Characterisation of strigolactone
profiles in root exudate from Zea
mays cultivars with different
Striga-susceptibilities
Abstract
In sub-Saharan Africa, parasitic Striga infestations endanger the livelihood of 300 million people. The most widely grown maize (Zea mays) varieties suffer approximately 70% yield losses annually due to Striga parasitism. Maize crop yield is determined in part by nutrient uptake in the roots, which is governed by interactions in the rhizosphere. Phytohormones strigolactones perform rhizospheric signalling functions, inducing hyphal branching in beneficial Arbuscular mycorrhizae fungi as well as inducing seed germination in detrimental parasitic plants such as Striga. Strigolactones are a diverse set of canonical and non- canonical compounds displaying varying moiety of a tricyclic lactone connected to a highly conserved D-ring. Root exudate contains a strigolactone profile which differs per maize cultivar, consisting of multiple strigolactones. Different strigolactones such as non-canonical zealactones and zeapyranolactones display varying seed germination activity, with Compound 5 having shown no allelopathy as of yet. The aim of this study was to detect the strigolactone profile in several maize cultivars, specifically the Compound 5 to Zealactone ratio, using Solid-phase extraction (SPE) methods and LC-MS/MS. The effect of inducing nutrient deficiencies was investigated as well by examining the strigolactone profile and the Compound 5 to Zealactone ratio. Maize line 15 displayed a significantly higher compound 5 to other strigolactone ratio compared to other maize lines. Induced nitrogen deficiency resulted in a different compound 5 to zealactone ratio compared to phosphorous deficient and control treatment groups.
Introduction
In sub-Saharan Africa Striga infestations endanger the livelihood of over 300 million people, spanning nearly 100 million hectares in 42 countries, and account for crop yield losses of 20% to 80% (Ejeta, 2007; Lagoke et al., 1991; M’boob, 1989). In the most widely grown maize (Zea mays) varieties in Africa, yield losses caused by the parasitic Striga amount to approximately 70% (Kim, Adetimirin, Thé, & Dossou, 2002). Crop yield is determined in part by nutrient uptake in the roots, which is governed by interactions in the rhizopshere. Plants interact with the surrounding organisms in the soil they occupy. The region these interactions take place is defined as the rhizosphere where beneficial or detrimental processes on plant development occur (Kennedy & de Luna, 2005). These processes include the uptake of nutrients, inhibit the growth of rival plants, and initiate chemotaxis in other organisms (Zhang et al., 2010). The plants achieve this by the secretion of chemicals out of their roots. The root exudate consists of a diverse variety of compounds, including primary and secondary metabolites (Koo et al., 2005). The configuration of the exudate can affect the composition of the soil organisms (Bais et al., 2006). A subgroup of the compounds exuded perform rhizosphere signalling functions, including strigolactones (Waters, Gutjahr, Bennet & Nelson, 2017).
The first identified strigolactone strigol could induce germination of the root parasitic Striga, and was therefore dubbed strigol (Cook et al., 1966). Strigol was found to be related to a family of structurally similar compounds, subsequently labelled strigolactones (Al-Babili & Bouwmeester, 2015). Aside from inducing Striga germination, the strigolactones were discovered to stimulate hyphal branching in Arbuscular mycorrhizae (AM) fungi (Akiyama, Matsuzaki & Hayashi, 2005). Approximately 80 percent of land plants are in a symbiotic relationship with AM fungi, a relationship that is thought to have played a crucial role in land colonization by plants (Parniske, 2008). AM fungi are obligate biotrophs that form symbiotic relations with plants, aiding in the efficient uptake of phosphorous and nitrogen from the soil in exchange for carbon sources (Smith & Read, 2010). In Arabidopsis thaliana, strigolactones are involved in the root response to low phosphorous conditions,
demonstrating an increase in biosynthesis in nutrient deficiencies (Mayzlish-Gati et al., 2012). Strigolactones have also been identified to act as phytohormones, regulating shoot branching (Gomez-Roldan et al., 2008), as well as impacting root architecture and leaf senescence (Yamada & Umehara, 2015). Currently over 25 different naturally occurring strigolactone analogues have been discovered, divisible in canonical and non-canonical groups (Wang & Bouwmeester, 2018). The strigol-like and orobranchol-like strigolactone classes contain a tricyclic ABC lactone attached to a D-ring and make up the canonical sesquiterpene strigolactones (Ueno et al., 2011; Xie et al., 2013). The naturally occurring non-canonical strigolactones, such as zealactones, contain components structurally similar to the canonical strigolactones (Charnikhova et al., 2017). The non-canonical strigolactones do not contain the tricyclic ABC lactone but the D-ring is highly conserved (Jia et al., 2017) The biosynthesis of strigolactones has increasingly become elucidated, which is derived from the carotenoid pathway (Matusova et al.,2005). Subsequent reactions, taking place in the plastid, of all-trans-ß-Carotene by the isomerase D27 and the carotenoid cleavage dioxygenases CCD7 and CCD8, form carlactone, a central strigolactone precursor (Alder et al, 2012; Bruno et al., 2014). Carlactone is transported out of the plastid in the cytosol, where enzyme homologues of MAX1 catalyse the formation of carlactonoic acid (Zhang et al., 2014). The myriad of functions exhibited by strigolactones make it a prime target for efforts in synthetic biology aiming to alter plant function for agricultural or industrial applications (Wurtzel, 2018). New plant mutants are designed with altered rhizospheric activity found in different strigolactone analogues (Boyer et al., 2014). A commonly used synthetic strigolactone is GR24 (Besserer et al., 2008). Striga species depend on the detection of allelopathic strigolactones by the seeds to identify hosts. An increase in strigolactone secretion has been shown to aggravate Striga infestation (Jamil et al., 2012). Strigolactones are primarily synthesised in plant roots (Al-Babili & Bouwmeester, 2015). The ABC transporter PhPDR1 carries strigolactones over membranes (Kretschmar et al., 2012). A change in the transport of strigolactones can influence the root colonization of AM fungi, affecting plant yield (Borghi et al., 2016; Liu et al., 2018). Striga plants are obligate parasites depending on root colonisation for survival (Xie, Yoneyama & Yoneyama, 2010). Since strigolactones have both beneficial and deleterious allelopathic properties aside from phytohormonal functions, host plant lines are under stress to evolve strigolactones in manners that maintain beneficial properties, such as inducing fungal branching, but decrease detection of parasites. The ensuing arms race with parasitic plants is put forward as a likely explanation for the presence of the abundant analogues (Al-Babili & Bouwmeester, 2015; Xie, Yoneyama & Yoneyama, 2010). Structural variation among the strigolactone profile in the exudate has been demonstrated to alter Striga seed germination activity (Yoneyama et al., 2015; Mohemed et al., 2018). Maize lines excrete multiple allelopathic chemicals, mainly strigolactones (Kato-Noguchi et al., 2000; Awad et al., 2006) The strigolactone profile in the exudate differs among several maize cultivars, displaying distinct allelopathic capabilities in parasitic seeds (Ye, Zhang, Zhang & Ma, 2020).
Multiple classical strigolactones make up the strigolactone profile in maize, such as sorgomol and 5-deoxystrigol (Awad et al., 2006; Yoneyama et al., 2005). Seven putative strigolactone compounds have been uncovered in maize so far and using LC-MS- and NMR spectrometry the structures of two isomers were characterized which were named as zealactones (Charnikhova et al., 2017; Xie et al., 2017). The structure of an additional strigolactone was elucidated, and named zeapyranolactones (Charnikhova, 2018). However, the structure of 4 other putative strigolactone compounds remains concealed. These compounds, coined compound 3-6 show varying activity in Striga seed germination when tested at concentration ranging from 0.3µM to 30µM. Compounds 4 and 5 did not induce any Striga seed germination at these concentrations, possibly due to their instability (Charnikova et al., 2017). Our colleagues’ work contributed data that describes a relationship between a relative upregulation of compound 5 compared with zealactone content and Striga-resistance for some maize lines (unpublished results). The allelopathic capabilities of several maize lines to Striga was studied and displayed a low seed germination inducing line as shown in Table 1 (unpublished results).
The aim of this study therefor is to initially detect the strigolactone profiles in maize, specifically the ratio of zealactone to Compound 5 between several maize lines. While strigolactones induce germination at very low concentrations they occur in small concentrations and may be unstable, making it harder measure them accurately even using high performance LC-MS system (Besserer at all., 2006; Boyer et al.,2012). Considering nutrient deficiencies raise the biosynthesis and secretion of strigolactones (Marzec, Muszynska & Gruszka,
2013; Ravazollo et al., 2019), the effects of phosphorous and nitrous starvation on the exudate will be measured.
Table 1. Genetic background of several maize lines with inducement of Striga seed germination measurements included.
Entry/Line Genetic background % Striga germination
4 Line derived from a cross of a susceptible line with a line that contains Z.diploperennis in its genome
High
12 Line derived from a Striga resistant composite High
15 Line derived from a Striga resistant synthetic Low <10%
19 Line derived from a backcross containing
Z.diploperennis as a source of Striga resistance
High
25 Line derived from a tropical population cross High
6 Line derived from a tropical population cross High
Materials & Methods
Plant growth and root exudate collectionThe Maize lines utilised were acquisitioned from collaborators. The lines used were maize 4, maize 6, maize 12, maize 15, maize 19 and maize 25. For the effect of phosphate and nitrogen starvation maize hybrid cv NK Falkone was used.
Materials
the Quantities in parenthesis represent quantity per single sample) 1. seed material
2. Seed disinfection solutions: 4% sodium hypochlorite with 0.2% Tween-20 (10 ml), 70% Ethanol (30 ml) 3. 15 ml falcon tubes (1x)
4. Rotating mixer
5. Sterile MilliQ water (80 ml) 6. 90mm petri dishes 7. Tweezers
8. Autoclaved 90 mm Whatman® glass microfiber filters, GF/A Grade / Qualitative filter paper 9. Parafilm
10. Aluminium foil 11. Incubator set to 30°C 12. Sand (45 ml)
13. 50 ml falcon tubes (3x)
14. Greenhouse compartment set to 28°C/25°C, 70% relative humidity, 16H photoperiod, 450 μmol. m-2. s-1
15. Modified Hoagland solution
16. Exudate collection solution: 5% ethanol–water (v/v) (30 ml) 17. Crushed-ice bath
18. Centrifuge
Protocol
1. Place the desired number of seeds in a 15ml falcon tube
2. Add 10 ml of 4% sodium hypochlorite with 0.2% Tween-20 and shake the tube for 45 min on a rotating mixer
3. After removing the bleach, incubate the seeds for 30 seconds in 10 ml of 70% Ethanol and wash with 10 ml of sterile MilliQ water (repeat 3 times this step)
4. Wash the seeds an additional 4 times with 10 ml of sterile MilliQ water
5. Moisten glass fibre filter papers with 3 ml sterile MilliQ water, then place 10-15 seeds on each filter paper 6. Seal the plates with parafilm and wrap them with aluminium, then incubate them for 2 days at 30°C. 7. Select the best seedlings and transfer them to 50 mL falcon tubes filled with 45 mL of sand
8. Move the planted tubes to the greenhouse, then select the better growing plants after 2 days (note replicates)
9. Water the plants every 2 days with 3-4 ml of half strength Hoagland solution 10. Grow the plants for 2 weeks
11. Elute the exudates with 30-40 ml modified half strength Hoagland solution (without phosphate or without nitrogen)to remove the existed compounds
12. Water the plants every 2 days with 3-4 ml of modified half strength Hoagland solution 13. Elute the exudates with 30-40 ml of 5% ethanol–water and collect 20 ml
Strigolactone collection from root exudates Protocol
Collection by Manifold with C18 fast column (500 mg/3 mL)
1. first of all, label the glass vials properly (sample name, plant, treatment, rep, own name, date, etc) 2. label the C18 column and put them on manifold
3. add 3 mL methanol in each column and allow it to drain (for pre-conditioning) 4. add 3 mL demi water/MQ water and allow it to drain
5. make pipe connection with manifold, big bottle for waste collection 6. start to add clean root exudates in each column
7. start vacuum pump (mild pressure)
8. finish the root exudates by running from respective column
9. remove any water until dryness with extra pumping, wash the column with 3-6 mL MQ water 10. clean and dry the manifold and put glass vials (10 or 4 mL) under each column with labels 11. add 2.5 mL 100% acetone in each column and elute strigolactones in above mentioned glass vials 3. evaporate acetone from sample up to dryness in speed vacuum/nitrogen (no water at all)
4. dissolve with 200 uL 25% acetonitrile, vortex
12. filter with ministart syringe filter in LC/MS analysis vials Data Analysis
The UPLC-MS-MS (MRM) chromatogram output channels utilised, as routinely used in our lab for strigolactone analysis (Kohlen et al., 2012), were: zealactone_377.2 > 97, zeapyranolactone_377.2 > 97, Compound3_393.2 > 96.8, Compound4_375.2 > 96.96, Compound6_361.2 > 96.96, Compound5_331.2 > 97 and Side_281 > 231. The channels for strigolactones Zealactones and Zeapyranolactones, as well as putative strigolactones Compound 3-6 were chosen for the present of a fragment of m/z 97 Da. This fragment is indicative for the presence of the D-ring, as is commonly found and highly conserved in strigolactones. Putative compound Side_281 fragmented into m/z 231 Da and m/z 249 Da pieces, of which the m/z 231 Da piece was analysed.
The degree of difference between the peak area and line, and between the ratio and maize line was computed using a One-way analysis of variance tests (ANOVA). The homogeneity of variance was checked with the Levenes test of equality of variance, and a Tukey’s range test was performed as posthoc test.
The data analysis and figure generation were performed using R software version 3.4.2, packages sciplot and car in app R-studio version 1.3.959.
Results
The UPLC-MS-MS(MRM) chromatograms displayed a variance in peak area measured in the channels associated with the smaller strigolactone subunits , m/z ≈ 97. The peak area measurements per line are depicted for the different strigolactones as mean value with standard error bars included(fig. 1A-G). Significant differences between the mean area per line were established, for the main strigolactone isomer depicted in figure 1A, p = 0.0053. The results from a Tukey’s range test are depicted in Table 2, displaying p-values for differences between the lines. The amount of Zealactone exuded by maize line 15 was significantly less than the Zealactone exudation for maize lines 12 and 25. The area measurements displayed a similar exudation pattern amongst the different strigolactone isomers except for compound 5, where line 15 displayed a deviating increase. To investigate the similarity in the isomeric exudation patterns found, The mean measurements per line were averaged per channel and checked for significant differences. A one-way analysis of variance (ANOVA) established significant differences between the maize lines for the average area measurements of the channels combined with p = 0.0275 (fig. 1H).
Figure 1. Bar graphs displaying the mean area measurements of UPLC-MS-MS(MRM) chromatograms for maize lines 12, 15, 19, 25, 4 and 6 for putative strigolactones A) Zealactone_377.2 > 97, P-value = 0.0053, B) Zeapyranolactone_377.2 > 97, C) Compound3_393.2 > 96.8, D) Compound4_375.2 > 96.96, E) Compound5_331.2 > 97, F) Compound6_361.2 > 96.96, G) Side_281 > 231 and H) combined, P-value = 0.0275. Bars represent the mean of replicates ± SE.
Table 2. P-values of differences between the mean peak area per maize line. Differences found with posthoc Tukey’s range test of a one-way ANOVA, differences are marked with a *, ** or *** * when a significance level of p < 0.05, p < 0.01 or p < 0.001 is found respectively.
Line Maize 12 Maize 15 Maize 19 Maize 25 Maize 4 Maize 6
Maize 12 x * Maize 15 0.02036508 x * Maize 19 0.09393226 0.99998852 x Maize 25 0.99244794 0.02673838 0.07854292 X Maize 4 0.40991716 0.99997116 0.99984607 0.29751169 x Maize 6 0.74365490 0.27957287 0.56707701 0.55603774 0.51407224 x
Line effect on the ratio compound5 to other strigolactones.
The ratio of compound 5 to zealactone and other strigolactone isomers is calculated by dividing the peak area measurements for compound 5 by the peak area measurements for the alternative strigolactones. The mean ratio of compound 5 to zealactone per line is illustrated in figure 2A, with significant differences found between the lines, p-value = 5.74e-13. Significant differences between the ratios per line were also found for the ratio of compound 5 to the mean of the ratios from the other strigolactones combined, which is illustrated in figure 2B, p=1.46e-10. The p-values for the differences of the ratio of compound 5 to Zealactone differences between the lines are shown in Table 3. The p-values for the differences of the ratio compound 5 to the other strigolactones combined are shown in Table 4. Maize line 15 displays relatively the highest compound 5 to zealactone ratio, as well as the highest ratio of compound 5 to the average of the other strigolactone isomers combined.
Figure 2. Bar graphs displaying per maize line the mean ratio of compound 5 to A) zealactone and B) the average of all channels except compound5_331.2 > 97 combined. A One-way ANOVA found significant differences of p = 5.74e-13 for (A) and p = 1.46e-10 for (B). Bars represent the mean of replicates ± SE.
Table 3. P-values of differences between the ratio of Compound 5 to Zealactone per line. Differences found with posthoc Tukey’s range test of a one-way ANOVA, differences are marked with a *, ** or *** * when a significance level of p < 0.05, p < 0.01 or p < 0.001 is found respectively.
Line Maize 12 Maize 15 Maize 19 Maize 25 Maize 4 Maize 6
Maize 12 x *** *
Maize 15 3.835154e-12 x *** *** ** ***
Maize 19 4.023052e-01 2.576828e-12 x *** ** ***
Maize 25 2.137489e-03 3.344357e-06 9.068473e-05 X
Maize 4 8.481832e-02 1.284526e-03 8.815941e-03 9.999997e-01 x
Maize 6 1.782516e-02 5.222758e-10 3.920417e-04 5.443597e-01 9.064848e-01 x
Table 4. P-values of differences between the ratio of Compound 5 to the mean of other strigolactones combined per line. Differences found with posthoc Tukey’s range test of a one-way ANOVA, differences are marked with a *, ** or *** * when a significance level of p < 0.05, p < 0.01 or p < 0.001 is found respectively.
Line Maize 12 Maize 15 Maize 19 Maize 25 Maize 4 Maize 6
Maize 12 x ***
Maize 15 2.962557e-10 x * *** ***
Maize 19 9.999976e-01 6.640428e-07 x
Maize 25 5.370250e-01 8.572089e-04 7.396533e-01 x
Maize 4 8.673250e-01 3.409314e-01 9.096740e-01 9.999639e-01 x
Maize 6 1.013444e-01 4.410651e-05 3.441061e-01 9.990324e-01 1.000000e+0 0
x
Nutrient effect on peak area
The effect of nutrient treatment on peak area was examined by grouping samples of NK Falcone seedlings into a control, a nitrogen deficiency and a phosphorous deficiency treatment set. The peak areas measured by the UPLC-MS-MS(MRM) of the NK Falcone maize line did not display significant differences per treatment group for the Zealactone measurements p = 0.0843, nor for the combined channel measurements in a one-way analysis of variance(ANOVA) , p = 0.385. Bar charts of the mean areas per treatment group for the putative strigolactones separate and combined are depicted in figure 3A-H, with standard error included. Whilst no significant difference was found, p = 0.141, a trend is noticeable that depicts a decrease in the areas measured for the nitrogen deficiency treatment group in compound 5 (Fig, 3E).
Nutrient effect on the ratio compound5 to other strigolactones
Significant differences in the ratio of Compound 5 to Zealactone and Compound 5 to the average of the channels except Compound5_331.2 > 97 combined were found, p = 5.01e-06 and p = 0.0112 respectively. The nitrogen deficiency treatment group had a significantly lower compound 5 to zealactone ratio than the control or phosphorous deficiency treatment as shown in Table 5. The nitrogen deficiency treatment group was found to have a significantly lower ratio of compound 5 to the other strigolactones combined than the control or phosphorous deficiency treatment as well.
Figure 3. Bar Graphs displaying the mean area measurements of UPLC-MS-MS(MRM) chromatograms for control, nitrogen deficiency and phosphorous deficiency treatment groups of maize line NK Falcone. Measurements of strigolactones A) Zealactone_377.2 > 97, p = 0.0843 B) Zeapyranolactone_377.2 > 97, C) Compound3_393.2 > 96.8, D) Compound4_375.2 > 96.96, E) Compound5_331.2 > 97, F) Compound6_361.2 > 96.96, G) Side_281 > 231 and H) combined, p = 0.385. Bars represent mean of replicates ±SE.
Figure 4. Bar graphs displaying for control, nitrogen deficiency and phosphorous deficiency treatment groups the mean ratio of compound 5 to A) zealactone and B) the average of all channels except compound5_331.2 > 97 combined. A One-way ANOVA found significant differences between ratios per treatment of p = 5.01e-06 for (A) and p = 0.0112 for (B). Bars represent mean of replicates ± SE.
Table 5. P-values of differences between the ratio of Compound 5 to Zealactone and of Compound 5 to the mean of other strigolactones combined per treatment group. Differences found with posthoc Tukey’s range test of a one-way ANOVA, differences are marked with a *, ** or *** * when a significance level of p < 0.05, p < 0.01 or p < 0.001 is found respectively.
Treatment group comparison Ratio compound 5 to Zealactone Ratio compound 5 to Combined Control – Nitrogen deficiency 3.056253e-5 *** 0.03302228 *
Control – Phosphorous deficiency 9.908700e-01 0.99762429
N deficiency – P deficiency 1.460804e-05 *** 0.02090983 *
Discussion
Significant differences between the lines were found on the area of the strigolactones. This indicates that the amount of strigolactones exuded differs per maize line. Significant differences were also found in the ratio of compound 5 to zealactone as well as the ratio of compound 5 to the other strigolactones combined between the lines. This indicates that the share of compound 5 in the strigolactone profile differs per line. Maize line 15 in particular displayed a large increase in the fraction size of compound 5 in the exudation profile compared to other maize lines. The Striga seed germination data provided by collaborators in table 1 show low allelopathic capabilities of maize line 15 compared to other maize lines. These insights combined could indicate a relationship between the fraction of compound 5 in the strigolactone exudation profile and the Striga seed germination capabilities of maize.
The effect of nutrient deficiencies on the strigolactone profile was examined by comparing the amount of strigolactones and the ratio of compound 5 to zealactone and other strigolactones combined within treatment groups of maize line NK Falcone samples. No significant difference in the area of strigolactones exuded was found between the treatment groups contrary to findings in previous research by Marzec, Muszynska & Gruszka, 2013, and Ravazollo et al., 2019. The experimental setup in this study utilised falcon tubes for hydroponic growth, and in supplying the varying Hoagland solutions alternated by water, small amounts of fluids were used as not to flush out the exudate. This might have put the samples from all treatment groups under similar stress which would negate exudate volume differences in the treatment groups. Significant differences between the ratios of compound 5 to zealactones and other strigolactones combined were found however, and indicate a decrease in exudation of compound 5 in response to nitrogen deficiency. This effect was not illustrated in the exudate of samples experiencing phosphorous deficiency or the control group. This implies a specific response of strigolactone profile alterations to nitrogen starvation instead of a general nutrient starvation.
This study set out to investigate the root exudation profile of strigolactones in multiple maize lines, and whether nutrient deficiencies affect these profiles. Since it is postulated that selection pressure by Striga parasitism
affects the differentiation of strigolactones (Al-Babili & Bouwmeester, 2015), It is imaginable that a decrease in allelopathic capabilities of the exuded strigolactones allows for an increase in overall exudation of strigolactones. Inquiries into the relationship between the amount of strigolactones exuded the ratio of compound 5 to other strigolactone isomers and Zealactone in particular could be made in further research. That research might indicate that the relationship between the line and area depends on the ratio of compound 5 to other strigolactones, as well as to zealactone in particular with a two-way analysis of variance(ANOVA). The patterns and ratio data for the line subset suggest a positive relationship, however for the nutrient effect the opposite seems likely. This would indicate an increase in zealactones exuded during stress but an overall increase of isomers during standard conditions.
Future research into this interaction therefore is warranted for the biological implication it may exemplify. The processes which lead to the altered strigolactone amounts and profile measured call for increased clarity. While the differences in strigolactone profiles amongst the maize lines may find their root in transcriptional, translational, proteasomal or epigenetic differences, the reoccurring exudation pattern of the different strigolactones except for compound 5 suggest a variation in downstream deviations such as enzyme cleavage prevalence. This connection might be explored by suppressing or enhancing the enzymatic capabilities of maize Strigolacone genes such as MAX1 which is involved in differentiation of non-canonical strigolactones(Jia et al., 2019). This study provides insights into the strigolactone profile of maize which combined with seed germination data adds to the understanding of underlying principles of plant resistance to parasites. This could in turn facilitate new advances of crop breeding in order to increase efficiency of crop production, potentially affecting global food supplies.
References
1. Akiyama, K., Matsuzaki, K., & Hayashi, H. (2005). Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. Nature, 435(7043), 824–827. https://doi.org/10.1038/nature03608
2. Al-Babili, S., & Bouwmeester, H. J. (2015). Strigolactones, a novel carotenoid-derived plant hormone. Annual review of plant biology, 66, 161-186.
3. Alder, A., Jamil, M., Marzorati, M., Bruno, M., Vermathen, M., Bigler, P., ... & Al-Babili, S. (2012). The path from β-carotene to carlactone, a strigolactone-like plant hormone. Science, 335(6074), 1348-1351.
4. Awad, A. A., Sato, D., Kusumoto, D., Kamioka, H., Takeuchi, Y., & Yoneyama, K. (2006). Characterization of strigolactones, germination stimulants for the root parasitic plants Striga and Orobanche, produced by maize, millet and sorghum. Plant Growth Regulation, 48(3), 221.
5. Bais, H. P., Weir, T. L., Perry, L. G., Gilroy, S., & Vivanco, J. M. (2006). THE ROLE OF ROOT EXUDATES IN RHIZOSPHERE INTERACTIONS WITH PLANTS AND OTHER ORGANISMS. Annual Review of Plant Biology, 57(1), 233–266. https://doi.org/10.1146/annurev.arplant.57.032905.105159
6. Bender, S. F., & van der Heijden, M. G. A. (2014). Soil biota enhance agricultural sustainability by improving crop yield, nutrient uptake and reducing nitrogen leaching losses. Journal of Applied Ecology, 52(1), 228–239. https://doi.org/10.1111/1365-2664.12351
7. Besserer, A., Bécard, G., Jauneau, A., Roux, C., & Séjalon-Delmas, N. (2008). GR24, a Synthetic Analog of Strigolactones, Stimulates the Mitosis and Growth of the Arbuscular Mycorrhizal Fungus Gigaspora rosea by Boosting Its Energy Metabolism. Plant Physiology, 148(1), 402–413.
https://doi.org/10.1104/pp.108.121400
8. Besserer, A., Puech-Pagès, V., Kiefer, P., Gomez-Roldan, V., Jauneau, A., Roy, S., … Séjalon-Delmas, N. (2006). Strigolactones Stimulate Arbuscular Mycorrhizal Fungi by Activating Mitochondria. PLoS Biology, 4(7), e226. https://doi.org/10.1371/journal.pbio.0040226
9. Borghi, L., Liu, G.-W., Emonet, A., Kretzschmar, T., & Martinoia, E. (2016). The importance of
strigolactone transport regulation for symbiotic signaling and shoot branching. Planta, 243(6), 1351– 1360. https://doi.org/10.1007/s00425-016-2503-9
10. Boyer, F.-D., de Saint Germain, A., Pouvreau, J.-B., Clavé, G., Pillot, J.-P., Roux, A., … Rameau, C. (2014). New Strigolactone Analogs as Plant Hormones with Low Activities in the Rhizosphere. Molecular Plant, 7(4), 675–690. https://doi.org/10.1093/mp/sst163
11. Bruno, M., Hofmann, M., Vermathen, M., Alder, A., Beyer, P., & Al-Babili, S. (2014). On the substrate-and stereospecificity of the plant carotenoid cleavage dioxygenase 7. FEBS letters, 588(9), 1802-1807.
12. Charnikhova, T. V., Gaus, K., Lumbroso, A., Sanders, M., Vincken, J. P., De Mesmaeker, A., ... &
Bouwmeester, H. J. (2017). Zealactones. Novel natural strigolactones from maize. Phytochemistry, 137, 123-131.
13. Cook, C. E., Whichard, L. P., Turner, B., Wall, M. E., & Egley, G. H. (1966). Germination of witchweed (Striga lutea Lour.): isolation and properties of a potent stimulant. Science, 154(3753), 1189-1190.
14. Ejeta, G. (2007). THE STRIGA SCOURGE IN AFRICA: A GROWING PANDEMIC. Integrating New Technologies for Striga Control, 3–16. https://doi.org/10.1142/9789812771506_0001
15. Isolation and identification of allelochemicals in maize seedlings. Plant Prod. Sci. 2000, 3, 43–46
16. Jamil, M., Kanampiu, F. K., Karaya, H., Charnikhova, T., & Bouwmeester, H. J. (2012). Striga hermonthica parasitism in maize in response to N and P fertilisers. Field Crops Research, 134, 1-10.
17. Jia, K. P., Baz, L., & Al-Babili, S. (2018). From carotenoids to strigolactones. Journal of experimental botany, 69(9), 2189-2204.
18. John Fox and Sanford Weisberg (2011). An {R} Companion to Applied Regression, Second Edition. Thousand Oaks CA: Sage. URL: http://socserv.socsci.mcmaster.ca/jfox/Books/Companion
19. Kato-Noguchi, H.; Sakata, Y.; Takenokuchi, K.; Kosemura, S.; Yamamura, S. Allelopathy in Maize I.:
20. Kennedy, A. C., & de Luna, L. Z. (2005). RHIZOSPHERE. Encyclopedia of Soils in the Environment, 399– 406. https://doi.org/10.1016/b0-12-348530-4/00163-6
21. Kim, S. K., Adetimirin, V. O., Thé, C., & Dossou, R. (2002). Yield losses in maize due to Striga
hermonthica in West and Central Africa. International Journal of Pest Management, 48(3), 211–217. https://doi.org/10.1080/09670870110117408
22. Kohlen, W., Charnikhova, T., Lammers, M., Pollina, T., Tóth, P., Haider, I., ... & López Ráez, J. A. (2012). ‐ The tomato CAROTENOID CLEAVAGE DIOXYGENASE 8 (S l CCD 8) regulates rhizosphere signaling, plant architecture and affects reproductive development through strigolactone biosynthesis. New
Phytologist, 196(2), 535-547.
23. Koo, B.-. J., Adriano, D. C., Bolan, N. S., & Barton, C. D. (2005). ROOT EXUDATES AND
MICROORGANISMS. Encyclopedia of Soils in the Environment, 421–428. https://doi.org/10.1016/b0-12-348530-4/00461-6
24. Kretzschmar, T., Kohlen, W., Sasse, J. et al. A petunia ABC protein controls strigolactone-dependent symbiotic signalling and branching. Nature 483, 341–344 (2012). https://doi.org/10.1038/nature10873
25. Lagoke, S. T. O., Parkinson, V., & Agunbiade, R. M. (1991). Parasitic weeds and control methods in Africa. Combating Striga in Africa, 3-14.
26. M’BOOB, S. S., 1989. A regional programme for Striga control in West and Central Africa. In T. O. Robson and H. R. Broad (eds), Striga Ð Improved Management in Africa. Proceedings of the FAO/OAU Government Consultation on Striga Control, 20 ± 24 October 1986 (Rome: Food and Agricultural Organization of the United Nations), pp. 190 ± 194.
27. Manuel Morales, with code developed by the R Development Core Team, with general advice from the R-help listserv community and especially Duncan Murdoch. (2020). sciplot: Scientific Graphing
Functions for Factorial Designs. R package version 1.2-0. https://CRAN.R-project.org/package=sciplot
28. Marzec, M., Muszynska, A., & Gruszka, D. (2013). The Role of Strigolactones in Nutrient-Stress Responses in Plants. International Journal of Molecular Sciences, 14(5), 9286–9304.
https://doi.org/10.3390/ijms14059286
29. Matusova, R., Rani, K., Verstappen, F. W., Franssen, M. C., Beale, M. H., & Bouwmeester, H. J. (2005). The strigolactone germination stimulants of the plant-parasitic Striga and Orobanche spp. are derived from the carotenoid pathway. Plant physiology, 139(2), 920-934.
30. Mayzlish-Gati, E., De-Cuyper, C., Goormachtig, S., Beeckman, T., Vuylsteke, M., Brewer, P. B., … Koltai, H. (2012). Strigolactones Are Involved in Root Response to Low Phosphate Conditions in Arabidopsis. Plant Physiology, 160(3), 1329–1341. https://doi.org/10.1104/pp.112.202358
31. Mohemed, N., Charnikhova, T., Fradin, E. F., Rienstra, J., Babiker, A. G. T., & Bouwmeester, H. J. (2018). Genetic variation in Sorghum bicolor strigolactones and their role in resistance against Striga
hermonthica. Journal of Experimental Botany, 69(9), 2415–2430. https://doi.org/10.1093/jxb/ery041
32. Parniske, M. (2008). Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nature Reviews Microbiology, 6(10), 763-775.
33. R Core Team (2017). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/.
34. Ravazzolo, L., Trevisan, S., Manoli, A., Boutet-Mercey, S., Perreau, F., & Quaggiotti, S. (2019). The Control of Zealactone Biosynthesis and Exudation is Involved in the Response to Nitrogen in Maize Root. Plant and Cell Physiology, 60(9), 2100–2112. https://doi.org/10.1093/pcp/pcz108
35. RStudio Team (2018). RStudio: Integrated Development for R. RStudio, Inc., Boston, MA URL http://www.rstudio.com/.
36. RStudio Team (2020). RStudio: Integrated Development Environment for R. RStudio, PBC, Boston, MA URL http://www.rstudio.com/.
37. Smith, S. E., & Read, D. J. (2010). Mycorrhizal symbiosis. Academic press.
38. Ueno, K., Nomura, S., Muranaka, S., Mizutani, M., Takikawa, H., & Sugimoto, Y. (2011). Ent-2 -epi-′ orobanchol and its acetate, as germination stimulants for Striga gesnerioides seeds isolated from cowpea and red clover. Journal of agricultural and food chemistry, 59(19), 10485-10490.
39. Wang, Y., & Bouwmeester, H. J. (2018). Structural diversity in the strigolactones. Journal of experimental botany, 69(9), 2219-2230.
40. Waters, M. T., Gutjahr, C., Bennett, T., & Nelson, D. C. (2017). Strigolactone Signaling and Evolution. Annual Review of Plant Biology, 68(1), 291–322. https://doi.org/10.1146/annurev-arplant-042916-040925
41. Wurtzel, E. T. (2018). Changing Form and Function through Carotenoids and Synthetic Biology. Plant Physiology, 179(3), 830–843. https://doi.org/10.1104/pp.18.01122
42. Xie, X., Kisugi, T., Yoneyama, K., Nomura, T., Akiyama, K., Uchida, K., … Yoneyama, K. (2017). Methyl zealactonoate, a novel germination stimulant for root parasitic weeds produced by maize. Journal of Pesticide Science, 42(2), 58–61. https://doi.org/10.1584/jpestics.d16-103
43. Xie, X., Yoneyama, K., & Yoneyama, K. (2010). The strigolactone story. Annual review of phytopathology, 48, 93-117.
44. Xie, X., Yoneyama, K., Kisugi, T., Uchida, K., Ito, S., Akiyama, K., ... & Yoneyama, K. (2013). Confirming stereochemical structures of strigolactones produced by rice and tobacco. Molecular plant, 6(1), 153-163.
45. Yamada, Y., & Umehara, M. (2015). Possible Roles of Strigolactones during Leaf Senescence. Plants, 4(3), 664–677. https://doi.org/10.3390/plants4030664
46. Ye, X., Zhang, M., Zhang, M., & Ma, Y. (2020). Assessing the Performance of Maize (Zea mays L.) as Trap Crops for the Management of Sunflower Broomrape (Orobanche cumana Wallr.). Agronomy, 10(1), 100. https://doi.org/10.3390/agronomy10010100
47. Yoneyama, K., Arakawa, R., Ishimoto, K., Kim, H. I., Kisugi, T., Xie, X., … Yoneyama, K. (2015). Difference inStriga-susceptibility is reflected in strigolactone secretion profile, but not in compatibility and host preference in arbuscular mycorrhizal symbiosis in two maize cultivars. New Phytologist, 206(3), 983– 989. https://doi.org/10.1111/nph.13375
48. Zhang, F., Shen, J., Zhang, J., Zuo, Y., Li, L., & Chen, X. (2010). Rhizosphere Processes and Management for Improving Nutrient Use Efficiency and Crop Productivity. Advances in Agronomy, 1–32.
https://doi.org/10.1016/s0065-2113(10)07001-x
49.Zhang, Y., van Dijk, A., Scaffidi, A. et al. Rice cytochrome P450 MAX1 homologs catalyze distinct steps in strigolactone biosynthesis. Nat Chem Biol 10, 1028–1033 (2014).
