D.sub.6-ABA .sup.h 4.5 269.17 159.00 (10) RT: retention time CE: collision energy Glc: glucose GIcU: glucuronic acid Rha: rhamnose Hex: hexose Pen: pentose .sup.a Resolution: 0.7 .sup.b [M + H].sup.+ or [M H].sup. if not stated differently .sup.c Resolution: 2 .sup.d Quantifiers are depicted in bold .sup.e [M + H-Glc].sup.+ .sup.f Verified by high resolution MS .sup.g Verified by NMR .sup.h Optimized with commercial available standards .sup.i Transitions predicted based on structural similar compounds and literature information
Adjusted Method for Targeted Blumenol Analysis in N. attenuata

(42) The AMF-indicative markers in N. attenuata, Compound 1 and 2, and the internal standard (D.sub.6-ABA) have been analyzed. Accordingly, the gradient program was adjusted as follows: 0-1 min, 10% B; 1-1.2 min, 10-35% B; 1.2-3 min, 35-42% B; 3-3.4 min, 42-100% B; 3.4-4.4 min, 100% B; 4.4-4.5 min, 100-10% B and 4.5-5.5 min, 10% B. The MRM settings are shown in Table 5.

(43) TABLE-US-00005 TABLE 5 MRM-settings for the analysis of selected blumenols in N. attenuate Q1 Nr. Compound Name RT [m/z] .sup.a, b Q3 [m/z] .sup.c, d (CE [V]) 1 11-hydroxyblumenol C-Glc .sup.f, g 2.82 +389.22 227.16 (2.5), 209.15 (7.5), 191.14 (12.5), 163.10 (15), 149.10 (17.5) 2 11-carboxyblumenol C-Glc .sup.f, g 3.22 +403.22 241.16 (2.5), 223.15 (7.5), 177.10 (15), 195.14 (12.5) .sup.+241.16 .sup.e 223.15 (5), 177.10 (15), 195.14 (10) D.sub.6-ABA .sup.h 4.0 269.17 159.00 (10) RT: retention time CE: collision energy Glc: glucose Hex: hexose Pen: pentose .sup.a Resolution: 0.7 .sup.b [M + H].sup.+ or [M H].sup. if not stated differently .sup.c Resolution: 2 .sup.d Quantifiers are depicted in bold .sup.e [M + H-Glc].sup.+ .sup.f Verified by high resolution MS .sup.g Verified by NMR .sup.h Optimized with commercial available standards
Determination of the AMF Colonization Rate

(44) To determine the fungal colonization rates and mycorrhizal structures, root samples were stained and analyzed by microscopy. For WGA-Alexa Fluor 488 staining, roots were first washed with distilled water and then soaked in 50% (v/v) ethanol overnight. Roots were then boiled in a 10% (w/v) KOH solution for 10 minutes. After rinsing with water, the roots were boiled in 0.1 M HCl solution for 5 minutes. After rinsing with water and subsequently with 1 phosphate-buffered saline solution, roots were stained in 1 phosphate-buffered saline buffer containing 0.2 mg mL.sup.1 WGA-Alexa Fluor 488 overnight in the dark. Zeiss confocal microscopy (LSM 510 META) was used to detect the WGA-Alexa Fluor 488 (excitation/emission maxima at approximately 495/519 nm) signal. Trypan blue staining was performed as described by Brundrett et al. ((1984) A new method for observing the morphology of vesicular-arbuscular mycorrhizae. Canadian Journal of Botany 62, 2128-2134) to visualize mycorrhizal structures. For the counting of mycorrhization, 15 root fragments, each about 1 cm long, were stained with either trypan blue or WGA-488 followed by slide mounting. More than 150 view fields per slide were surveyed with 20 object magnification and classified into 5 groups: no colonization, only hyphae (H), hyphae with arbuscules (H+A), hyphae with vesicles (V+H), and hyphae with arbuscules and vesicles (A+V+H). The proportions of each group were calculated by numbers of each group divided by total views.

(45) For the molecular biological analysis of colonization rates, RNA was extracted from the roots using the RNeasy Plant Mini Kit (Qiagen) or NucleoSpin RNA Plant (Macherey-Nagel) according to the manufacturer's instructions and cDNA was synthesized by reverse transcription using the PrimeScript RT-qPCR Kit (TaKaRa). Quantitative (q)PCR was performed on a Stratagene Mx3005P qPCR machine using a SYBR Green containing reaction mix (Eurogentec, eurogentec.com; qPCR Core kit for SYBR Green I No ROX). We analyzed the R. irregularis specific housekeeping gene, Ri-tub (GenBank: EXX64097.1), as well as the transcripts of the AMF-induced plant marker genes RAM1, Vapyrin, STR1 and PT4. The signal abundance was normalized to NaIF-5a (NCBI Reference Sequence: XP_019246749.1).

(46) Transcript Analysis of the Apocarotenoid Pathway

(47) The transcript analysis of the methylerythritol 4-phosphate (MEP) and (apo)carotenoid pathway was conducted based on RNA-seq by using N. attenuata roots with or without R. irregularis inoculations. The data analysis methods are based on the previously published pipeline of Ling et al. ((2015) Insect herbivory elicits genome-wide alternative splicing responses in Nicotiana attenuata. The Plant Journal 84, 228-243). Representative values for transcripts abundances are TPM (Transcripts per kilobase of exon model per million mapped reads).

(48) Blumenol Transfer Experiment

(49) To analyze the root-to-shoot transfer potential of blumenols, we placed three N. attenuata seedlings, previously germinated on petri dishes with GB5 Agar for approximately 10 days, into 0.5 mL reaction tubes. The roots were placed into the tube, while the shoot projected out of the tube. The tubes were carefully covered with parafilm, which held the seedlings in place and isolated roots from shoots (see FIG. 3C). The tubes were filled with tap water supplemented with 0.5% v/v plant extracts enriched in Compounds 1 or 2 (unknown concentration; purified fractions), or a commercial available standard of Compound 6 (25 ng L.sup.1 end concentration; Roseoside; Wuhan ChemFaces Biochemical Co., Ltd.). Compound 1 or 2 were prepared from a mix of leaf tissues from different plant species (M. truncatula, Z. mays, S. lypersicum and N. attenuata) by methanol extraction followed by purification by SPE (Chromabond HR-XC column) and HPLC (Agilent-HPLC 1100 series; Phenomenex Luna C18(2), 25010 mm, 5 m; equipped with a Foxy Jr. fraction collector). As a control, we used tap water supplemented with the respective amounts of MeOH. The seedlings were incubated for 1 d in a Percival climate chamber (16 h of light at 28 C., and 8 h of dark at 26 C.). During sample collection, roots and shoots were separated and the roots were rinsed in water (to reduce the surface contamination with the incubation medium). While the shoots were analyzed separately, the roots of all seedlings from the same treatment were pooled. Sample extraction was conducted as described above.

(50) QTL Analysis

(51) For quantitative trait loci (QTL) mapping, we used the AZ-UT RIL population described by Zhou et al. ((2017) Tissue-specific emission of (E)-alpha-bergamotene helps resolve the dilemma when pollinators are also herbivores. Current Biology 27, 1336-1341). The field experiments were conducted in 2017. Collected leaf samples were extracted as described with 80% MeOH spiked with D.sub.6-ABA as internal standard and analyzed with the method described under Adjusted method for targeted blumenol analysis in N. attenuata. The peak areas for Compound 2 were normalized by amount of extracted tissue, internal standard and log-transformed. Samples with missing genotype or phenotype information were removed. In total, 728 samples were used for QTL mapping analysis. QTL analysis was conducted according to Zhou et al. ((2017) Tissue-specific emission of (E)-alpha-bergamotene helps resolve the dilemma when pollinators are also herbivores. Current Biology 27, 1336-1341).

(52) Statistics

(53) Statistical analysis of the data was performed with R version 3.0.3 (R-project.org). The statistical methods used and the number of replicates are indicated in the figure legends.

EXAMPLE 2

Results

(54) We performed an untargeted metabolomics analysis of root tissues in a transgenic, line of Nicotiana attenuata, silenced in the calcium- and calmodulin-dependent protein kinase (irCCaMK), and empty vector (EV) plants co-cultured with or without Rhizophagus irregularis (FIG. 1A). By using irCCaMK plants, unable to establish a functional AMF association (Groten et al. (2015) Silencing a key gene of the common symbiosis pathway in Nicotiana attenuata specifically impairs arbuscular mycorrhizal infection without influencing the root-associated microbiome or plant growth. Plant, Cell & Environment 38, 2398-2416), we were able to dissect the AMF association-specific metabolic responses from those changes that result from more general plant-fungus interactions. Untargeted metabolome profiling of roots using liquid chromatography (LC) coupled time-of-flight mass spectrometry (qTOF-MS) resulted in a concatenate data matrix consisting of 943 mass features (m/z signals detected at certain retention times). A coexpression network analysis was conducted in which nodes represent m/z features and edges connect metabolite mass features derived from similar in-source fragmentation origins and sharing biochemical relationships (Li et al. (2015) Navigating natural variation in herbivory-induced secondary metabolism in coyote tobacco populations using MS/MS structural analysis. Proceedings of the National Academy of Sciences 112, E4147-E4155, Li et al. (2016) illuminating a plant's tissue-specific metabolic diversity using computational metabolomics and information theory. Proceedings of the National Academy of Sciences 113, E7610-E7618). For example, features of well-known compounds, like nicotine and phenylalanine, were tightly connected (FIG. 1B). A STEM clustering pipeline was performed to recognize patterns of metabolic accumulations in the genotypestreatment data matrix [(EV/irCCaMK)(/+AMF inoculation), respectively]. As a result, 5 of 8 computed distinct expression patterns were mapped onto the covariance network in FIG. 1B (shown in different grey levels). A tightly grouped cluster of unknown metabolites (FIG. 1B, upper left) occupied a distinct metabolic space. Metabolites grouped in this cluster were highly elicited upon mycorrhization in EV, but not in irCCaMK plants (FIG. 1C). It is also noteworthy that this group of compounds appeared to be de novo synthesized, as none were detected in non-inoculated plants (FIG. 1C). The structures of the compounds of this cluster were annotated based on tandem-MS and NMR data. Five metabolites were annotated as blumenols: 11-hydroxyblumenol-C-9-O-Glc (FIG. 1C; Compound 1), 11-carboxyblumenol-C-9-O-Glc (FIG. 1C; Compound 2), 11-hydroxyblumenol-C-9-O-Glc-Glc (Compound 3), blumenol-C-9-O-Glc-Glc (Compound 4) and blumenol-C-9-O-Glc (Compound 5).

(55) To trace these compounds throughout the plant, we used a more sensitive and specifically targeted metabolomics approach based on LC-triple-quadrupole-MS. The abundance of the five blumenol-C-glycosides continually increased with mycorrhizae development and was highly correlated with mycorrhization rate determined based on transcript abundances of classical marker genes (fungal house-keeping gene, Ri-tubuline; in-planta marker genes, Vapyrin, RAM1, STR1 and PT4; Park et al. (2015) Hyphal branching during arbuscule development requires Reduced Arbuscular Mycorrhiza1. Plant Physiology 169, 2774-2788).

(56) Compounds 1 and 2 showed a similar AMF-specific accumulation in the leaves, as observed in the roots (FIG. 1D). The other analyzed blumenols were not detected in leaves (Compounds 3 and 4) or showed a less consistent AMF-specific accumulation (Compound 5; due to its constitutive background level). The identity of Compounds 1 and 2 in the leaves was verified by high resolution qTOF-MS in a procedure which required additional sample purification and concentration steps due to their low abundance and high matrix effects in leaves, the likely reasons why previous un-targeted metabolomics attempts had failed to detect these signatures.

(57) Next, we determined the correlations among the contents of AMF-indicative foliar Compounds 1 and 2 and root colonization rates. In a kinetic experiment, both compounds increased their accumulations in the leaves of plants inoculated with R. irregularis (FIG. 2A). In contrast, the classical AMF-marker-genes, which are usually analyzed in the roots, did not respond in the leaves. In an inoculum-gradient experiment using increasing inoculum concentrations, proportionally higher Compound 1 and 2 levels were observed (FIG. 2B), accurately reflecting the differential colonization of roots across treatments (FIG. 2E). In addition to inoculations with single AMF-species (R. irregularis), we also tested mycorrhizal inoculum originally collected from the plant's native habitat, the Great Basin Desert in Utah, USA, which mainly consists of Funneliformis mosseae and R. irregularis. EV plants inoculated with this natural inoculum also accumulated Compounds 1 and 2 in leaves, while irCCaMK plants did not (FIG. 2C). When planted into the plant's natural environment in Utah, both EV and irCCaMK plants could be clearly distinguished by their leaf Compound 1 and 2 contents. T the signature from Compound 2 provided a better quality marker in these field-grown plants (FIG. 2D). The foliar content of these two compounds was highly correlated with the percentage of arbuscules in roots, the core structure of AMF interactions (FIG. 2F). In contrast, other biotic or abiotic stresses, including herbivory, pathogen infection and drought stress, did not induce the foliar accumulations of Compounds 1 and 2, as has been described for roots (Maier et al. (1997) Accumulation of sesquiterpenoid cyclohexenone derivatives induced by an arbuscular mycorrhizal fungus in members of the Poaceae. Planta 202, 36-42). An analysis of various plant tissues, including different leaf positions, stem pieces, flowers and capsules revealed that these AMF-specific signatures accumulated throughout the shoot (FIG. 2G). Taken together, we conclude that the contents of particular blumenols in aerial plant parts robustly reflect the degree of mycorrhization in N. attenuata plants.

(58) Blumenols are apocarotenoids originating from a side branch of the carotenoid pathway (Hou et al. (2016) Synthesis and function of apocarotenoid signals in plants. Trends in Plant Science 21, 792-803). Most of the genes likely associated with blumenol biosynthesis were upregulated in roots, but not in leaves of N. attenuata plants in response to mycorrhization (FIG. 3A). We inferred that the AMF-indicative leaf apocarotenoids are transported from their site of synthesis in colonized roots to other plant parts. This is consistent with the occurrence of blumenols in stem sap, which was collected by centrifuging small stem pieces. To clarify the origins (local biosynthesis vs. transport) of these AMF-indicative leaf blumenols, we genetically manipulated the carotenoid biosynthesis of N. attenuata plants. To minimize the effects of a disturbed carotenoid biosynthesis on the AMF-plant interaction, we used the dexamethasone (DEX)-inducible pOp6/LhGR system to silence the phytoene desaturase (PDS) expression in a single DEX-treated leaf position (Schfer et al. (2013) Real time genetic manipulation: A new tool for ecological field studies. The Plant Journal 76, 506-518). Treated leaves showed clear signs of bleaching indicating PDS silencing (FIG. 3B), but levels of the AMF-indicative Compounds 1 and 2 were not affected, consistent with their transport from other tissues, likely the highly accumulating roots. As a control, we analyzed the non-AMF-inducible Compound 6, showing constitutive levels in aerial tissues. In DEX-treated leaves, Compound 6 concentrations were reduced by nearly 40 percent, consistent with local production (FIG. 3B). To confirm the within-plant transport potential of blumenols, we dipped roots of seedlings into aqueous solutions of Compounds 1 or 2. After overnight incubation, the blumenol derivatives were clearly detected not only in roots, but also in shoots (FIG. 3C). We propose that the AMF-indicative blumenols (e.g., Compounds 1 and 2) are produced in colonized roots and transported to the shoot, while other AMF-independent blumenols (e.g., Compound 6) originate from local production and within-shoot transport (FIG. 3D).

(59) To test the potential of these foliar metabolites as a screening tool, we quantified them in a forward genetics experiment, an experiment which would be challenging with the classical screening tools of root staining or nucleic acid analysis. We focused our analysis on Compound 2 due to the superior quality of its signature in the leaves of field-grown plants. The experiment consisted of a recombinant inbred line population of two N. attenuata accessions (Utah, UT and Arizona, AZ)(Zhou et al. (2017) Tissue-specific emission of (E)-alpha-bergamotene helps resolve the dilemma when pollinators are also herbivores. Current Biology 27, 1336-1341) which differ in mycorrhization (FIGS. 4A-B) and accumulations of foliar Compound 2 in the glasshouse (FIG. 4C). A QTL analysis of 728 plants grown across a 7200 m.sup.2 field plot (FIG. 4D) revealed that the abundance of Compound 2 mapped to a single locus on linkage group 3 (FIG. 4E), which harbored a homologue of NOPE1, previously shown to be required for the initiation of AMF symbioses in maize and rice (Nadal et al. (2017) An N-acetylglucosamine transporter required for arbuscular mycorrhizal symbioses in rice and maize. Nature plants 3, 17073-17073). While clearly requiring additional follow-up work, these results highlight the value of these signature metabolites for HTP screenings, which form the basis of most crop improvement programs.

(60) The AMF-specific accumulation of blumenol-C-derivatives in roots is a widespread phenomenon within higher plants (Strack and Fester (2006) Isoprenoid metabolism and plastid reorganization in arbuscular mycorrhizal roots. New Phytologist 172, 22-34); however, how general are the observed blumenol changes in aerial parts across different combinations of plants and AMF species? We analyzed Solanum fycopersicum, Triticum aestivum and Hordeum vulgare plants with and without AMF inoculation and again we found an overlap in the AMF-specific blumenol responses in roots and leaves, consistent with the transport hypothesis. Further analyses lead to the identification of additional AMF-indicative blumenols in the leaves of Medicago truncatula, S. tuberosum and Brachypodium distachyon. We identified various types of blumenols that showed an AMF-specific accumulation in the shoot, including blumenol-B (Compound 7), which has not previously been reported in an AMF-dependent context (FIG. 4F). As reported for roots, the particular blumenol types were species-dependent, but the general pattern was widespread across monocots and dicots in experiments conducted at different research facilities. In tests with different fungal species (Rhizophagus irregularis, Funneliformis mosseae and Glomus versiforme), the observed effects were not found restricted to specific AMF taxa (FIGS. 4F and 5). In short, the method is robust.

EXAMPLE 3

(61) Optimization of AMF Marker Analysis for Rice (Oryza sativa) Plants

(62) Blumenol markers in (i) rice wild-type Nipponbare (NB) and (ii) two mutant genotypes deficient in Calcium- and Calmodulin-dependent protein kinase (CCaMK) which are unable to form a functional AMF association (ccamk-1 and ccamk-2) have been analyzed.

(63) Samples were harvested from two leaf positions, leaf 4 (L4) and leaf 5 (L5), of plants treated with three different AMF inoculation treatments: without AMF (mock), with a Rhizophagus irregularis inoculum prepared from colonized Tagetes roots (crude) or a R. irregularis inoculum from a pure culture on sterile carrot roots (plate). Frozen tissue samples (100 mg) were ground and extracted with 0.8 mL extraction buffer (80% methanol) containing 10 ng of D6-ABA as internal standard.

(64) After an initial screening for blumenol-related compounds we identified suitable markers indicating the colonization of rice plants with R. irregularis (FIGS. 6 and 7).

(65) The identified AMF marker compounds were quantified using compound-specific multiple-reaction-monitoring (MRM, Table A) on a triple-quadrupole UPLC-MSMS as described in Wang et al. 2018.

(66) TABLE-US-00006 TABLE A MRM settings used for the quantification of specific blumenol derivatives in rice leaves. RT Compound name [min] Quantifier m/z [CE] Qualifier m/z [CE] 11-hydroxyblumenol C-Glc 2.81 (+) 389.2 > 209.2 [7.5 V] 227.2 [2.5 V], 191.1 [12.5 V], 163.1 [15.0 V], 149.1 [17.5 V] 11-carboxyblumenol C-Glc 3.17 (+) 241.2 > 195.1 [10.0 V] 223.2 [5.0 V], 177.1 [15.0 V] 11-carboxyblumenol-MalGlc 3.60 (+) 489.2 > 195.1 [12.5 V] 241.2 [2.5 V], 223.2 [7.5 V], 177.1 [15.0 V] D.sub.6-abscisic acid (IS) 4.01 () 269.2 > 159.0 [10.0 V] RT: retention time [min]; CE: collision energy; IS: internal standard

(67) Similarly to other plant-AMF systems (i.e. Nicotiana attenuata), the abundances of 11-carboxy- and 11-hydroxyblumenol C glucoside were indicative of AMF colonization. Additionally, a malonylated derivative of the carboxyblumenol glucoside was showing a similar pattern of AMF-induced accumulation in wild-type NB rice plants which is abolished in the two ccamk mutants (FIG. 8).