The Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, International Atomic Energy Agency, Department of Nuclear Sciences and Applications, Plant Breeding and Genetics programme has supported the country's national crop improvement program helping to apply plant mutation breeding techniques to develop new cereal mutant lines since 1998. A Coordinated Research Programme CRP under close collaboration with the International Atomic Energy Agency (IAEA) was established in 2016 to support the *Striga* management in the country in order to develop new rice and maize mutant lines with enhanced *Striga* resistance. To realize gains from mutation breeding to cereal productivity in Madagascar, it is important to develop efficient methods for screening maize and rice after mutagenesis for both continued adaptation to local culture and select for any gained *Striga* resistance. The main objective of this document is to present established robust and efficient field and glasshouse screening protocols for identification of rice and maize mutants with resistance/tolerance to *Striga asiatica* under artificial *Striga* infestation.

#### **Protocols**

*Collecting* Striga asiatica *seeds.* In order to conduct field and pot experiments using artificial infestation sufficient to differentiate resistant maize and rice plants from susceptible ones, sufficient quantities of good quality *S. asiatica* seeds are needed. They must therefore be collected at the proper time and place and stored in a way that preserves their germinability. The life cycle of *S. asiatica* is similar to *S. hermonthica*, but the plants are smaller and flowers are scarlet rather than purple (Fig. 1). Seeds are best harvested from an area near where the screening takes place.  Seed is mature when the capsules turn brown. Bundles of stems should be collected in tight plastic mesh bags just after most

**Fig. 1** *Striga asiatica* plant development: **a** vegetative stage, **b** flowering stage, **c** post-flowering stage with green capsules, **d** mature stage where capsules are dried and seed is fully mature

capsules have turned brown (Fig. 2). In Middle West Madagascar where upland rice and maize are grown, this period generally occurs in late June. The bags are ideally hung in an open shelter with a roof and plenty of air circulation so that they can quickly dry. They should be kept in these bags, protected from excessive humidity and temperature until they are fully dry. Like *S. hermonthica*, seeds of *S. asiatica*  have a dormancy period such that they will not germinate in the season in which they were produced. Therefore, *Striga* seed should be collected at least one year previous to their intended use.

Once the harvested *Striga* stems are fully dry, they may be removed from the mesh bag and threshed over a pan by rolling them between the palms of your hands or by picking the capsules from the dried stems (Fig. 3). Any seed left in the bag after removing the stems can be added to the collection pan. The capsules can be gently crushed with a mortar and pestle to dislodge any remaining seeds. The material in the pan is then passed through a series of sieves to separate the seeds from the capsules and other debris. Sieve size should begin at 2 mm, followed by 300 µm then 250 µm and finally 180 µm until only *Striga* seed remains. The seed can then be transferred to a storage container such as the magenta boxes shown in Fig. 3 which are then put into a second container (large enough to fit the container of seed) into the bottom of which activated silica gel desiccant is placed to keep the seed dry during storage for up to several years at room temperature.

*Conditioning and germination test for* Striga asiatica *seeds*. Prior to all experiments, the *Striga* seed batch germination rate should be checked. As with *S. hermonthica*, *S. asiatica* seeds that are at least one year old (after-ripened) need to be surface sterilized under aseptic conditions and allowed to condition for two weeks before testing their germination in the laboratory. Under a laminar flow hood, line the bottoms of three sterile 100 mm petri dish each with two sterile 90 mm Whatman No. 1 filter paper circles. Transfer an aliquot of 50 mg dried *S. asiatica* seed to a 2.5 mL Eppendorf tube and add 1 mL of 70% ethanol. Cap the tube and shake for 1 min. Under a laminar flow hood, remove the alcohol and add 20% (v/v) of household bleach (5.25% NaOCl) in water. Keep the seeds in this solution for 15 min, shaking every 5 min. Rinse the seeds five times with sterile distilled water (ddH2O). Distribute the surface sterilized *Striga* seeds across the surface of the filter papers in the three petri dishes. Add 5 mL of ddH2O to each to soak the filter papers. Seal

**Fig. 2** Mature *Striga asiatica* plants collected and stored in a tight plastic mesh bag

**Fig. 3** Process of threshing and cleaning *Striga asiatica* seeds: **a** Dried *Striga* stems with capsules are removed from the tight plastic mesh bag in which they were collected, **b** seeds are gently threshed by hand over a shallow pan, **c** capsules are gently ground in a mortar and pestle to dislodge seed, **d** threshed material is sieved until only seed remains, **e** seeds are transferred to storage containers **f** the storage containers are placed in a desiccator over silica gel

the plates with parafilm and incubate at 30 °C for 14 days in a dark incubator to condition the *Striga* seed.

After the seeds have conditioned for two weeks, open the plates under a laminar flow hood and add 5 mL of 1 ppm GR24, a synthetic strigolactone that stimulates *Striga* germination. Seal the plates with parafilm. Incubate for 1–2 days at 30 °C in darkness. Count germinated *Striga* seeds under a dissecting microscope. Divide the number germinated by the total number of seeds in a viewing field in multiple fields across the three plates to determine germination rate of that batch of *S. asiatica* seed.

*Preparing an inoculum for pot and field screening.* If the *Striga* seed germination rate determined from the laboratory check of the batch is at least 50%, the batch is suitable for screening purposes in field or greenhouse. Before using it for such, mix it with an equal volume of fine dry sand and return to the desiccator until use (Fig. 4).

*Plant material used and irradiation treatment*. The upland rice varieties chosen for mutagenesis were B22, a line originally from Brazil but widely grown in Madagascar, and F154, a variety developed by FOFIFA, the Malagasy national agricultural research entity. The maize used were Plata and IRAT200, both yellow OPVs (Fig. 5). All chosen varieties are farmer-preferred (yield, growth habit, grain quality), but sensitive to *S. asiatica* and adapted to the Middle West *tanety* where *Striga* is particularly problematic.

**Fig. 4** *Striga asiatica* seed inoculum, 50:50 mix of cleaned *Striga* seeds with fine dry sand

**Fig. 5** Varieties mutagenized by gamma irradiation and derived populations screened in Madagascar. From left to right, yellow maize IRAT200 and PLATA and upland rice F154 and B22

In order to increase genetic variability within these rice and maize genotypes, 1000 seeds of each were gamma irradiated at various doses at the Plant Breeding and Genetics Laboratory of the Joint Food and Agricultural Organization of the United Nations International Atomic Energy Agency (FAO/IAEA) Division of Nuclear Techniques in Food and Agriculture at Seibersdorf, Austria using a 60Cobalt source. (van Harten 1998; FAO/IAEA 2018). Radiosensitivity tests of each variety was done prior to all experiments to determine the appropriate dosages (Chapter "Physical Mutagenesis in Cereal Crops"; van Harten 1998; Razafinirina 2011; Rakotoarisoa et al. 2017; FAO/IAEA 2018). Effective mutagenic but non-lethal doses chosen were 100, 200, 300 Gy for maize Plata and B22 rice varieties, and 100 and 200 Gy for maize IRAT200 and F154 rice varieties. Mutant populations derived from these mutagenized materials were screened and advanced in Madagascar first in the field and subsequently in greenhouse pots.

*Field screening*. Screening of early generations after mutagenesis (M2/M3) was done at the Station of Kianjasoa, Regional Research Center of the Middle West FOFIFA. The station is located 190 km from Antananarivo, the capital of Madagascar, in the district of Tsiroanomandidy of the Bongolava region. Coordinates are: 49°22' 62.1'' East longitude, 19°03' 20.3'' South latitude at 899 m above sea level. Both maize and upland rice production are constrained by *S. asiatica* in this region due to erratic rainfall and poor soils prone to erosion. Organic matter (1.7%), phosphorous (< 2.5 ppm) and nitrogen 0.25%) in the plots were all suboptimal. Screening was performed without nutrient inputs, typical of traditional farming in the region that favors *Striga* infestation (Andriamialiharisoa 2019; Rakotoarisoa et al. 2019). Soil was ploughed in advance by a tractor and hand weeded where necessary just before plots were marked and planting holes dug by hand hoe (Fig. 6).

The experiments were planted at the corresponding regular seasons for maize and rice in the region into 8 cm deep holes spaced 25 cm apart for rice and 70 cm for maize (see details below) into which 0.05 g of the sand/*Striga* inoculum (50:50, v/v,

**Fig. 6** Field preparation, **a** ploughing the land, **b** hand-weeding, **c** setting plots, **d** digging planting holes

approximately 5000 weed seeds) was added to ensure uniform and heavy infestation. This was covered by 3 cm of soil before sowing the cereal. Each crop seed was covered by 3 cm of additional soil (Fig. 7). Five meter row plots were used for sowing M2/ M3 rice seeds and 10 m for the maize seeds per variety, per initial irradiation dose, replicated in three plots for each rice entry and four plots per tested maize variety (Fig. 7). Plots were labeled with the name of the variety and the dose of irradiation. The same plot layout in the same area of the field was used in the subsequent (M3) generation screening with additional infestation to ensure high *Striga* pressure.

*Harvest and maintenance of M2/M3 seeds*. Assuming that the first generation screened in the field was at the M2 generation, certain individuals may be selected based on apparent improved performance (e.g., fewer emerged *Striga*, less host plant damage) over their unmutagenized counterparts (0 Gy). These selected individuals need to be advanced to the M3 for further testing. This requires self-pollination. For rice, no special techniques are needed since self-pollination occurs within each spikelet before anthers emerge. However, to ensure self-pollination, a small bag may be put over the panicle before anthers emerge. This bag should be left on until harvest. In maize, which is a naturally cross-pollinated species, self-pollination requires some effort. As the uppermost ear begins to emerge, usually when tassels have fully emerged and expanded, but before anthers emerge, cover the ear shoot with

**Fig. 7** Field screening of maize and rice for *S. asiatica* resistance: **a** artificial infesting planting holes with *Striga* inoculum, **b** sowing of cereal seeds, **c** labeling of planted plots (photo taken at maturity in maize plots), **d** labeling of the holes sown with rice or maize seeds of M2 or M3 families in the same row

a coin envelope or shoot bag before silks emerge. Leave the shoot covered for several days until silks emerge (usually when the first anthers are visible on the tassel). When dew dries from leaves in the morning, use a larger envelope or pollinating bag to collect pollen (gently bend into the pollinating bag with opening facing up and shake tassel inside the bag to release pollen). Briefly uncover shoot on the same plant and immediately invert bag of collected pollen over the silks. Cover the ear with the pollinating bag and secure by folding bottom edges around the stalk and stapling. Leave bag until harvest.

When selected plants are mature, cut rice panicles or maize ears and dry in paper envelopes within mesh bags labeled with plot number, plant number, variety, original radiation dose. Thresh or shell when harvested panicles or ears are fully dry and store seeds in labeled envelopes in plastic bags in a refrigerator (4–8 °C) until use.

*Details of the screening plot plantings*. Sow up to 20 M3 rice seeds from each selected M2 plant into a single 5 m row following the field layout and inoculation method described above with each planting hole spaced 25 cm apart. Plant one seed per hole. Leave 25 cm between rows such that spacing is 25 × 25 cm between plants. It is convenient to organize the M3 family rows by variety and radiation dose. Do the same for maize, but use 10 m long rows, with holes spacing of 70 cm within row and 70 cm between rows planting 14 M3 seeds from each selected M2. Repeat the same plantings in three replicate plots for rice and four for maize of each M3 family that was advanced from the selected M2 plants. You should therefore have up to 60 M3 plants from each selected M2 mutant and 56 M3 plants from each maize mutant to evaluate. Plant the corresponding control (parent variety-0 Gy) in two rows in the middle of each replicate plot as a check for phenotype comparison. After sowing, label each family row with the variety and original radiation dose as well as the number of the M3 family (Fig. 7).

Aside from clearing weeds before sowing (Fig. 6), hand weeding should be practiced during the crop vegetative stage at 30, 45 then 60 days after sowing (DAS), taking care not to damage *Striga* plants in latter days. A hoe may be shallowly used at the 30-day weeding since *Striga* is unlikely to emerge by then (Fig. 8). During the crop reproductive phase, weeds on plots should no longer be removed. *Striga* emergence counts and host sensitivity parameters (see below) are taken at flowering for both rice and maize. Selection of potential *Striga* resistant/tolerant mutant lineages to advance to further trials is based on comparisons with the unmutagenized (0 Gy) original parent line within each plot. Those lineages with apparently fewer *Striga*  and/or fewer symptoms of *Striga* infection (less leaf firing, larger ears and grain) than the unmutagenized check are tested in subsequent field or pot trials.

*Screening advanced rice mutant lineages in pots*. Once the number of selected *Striga* resistant mutants from field screening is reduced to a manageable number, further screening and verification of resistance/tolerance can proceed in greenhouse pots. This way, more detailed observations are possible including comparisons to the original unmutagenized line with limited impact of environmental influences encountered in field trials. We used a well-vented greenhouse during the normal cropping season to evaluate advanced mutant rice lineages (M4–M6) in both rice varieties (B22 and F154). Temperature remained in the range of 28–38 °C in the

**Fig. 8** Rice plots at **a** vegetative phase, **b** during hand weeding, **c** after weeding, **d** seed set

greenhouse during the pot trials. We did not find pot screening useful for maize as plants did not grow well in the greenhouse. Screening rice for *S. asiatica* resistance can be performed as follows.

In a 10 L plastic pot with drainage holes, put a thin sail (filter) in the bottom of each pot to prevent *Striga* seeds from coming out through the bottom holes (Fig. 9). Fill pots 2/3 to the top with local soil mixed with equal parts thin sand. Artificially infest pots by spreading 0.5 g of the *S. asiatica*/sand (50:50) inoculum described earlier in this chapter on the soil placed in the pot. This should put about 5000 germinable *Striga* seeds in each pot. Cover this *Striga* layer with enough of the same sand/soil mixture used in the bottom to raise the surface 4 cm. Add another *Striga*/sand layer as before and then add another 4 cm of the sand/soil mixture. Plant two rice seeds per pot and cover with an additional 3–4 cm of the sand/soil mixture and water until saturated. Continue watering to saturation two times a week for two weeks to ensure the rice seeds germinate and *Striga* seeds are conditioned. Thereafter, water once weekly until rice plants flower then stop watering to encourage *Striga* infection. Do not add fertilizer to pots, again to encourage *Striga* infection. This harsh selective pressure will help distinguish resistant from susceptible plants. Pull any weeds in pots besides *Striga* at 15, 30 and 45 days after sowing rice. Count the number of emerged *Striga* in each pot at these same intervals as well symptoms of infected rice plants (see below). Compare these measures on selected mutant lineages with those of infested and uninfested pots of unmutagenized counterparts. Arrange pots in a complete randomized design. Pots and soil may be reused in subsequent trials.

**Fig. 9** Pot preparation for glasshouse screening of mutant rice lineages for resistance to *Striga asiatica*

*General guidelines for screening maize and rice mutant lineages for* S. asiatica *resistance and tolerance*. A general assumption in this mutagenesis breeding project is that gained *S. asiatica* resistance will be expressed by fewer emerged *Striga* around mutant maize and rice plants relative to their unmutagenized counterparts (0 Gy). Also, because there is less *Striga* infection around such plants, symptoms in resistant mutants will be less severe than those of *Striga* infected controls. All of the original rice and maize varieties chosen for mutagenesis are particularly sensitive to *Striga*  infection which causes leaf chlorosis and firing (senescence), reduced shoot growth and fertility through reduced pollen production (smaller tassels in maize, smaller panicles in rice with fewer anthers) and reduced grain yield (smaller rice panicles and smaller or deformed maize ears). Since such symptoms, particularly reduced fecundity, can result from gamma irradiation itself, it is important to distinguish heritably reduced sensitivity to *Striga* from general reduced fitness due to mutation in genes controlling normal growth and reproduction. Mutants of the latter type are generally eliminated in early generation screenings. The goal of mutagenesis must be to improve *Striga* resistance and/or tolerance at no cost to overall plant productivity or adaptation to their target environment. Therefore, mutant lineages selected for advancement must show improved performance under *Striga* pressure relative to the original unmutagenized line from which they were derived with equal or better performance to the original line in uninfested conditions. It is therefore important to compare selected mutants with potential gained *Striga* resistance in both *Striga*  infested and non-infested conditions at some point during the screening process in their target environment.

Because all the original rice (B22 and F154) and maize (IRAT200 and Plata) varieties are particularly sensitive and susceptible to *S. asiatica*, with multiple parasites eventually emerging around host plants with fairly severe symptoms (prematurely dying leaves and low yield) under *Striga* infestation, selection for *Striga* resistance/ tolerance began in early generations (M2 and M3). Verification was then done on advanced progeny in the selected lineages (M4 and M5). Under the severe selection pressure described earlier for field screening (*Striga* inoculum added to each planting hole and no fertilizer applied to plots), seeds were only harvested from plants of mutant lineages having fewer emerged *Striga* and less affected leaf area and more grain than unmutagenized counterparts for advancement to subsequent trials (field or pot) to verify their gained resistance and tolerance. In these verification trials, specific phenotypic characters were observed and recorded including the *S. asiatica* plants number emerging above the soil per plant, counted at host flowering, the survival rate per genotype (number of surviving plants divided by the total number of germinated seeds multiplied by 100), the maximum plant height, the number of rice tillers and fertile panicles or ear numbers produced per maize plant, the 100 (for maize) or 1000 (for rice) seed weights, the number of leaves showing premature senescence expressed as a percentage of total number of leaves per plant and for maize, the ear shape. Figures 10 and 11 show examples of observed phenotypes in maize trials and Fig. 12 shows rice examples.

From these detailed observations, advanced mutant lineages can be classified according to their performance under *Striga* infestation. We used modifications of those classes described by others (Kim 1991; Sinebo and Drennan 2001) without clear distinction between resistance and tolerance characters since the latter under

**Fig. 10** Examples of maize performance under *S. asiatica* infestation. **a** Susceptible maize plant with most leaves affected, **b** a highly susceptible maize plant with premature shoot death, **c** a highly resistant maize

**Fig. 11** Various maize ear types observed under *S. asiatica* infestation to classify resistance. **a** Ears from a moderately resistant mutant having small ears with empty rows and kernels arranged in disorder, **b** ear from a resistant mutant with < ¼ affected with slight blackening and curving at tip, **c** ear from a highly resistant mutant, large with full, well-arranged kernels. **d** Multiple ears from an advanced resistant mutant line, IRAT200Gy-L9-2-1-X22, harvested from uninfested plots in yield trials after selection

**Fig. 12** Examples of rice performance in greenhouse pots. **a** original F154 rice line (left) in *Striga*  infested pot, resistant F154 mutant (center) in *Striga* infested pot and original F154 rice line (right) in uninfested pot. **b** From left to right, original B22 rice line, a moderately resistant B22 mutant, a susceptible B22 mutant, a resistant B22 mutant in pots infested with *Striga asiatica.* The fifth pot contains the original B22 without *Striga*. All mutant lineages are at the M6

the high infestation conditions employed was largely a function of the former. Table 1 gives a summary of this classification system.

Resistance among the selected mutant lineages in this mutation breeding exercise may involve genes contributing to both tolerance and resistance to *S. asiatica.* Distinguishing alleles resulting specifically in tolerance would require detailed comparisons of the same mutants grown with and without *Striga* (see Chapter "Screening for Resistance to Striga Hermonthica in Mutagenized Sorghum and Upland Rice in Burkina Faso"). The end result, however, was that selected maize (Table 2) and rice (Table 3) mutants show improved yield under *Striga* infestations over their original varieties. Generally, these mutants appear to be resistant to *S. asiatica*, in that they supported fewer parasites than their unmutagenized counterparts. Several of the M5 selections are being propagated for evaluation in replicated field trials for possible new variety release in Madagascar.

#### **Conclusion**

The protocols described in this chapter were successfully used to select *Striga* resistant mutants from gamma irradiated maize and upland rice varieties adapted to the *tanety* of Middle West Madagascar where *S. asiatica* is a serious production constraint. Introducing *Striga* resistance through mutagenesis in cultivars adapted to these conditions can help to sustain maize and expand rice production in the country, especially if used in combination with conservation agricultural practices that reduce erosion and improve soil fertility in this fragile agro-ecology. Mutation breeding with selection based on the protocols described has resulted in 19 maize varieties in two farmer-preferred backgrounds that are superior to the original varieties in terms of resistance and/or tolerance to the local strain of *S. asiatica*. Similarly, 34 *Striga* resistant/tolerant upland rice varieties in two popular genetic backgrounds were identified and advanced through a combination of field and greenhouse screenings among the progeny of mutagenized seed. Further characterization in both laboratory assays (Chapters "An Agar-Based Method for Determining Mechanisms of Striga Resistance in Sorghum"–"Striga Germination Stimulant Analysis") and multi-location field trials of the selected *S. asiatica* resistant maize and rice varieties continues with much promise for improved cereal production in Madagascar. Similar efforts are described in Chapter "Screening for Resistance to Striga Hermonthica in Mutagenized Sorghum and Upland Rice in Burkina Faso" for sorghum and rice in Burkina Faso and in Chapter "Mutation Breeding for Resistance to Striga Hermonthica in Sorghum and Rice for Sustainable Food Production in Sudan" for sorghum and rice in Sudan.

**Table 1** Adapted method for assessing the resistance of tested maize and rice genotypes under severe *Striga asiatica* infestation

(continued)

**Table 1** (continued)

<sup>a</sup>*h*—highly resistant, *R*—resistant, *S*—susceptible and *HS*—highly susceptible

**Table 2** Total number of maize plants selected and advanced for further testing at each generation after mutagenesis

**Table 3** Total number of rice plants selected and advanced for further testing at each generation after mutagenesis

**Acknowledgements** The authors gratefully acknowledge the financial support and collaboration of the International Atomic Energy Agency in the context of the CRP D25005. We appreciate all experts who have shared their experience and knowledge with us. Many thanks to the FOFIFA leaders, the heads of the station at Kianjasoa, as well as technicians and laborers there for their kind collaboration and to all student researchers who have contributed to this research activity. We acknowledge and appreciate the aid of IAEA officer Mr. Ljupcho Jankuloski for his support during the implementation of the national Technical Cooperation (TC) projects and to TC African for the support during the national TC project.

#### **References**

FAO/IAEA (2018) Manual on mutation breeding, 3rd edn. In: Spencer-Lopes MM, Forster BP, Jankuloski L (eds) Food and agriculture organization of the United Nations. Rome, Italy

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# **An Agar-Based Method for Determining Mechanisms of** *Striga* **Resistance in Sorghum**

**Patrick J. Rich**

**Abstract** This chapter describes an agar-based method for co-culturing sorghum and *Striga* that can be used to determine *Striga* germination stimulant activity, haustorial initiation activity and other pre-attachment and early post-attachment resistance mechanisms in sorghum.

### **Keywords** *Striga* · Sorghum · Bioassay · Mechanisms of resistance · GR24

#### **Introduction**

Having evidence of gained *Striga* resistance in mutagenized cereals through field and pot studies, it is useful to determine what mechanism underlies the resistance reaction. This is particularly important in the context of sustainability and integrated *Striga*  management, whereby the crop resistance is combined with other control methods, e.g., soil nutrient enhancement through fertilizers, water conservation practices, postemergent weed control practices or with other resistance traits (Rich 2020). Extended reliance on a singular host plant resistance trait may lead to selection of virulent *Striga*  populations that are able in a few generations to overcome the acquired resistance from mutagenized crop varieties. Determining the underlying mechanism leading to the reduced number of parasites emerging around field and pot grown plants is most easily done in a medium in which the *Striga*-host interactions can be observed at their earliest stages with repeated, non-destructive observations.

P. J. Rich (B)

Department of Agronomy, Purdue University, 915 W. Mitch Daniels Blvd., West Lafayette, IN 47907, USA e-mail<EMAIL_ADDRESS>
<sup>©</sup> IAEA: International Atomic Energy Agency 2024 A. M. A. Ghanim et al. (eds.), *Mutation Breeding and Efficiency Enhancing Technologies for Resistance to Striga in Cereals*, https://doi.org/10.1007/978-3-662-68181-7\_6

#### **Protocols**

This section tells how to prepare plates of *Striga* seed embedded in 0.7% agar beginning with surface sterilization and conditioning of the *Striga* seed. It builds on an agar gel assay for determining *Striga* germination stimulant activity published decades ago by Hess et al. (1992). In this assay, since one is observing a limited number of *Striga* seeds, it is important to choose a weed seed source that germinates fairly well, at least 30% in 10−8 M GR24. Ideally, the source of *Striga* seed should come from the target environment in which the resistant cultivar is to be grown. The following procedures are intended for determining sorghum reactions to *Striga asiatica* or *S. hermonthica*. They may be adaptable to maize or rice but modifications to suit screening of those crops is not covered in this chapter.

*Conditioning* Striga *seed*. Prepare a Benomyl (a benzimidazole fungicide, Sigma-Aldrich) 100× stock solution by dissolving 0.15 g Benomyl wettable powder in 10 mL DMSO. Heat (50–70 °C) to completely dissolve the fungicide in the solvent. Store at room temperature in a brown glass bottle for up to three months.

Prepare Metricide 28® solution (2.5% glutaraldehyde; Metrex Research Corp.). A small volume can be activated by adding 1.2 mL of activator to 100 mL of the bulk Metricide if only a few batches of *Striga* will be conditioned over the period. In a biosafety cabinet or on a lab bench, but outside the (laminar flow) transfer hood, remove an aliquot of cleaned (dry but sieved to remove plant material other than seed) *Striga* seed from bulk to a 50 mL flask. This should be done on a heavy white blotter soaked with bleach to contain any dry seed which might miss the flask during the transfer. The normal amount for this size flask is usually six smidgen scoops, enough for about 100 plates. You should be able to get about 15–18 100 mm plates for each smidgen of clean weed seed. Less can be used, but if you use more than six scoops, you will need to perform the surface sterilization steps in a 125 mL flask, doubling the volumes of sterilants (ethanol and Metricide), washes and conditioning solution. Add 25 mL 75% ethanol to the seed in the flask. Wash the outside of flask with bleach.  Let the seed settle in the flask.   Add this back to the flask of seed in the 75% ethanol.

Add 25 mL of activated Metricide. As before, agitate for 2 min by sucking seeds and sterilant in and out of the pipette under the surface of the liquid. Remove the bubbles from the surface of the Metricide solution with the pipette before emptying the liquid to the waste flask in the same manner as the alcohol was removed, pouring off most, then sucking remainder off with the pipette. Rinse 2× with sterile distilled deionized water (ddH2O), each time agitating with the pipette for about a minute then removing the liquid as before.

Add 25 mL ddH2O and 25 µL of the 100× Benomyl stock to flask. This is the conditioning solution. Cover the mouth of the flask with Aluminium foil and put in darkness in an incubator set at 29 °C. **Change Benomyl solution after 1 day** and then every 2–3 days after until the seed is embedded in agar.

*Pouring the plates*. You may embed the *Striga* seed in agar after 5 days in the conditioning solution and allow the conditioning to finish in the agar. It is usually most convenient to do this a day or two before planting the sorghum. **Do not use the seed until it has been in a combination of solution and agar for a total of 10 days**. Germinability is maximal from 10 to 15 days after the conditioning start date.

Prepare agar (0.7%) to embed *Striga* seeds (six smidgen scoops of seed should yield about 100 plates). Prepare bottles of agar in 1 L Pyrex bottles, each containing 900 mL water + 6.3 g BactoAgar. Each bottle should be enough to pour twenty-five 15 × 100 mm plates (35 mL/plate) or fifteen 25 × 100 mm plates (60 mL/plate). Autoclave the bottles of agar on a liquids cycle with 15–20 min sterilization time, then allow to cool in a water bath to 50 °C (turn on water bath when autoclave cycle starts).

To distribute the seed properly and uniformly on each plate takes some practice. Ideally you want around 20 *Striga* seeds per square cm (more if germinability is low). Use a glass Pasteur pipette, the same kind used during the initial surface sterilization and changing of Benomyl. After changing the Benomyl solution, suck as much seed into the pipette from the conditioning flask as possible with the aid of an amber bulb and let it just settle to the narrow tip. Under sterile transfer hood, set out 20 plates (or whatever amount conveniently fits in the front of your hood) and open them. Add a drop of settled *Striga* seed from your pipette by touching it to the center of the opened plate bottom and barely squeeze the bulb to deliver an amount of wet seed about the size of a sorghum seed to each plate, trying to be consistent between plates. The amount of *Striga* is the same whether you are using 15 or 25 mm Petri dishes.

After distributing seed to all plates in a set, add more of the conditioning solution from the flask of conditioned seed to dilute the drop of seed. Compare the density of seed in each drop and add or remove seed from plates until all look to have a similar amount of seed (Figs. 1 and 2). Be sure not to let any seed dry out during this distribution process. After seed has been put in all the plates in roughly equal portions, remove the bottle of sterile agar cooled in a water bath to 50 °C, spray the outside with 75% ethanol and then bring it under the hood and open it. In a smooth and steady manner, pour the liquid agar directly over the *Striga* seed keeping the mouth of the bottle as close to the plate as possible and not interrupting the flow until just after the agar reaches the sides of the plate. The volume of agar should be about 35–40 mL for 15 × 100 mm plates or 60 mL for 25 × 100 mm plates (the level of the agar in the plate should appear to be about 2/3–3/4 full, Fig. 3) and the *Striga*  seeds distributed evenly across the plate (Fig. 4). Using the deeper plates will allow

**Fig. 1** Try to distribute *Striga* seed equally among plates. Keep the seeds always covered with liquid

the sorghum shoot more space to grow if you plan on keeping them long enough for early post-attachment observations (21 days or longer after plating sorghum). Allow plates to cool 10–15 min before covering. Continue this process until all the plates you need for that set are poured.

Record the conditioning start date and the date the seed was embedded in agar for each batch of poured plates. Stack 18–20 plates, making sure lids of each are securely fitted. They can be left in the laminar flow hood so long as the blower is turned off. The blower will dry them out. They can also be left on a lab bench if they will be used within a day or two. If they were poured early and won't be used for a few days, they should be closed inside a plastic bag, most conveniently the sleeve in which the new plates were originally packaged, to maintain high humidity during the remaining conditioning period and placed in a dark 29 °C incubator until they are ready to use (between 10 and 14 days from the conditioning start date of the *Striga*  seed).

*Surface sterilization of sorghum seed.* Determine which accessions you will test according to how many *Striga*-in-agar plates you have prepared and what you can reasonably read at each observation period. Always include at least one check entry with known high *Striga* germination stimulant activity (most important), and another with low *Striga* germination stimulant activity. These will allow you to compare between sets of experimental entries (accessions) as you screen your collection. I

**Fig. 2** In a drop of conditioning solution with a 1 cm diameter, the seed density should look similar to this

**Fig. 3** The agar level should fill the bottom of the petri dish 2/3–3/4 full

usually try to keep the number of accessions to about 30 in a set so that with three plates per accession, I am not overwhelmed on observation days and I can fit all 90 or so plates on a large tray on a single shelf in our incubator.

Deglume sorghum seeds and remove any debris or immature, uncharacteristic or damaged kernels. Record seed source (year and field row) and assign each entry a simple number. Count out 10–20 seeds and place in a 6 dram glass vial, 15 mL Falcon® tube or similar glass or plastic container. Having them all together in a rack is a convenient way to handle multiple entries. Once all entries are placed in vials, add approximately 5 mL of a freshly prepared 50% bleach/0.2% Tween® 20 (a polysorbate-type nonionic surfactant, Sigma-Aldrich) solution from a squirt bottle (for 500 mL, combine 250 mL household bleach [5.25% NaOCl] with 250 mL ddH2O and add to this 1 mL of Tween 20, use this within 1–2 days). Cap the vials. Shake them either individually or together in the rack.

**Fig. 4** If poured properly, seed will be evenly distributed in the agar at about this density. Ideally the seed density will average about 20 seeds/cm<sup>2</sup>

Soak seeds in this solution for about 30 min (but don't leave more than 60 min). Shake them occasionally (two or three times) during this surface sterilization to assure uniform contact of bleach solution and break surface tension. Seeds which do not sink to the bottom may be bug eaten or damaged and will likely not germinate so they should be discarded. After the 30 min soak, move the vial(s) to a sterile transfer hood. Pour off the bleach solution into a waste container, a large beaker or better yet, a sidearm flask fitted with a funnel and lined with filter paper. Be careful not to pour out the seeds. If any are still floating, you will likely lose them but that's okay since any that float are probably not viable. After pouring off the bleach, fill the vial with sterile ddH2O from another squirt bottle. Pour this off and repeat this water wash two more times to remove the bleach solution. Proceed with these washes for each vial of seeds. Leave the caps off.

Prepare a 5% w/v Captan 50 W (a phthalimide fungicide) solution by adding 25 g Captan powder to 500 mL sterile ddH2O in a squirt bottle (use within 1 week). Wear gloves and a mask when weighing out Captan powder. The powder doesn't really dissolve in the water but rather forms a slurry. It is therefore important to shake the bottle often to keep the slurry at the appropriate 5%. Wear gloves when handling the Captan solution. Add approximately 5 mL of this Captan slurry to each vial, remembering to agitate the squirt bottle between vials. Recap the vials and let the seeds soak in this solution overnight to imbibe. They can be removed from the transfer hood as long as they are capped.

*Germination of sorghum seed*. Next day (or after at least 5 h of soaking in Captan) under a laminar flow hood, pour off Captan slurry (into the Captan waste container) after shaking the tube to resuspend the Captan. Take care not to dump the seeds. Add 5 mL sterile distilled water. Pour seeds and water into labelled sterile 100 mm petri dishes, each containing two sterile 90 mm germination paper circles (#SD3-3/8 from www.anchorpaper.com) in the bottom and another wetted one in the lid. Replace the plate cover (lined with germination paper). You may use autoclaved glass petri dishes so that they can be reused. If the seed are clumped together, spread them across the filter paper with gentle shaking or use forceps cleaned with alcohol to move them. Stack plates inside a plastic box with a loose fitting lid to retain high humidity but also allow air flow. Add wetted paper towels to the box to help maintain high humidity. Place this box in a warm dark place (30 °C incubator) overnight or until the seed has germinated and the radicle is around 1 cm long. Unfortunately, this varies among accessions so you may have to adjust the time in the incubator. One cm is ideal, but seedlings with roots as long as 2 cm and as short as 0.5 cm are still okay.

*Plating sorghum into agar with embedded* Striga. Next day, move plates containing germinated seeds to the laminar flow hood. Remove 100 mm plates containing *Striga* in agar (at least 8 days after conditioning start date, but not more than 18 days—optimal is 10–12 days). Remove atomizer containing 10−5 M GR24 (see inset "*How to prepare and keep a clean and active GR24 solution for applying to agar plates*") from refrigerator so that it can come to room temperature. Open each plate (under the hood) and wipe out any condensation that may have formed from inner lid surface. Label agar plates at the edge on both the top and bottom with the appropriate sorghum entry number. Use alcohol wiped forceps to puncture the agar and split it gently apart enough to accommodate the sorghum seed and radicle. Make this "planting hole" over the place where you marked the entry number. Gently pick up the germinated sorghum with the forceps, holding it by the kernel so as to not damage the emerging shoot or root (be especially careful with longer roots, as they can easily bend and break). Plant it into the gap you made in the agar such that the root reaches beneath the agar to where the *Striga* seeds are embedded and that it points toward the center of the plate. The kernel should be at least 1 cm from the edge of the plate. Use the forceps to make the split in the agar close over the remaining root, again being very careful not to damage it. Done properly, the root will grow across the plate among the *Striga* seed and the shoot will grow in the space between the top surface of the agar and the lid. Prepare at least three plates per sorghum accession being tested, more if the accession is heterogeneous (like a landrace or segregating population). I prefer to prepare four plates of each from which the best three best will be used for measurements. Wash forceps in 100% ethanol between each accession. Leave one plate with the same batch(es) of *Striga* blank (no sorghum) to test the germinability of each *Striga* batch. After all accessions are plated, move in stacks of three on trays to shelves in the 29 °C incubator where they will remain for 3–4 days. After returning other plates to the incubator, open the blank plate (no sorghum) and lean it against the back of the hood. Get the atomizer containing 10−5 M GR24 (that you removed from the 4 °C earlier, it should now be at room temperature, see inset "*How to prepare and keep a clean and active GR24 solution for applying to agar*

*plates*"), swirl it to mix and spray the exposed agar with two firm squeezes (three if you are spraying a 25 mm deep plate) of the bulb. Make sure you are holding the spray bottle upright so that the bottom of the atomizer reservoir is parallel to the surface of the transfer hood when you squeeze the bulb. This should deliver around 35 µL of the solution so that once it diffuses through the 35 mL (or 60 µL to 60 mL in deep plates) of agar, gets diluted to around 10−8 M. Return spray bottle to the 4 °C refrigerator. Replace lid on the sprayed blank plate and return to incubator, stacking it along with those planted with sorghum no higher than three plates to a stack. Check the plates in three days. As long as the roots of the sorghum have grown across the plate, most being *at least* 5 cm long, they are ready to read.

*Reading* Striga *germination at Day 3*. With the blank plate(s), that is a plate run in the same batch as the ones being read that contains only *Striga* with no sorghum sprayed with GR24, randomly position the plate in at least ten different areas under a dissecting microscope, counting both the number of *Striga* seeds and the number of germinated *Striga* in individual 2 × 2.5 cm fields. If the *Striga* seed density is ideal, you will be counting about 100 seeds in each 5 cm2 field. These counts may be taken later by imaging the ten fields. Observe all plates through the bottom of the plate because the root and *Striga* seed are closest to this surface and you won't have to look through the layer of agar. Record each of these counts on the data sheet. Aim at counting around 1000 seeds. Calculate the *Striga* batch germination rate for this set of plates by adding all the counts (from each of the ten field counts) for no. germinated *Striga* and dividing this by the total number of *Striga* seeds (in the ten fields). This should be at least 30%. If it is less, the *Striga* seed is not responsive enough to give you a meaningful measure of *Striga* germination stimulant activity on your sample (sorghum) plates and so you shouldn't waste more time reading those.

Provided you have > 30% germination on your blank plates, the plates containing sorghum are checked under the dissecting microscope after 3 days. Before taking them to the microscope, wipe any condensation inside the plate lids with clean paper towels (plates should only be opened under the transfer hood). Select the best three plates out of the four prepared for each entry (more if you're looking at a heterogeneous group) for microscopic observations. Selections should be based on health of the host plant and whether its root has properly penetrated the agar. Assign each a number (− 1, − 2, − 3) and mark it on the top and bottom of each plate next to the entry number marked when planting the sorghum. Record the condition of each host plant on the data sheet (Fig. 5). Use the descriptor "G" (good) for normal looking sorghum or "F" (fair) if the root or shoot is especially small or there is some other problem. Record any problems with host condition in the notes column of the data sheet. If the sorghum seedling is damaged or didn't grow, and its main root is < 3 cm long, return it to the incubator if you have less than three plates for that entry. Check it again in a day or two and use it only if the root exceeds 3 cm.

Observe plates at about 10× magnification (again through the bottom of the plate). Look for germinated *Striga* along the sorghum root beginning 2 cm from the kernel. Mark a line at the basal 2 cm and only search for germinated *Striga*  beyond this mark (toward the root apex). Mark a small dot on the plate over the seed coat of the three germinated *Striga* furthest from each host root (Figs. 6 and An Agar-Based Method for Determining Mechanisms of *Striga* … 93

**Fig. 5** Example data sheet for recording MGD

7). Try to avoid areas within 2 cm of the kernel if the main root happens to curl back toward it. Using a transparent ruler, record the distances of these marks (to the nearest 0.5 mm) to the nearest sorghum root (main root or branch). If less than three germinated *Striga* are seen, record this on the sheet. Maximum germination distance (MGD) is the average of these three measurements on each seedling. If less than three germinated weed seeds are observed, enter "0" in the calculations for average. For example, if you observe only one germinated *Striga* in the measuring zone and the distance from that to the nearest root is 1 mm, then the MGD is 0.3 mm [(1 + 0 + 0)/3]. MGD for the accession is the average of at least three good seedlings. Also calculate the standard deviation between seedling MGDs of a given accession to give an indication of the variability between them. The MGD is a measure of *Striga* germination stimulant activity. Generally, a sorghum seedling with an MGD > 10 mm has high *Striga* germination stimulant activity. A seedling with an MGD < 5 mm has low *Striga* germination stimulant activity. Those with MGDs between 5 and 10 are borderline lows and may indicate influence of germination inhibitors (see next section). All measures should be considered in the context of the standard high and low checks run with the set of plates. Sometimes the 10 mm threshold will need adjusting slightly, particularly for those borderline accessions or with low responsive *Striga* seed sources. A sorghum line truly having the resistance mechanism of low *Striga* germination stimulant activity should consistently show MGDs below 10 mm from set to set.

*Some optional measures—extending the agar assay*. Near host germination rates can also be recorded at Day 3 to get another measure of *Striga* germination stimulant activity. A standard area is selected for all observed seedlings so that comparisons of

**Fig. 6** Example of MGD measures at Day 3. Blue dots were marked on bottom of plate on what appear to be the furthest germinated *Striga* seeds. Distances of the three furthest to the nearest root are measured and averaged for the MGD [in this case, (8 + 8 + 10)/3 = 8.7 mm]

*Striga* germination stimulant activity as well as any possible germination inhibition and influence on haustorial formation can be made. This measure generally correlates well with MGD except in cases where germination inhibitors are present in the sorghum root exudate. It is also useful for comparing to in vitro germination assays using collected sorghum exudates. To prepare for these counts, cut a template from a transparent ruler that is 2 × 2.5 cm. Use this or another ruler to make a small mark 2 cm from the sorghum kernel along the main root. Use the template to trace a rectangle on the bottom of the plate, keeping one of the narrow edges (a 2 cm side of the rectangle) on the 2 cm mark along the main root you made from the kernel. Try to keep the main root in the center of this rectangle so that it splits it in half, that way the rectangular space you will observe under the microscope will cover a 5 cm<sup>2</sup> area along the main root from 2 to 4.5 cm from the kernel and roughly 1 cm on either side of the main root. After marking all your plates with these rectangles, look at them each under the microscope, setting the magnification to around the 1.4 mark, or there about, such that the edges of your drawn rectangle match the edges of your viewing field. Once you've set this field of view for the first plate and focused and set the illumination to clearly see the main sorghum root and surrounding *Striga*, you shouldn't have to change it for the other plates. Snap a photo of each plate, keeping track of which image goes with which plate. Make sure you always orient each plate

**Fig. 7** Another example of MGD measure at Day 3, this time on a sorghum seedling with low *Striga* germination stimulant activity [(0 + 0 + 0.5)/3 = 0.2 mm]

as you image them so that the basal end of the main root (toward the kernel) is always to the right of the captured image. These images contain all the *Striga* within about 1 cm on each side of the main root in this zone and from these counts of germinated and total number of *Striga* seed can be made later. Record these on the data sheet (Fig. 8). Because germinability varies between conditioned batches of *Striga* seed, it is a good idea to correct germination rates counted in your images by dividing the germination rates by the batch germination rate determined from the blank plate that you treated with GR24 at Day 0 and from which you imaged 20 random 5 cm2 fields on Day 3. This "corrected" germination rate allows you to more meaningfully compare plates run in separate batches or with different *Striga* seed sources, as it tells you the proportion of viable seeds that responded to the sorghum root germination stimulants.

To overcome differences in *Striga* germination stimulant activity between sorghum accessions for post-germination measures, you can spray all the plates (after imaging) with 10−5 M GR24 (final conc. = 10−8 M, after diffusing through the agar) as you did earlier for the blank plate, under the hood and return them to the incubator for two more days.

**Fig. 8** Example data sheet for recording Day 3 measures of germination rates. Germination rates are corrected for germinability of *Striga* seed source used

#### **How to prepare and keep a clean and active GR24 solution for applying to agar plates**

1. Prepare a GR24 "stock solution"

Dissolve 0.024 g GR24 (a synthetic strigolactone, molecular wt. = 298.3) in 10 mL DMSO

0.024 g/0.01 L = 2.4 g/L ≈ 8 × 10−3 M (in 100% DMSO)

Keep this frozen in a brown glass bottle at − 20 °C when not in use. Thaw thoroughly before using.  Prepare the GR24 "application solution"

Add 250 µL "stock solution" to 100 mL ddH2O in a clean brown glass bottle 8 × 10−3 M GR24 × 0.25 mL/100 mL = 2 × 10−5 M (in 0.25% DMSO)

Use this solution to fill the atomizer (15-RD glass atomizer with plastisol bulb, DeVilbiss Healthcare LLC) which holds about 25 mL. Keep the remainder (not put in the atomizer) frozen at − 20 °C when not in use. Thaw thoroughly before filling the atomizer and shake after thawing to redissolve any GR24 which may have precipitated. Keep atomizer in a refrigerator (4 °C) when not in use. Bring to room temperature before spraying plates

3. Spraying the plates

Open agar plates only under a laminar flow hood to avoid surface contamination. GR24 is usually applied after measuring MGD and imaging for germination counts 3 days after plating the sorghum in the agar embedded with Striga. Only open a few plates at a time when spraying to avoid drying them out under the airflow of the hood. Lean them at the back of the hood so that the surface of the agar is perpendicular to the work surface. Take care not to damage the sorghum shoot which may fall from the bounds of the plate edges. Holding the glass reservoir of the atomizer in one hand such that the delivery nozzle is 10–15 cm from the exposed agar surface, give the bulb two firm squeezes with the other hand. This should deliver about 35 µL (≈ 17 µL per squeeze) to the agar. If you are using 25 mm deep plates containing 60 mL agar, spray with three firm squeezes of the bulb. It is important to keep the bottom of the atomizer reservoir parallel to the work surface of the hood. Tilting it excessively will not deliver the proper volume. Assuming the volume of the agar is 35 mL (60 mL in deep plates), the final concentration of GR24, once it diffuses through to the embedded Striga seed, will be in the range of 10−8 M, which is ideal to germinate all germinable weed seed. The final DMSO concentration is negligible and will not harm the plants. Replace the lids on the sprayed plates promptly, returning them to a horizontal position. Carefully guide the sorghum shoot back into the confines of the plate if it came out during spraying. Make sure not to pinch it between the bottom plate sides and lid. Wait two days before imaging for post-GR24 germination counts. Return atomizer to refrigerator. A full atomizer reservoir should be enough to spray 100 plates

Two days after spraying the plates with GR24 (Day 5), mark a second 2 × 2.5 cm rectangle on the bottoms of the plates using the template you used on Day 3 (Fig. 9). This time, however, choose an area as far from any roots (especially the main root) as possible on the plate, avoiding that area within 2 cm of the kernel. Use a different color for this rectangle than what was used on Day 3. Where you place this second rectangle will vary from plate to plate, the idea being that you observe *Striga* seeds minimally influenced by any sorghum roots so that you can compare these to that *Striga* within 1 cm on each side of the main root from 2 to 4.5 cm. Since you sprayed the plates with GR24, most weed seed on the plate, whether near or far from the sorghum root, ought to have received adequate germination stimulant. Reimage the rectangle you marked on Day 3, the "near-host-root", and snap another image of the new "far-from-root" 2 × 2.5 cm area for each plate. Again, keep the plates oriented in the same way (kernel to the right) as you did on Day 3 and keep track of each image file name with your "near and far" images of each plate.

By comparing these areas, in terms of *Striga* germination rate particularly, you can measure any inhibition that a sorghum root might have on *Striga*. Germination counts are made on your Day 3 and Day 5 images by dividing the number of germinated *Striga* seeds in the image by the total number of seeds and express as a percentage to determine the germination rate. You may also want to determine the percent germinated *Striga* near the root with haustoria at both Days 3 and 5. To do this, count the number of germinated *Striga* with haustoria. Only those *Striga* radicles that are

**Fig. 9** Agar gel assay plates marked for "Near & Far" measures. After reading the germination distance (MGD) at Day 3 and imaging the area 2–4.5 cm from the kernel for observations and germination counts (blue rectangles), plates are sprayed with 35–40 µL of 10−5 M GR24 (final conc. <sup>=</sup> <sup>10</sup>−8 M) to germinate remaining *Striga* seed on plate that was not stimulated by the host root and returned to the incubator for two more days. At Day 5, before reimaging the "near" area (blue rectangle), a second (red) rectangle is drawn on the plate as far from the main root as possible tracing the same 2 × 2.5 cm template cut from a transparent ruler (bottom). Both areas are then imaged and germination rates within each compared for any indication of germination inhibition

wider at their apical end relative to their basal width are counted as having haustoria. Divide this count by the number of *germinated* weed seeds in the area (Fig. 10).

To determine if there is any germination inhibition near the host root, use the two images for each plate taken after GR24 treatment, the Day 5 images. Count the number of germinated seeds and total seeds in each rectangle to determine the germination rates in each place. Then divide the germination rate calculated for the "near-host-root" area by the germination rate of the "far-from-root" area to obtain the "germination index" (GI). To avoid effects of peculiarities on certain plates where root-free areas are hard to find, average the far-from-host germination counts for all plates in a set (all plates from all entries tested in that set) and use this value as the germination count by which to divide each near-host-root rates to determine the GI. Average these germination indices for all plates observed for a given accession (Fig. 11). Example images from which these counts are taken are shown in Figs. 12 and 13. A GI wildly different than one could indicate some inhibition of the sorghum accession on *Striga* germination. Most sorghum does show some germination inhibition by this measure, that is, GI in the range of 0.7–1.0. Sorghum accessions that consistently show a GI ≤ 0.65 in this assay, especially with multiple *Striga* seed sources, we consider to have a germination inhibition mechanism of *Striga* resistance. The impact of this trait on overall resistance and its chemical nature remains to

**Fig. 10** Example data sheet for recording Day 5 proportion of germinated *Striga* forming haustoria. Red arrows on example image indicate germinated *Striga* with haustoria

be determined. It is, however, clearly distinct from low *Striga* germination stimulant activity.

You may also note some other allelopathic influences of certain accessions on surrounding *Striga* in the Day 5 "near" images. Do haustoria form in germinated *Striga* at similar rates? Do these haustoria look unusual? Do the *Striga* come out of their seed coats? Are *Striga* radicals unusually short or opaque near the sorghum

**Fig. 11** Example data sheet for measuring germination inhibition

**Fig. 12** Image of "near-host-root" area. Image taken at Day 5 (2 days after GR24 treatment) covering an area of 2 × 2.5 cm. This includes *Striga* within approximately 1 cm of the host main root over a length of 2 cm (right) to 4.5 cm (left). Note that most germinated *Striga* in this area have haustoria. Toward the basal end (right) an orange colored exudate can be seen being absorbed by surrounding *Striga*

root? Do you notice colored exudates (yellow, orange or red) bleeding from the sorghum root and being absorbed by the surrounding *Striga* (Fig. 12)?

You may also want to count the number of lateral branches on the sorghum root, their lengths, etc. as an indicator of rooting habit. These kind of measurements are easier if you can get a digital image (or images) of the entire plate. If the microscope you have won't quite zoom out that far, a smart phone camera using the "square" setting is a good way to image the entire plate. Even better if you can compare two images of the same root, perhaps at Day 3 and Day 5, to get an idea about root growth rate over that specific time. Software that comes with some microscopes or root analysis software like ImageJ® (available for free from the United States National Institutes of Health at https://imagej.nih.gov/ij/) can assist with such measures.

*Even more from the agar plates*. If you use deeper (25 mm) plates with thicker agar layers (60 mL) you should be able to keep the plates for observing any *Striga*  attachments on the sorghum seedlings. Leaving the plates stacked only two or three high on a lab bench under fluorescent lights can keep the sorghum seedlings alive and growing for three weeks. As long as the plate remains free from mold or bacterial contamination and the sorghum shoot remains green, plates remain useful. Wiping

**Fig. 13** Image of "far-from-root" area from the same plate. Image taken at Day 5 (2 days after GR24 treatment) from the same plate covering a 2 × 2.5 cm area away from the sorghum root. Note germinated *Striga* here lack haustoria since they are not near enough to the host to receive the haustorial initiation signal

out condensation inside the lid of the plate where the sorghum shoot touches also helps to keep them going. If, after 10 days from when you planted the sorghum in the agar, the sorghum shoot is green and the agar layer hasn't dried out (still comes to at least the half full mark in the bottom of the plate), you can check them under the microscope for attachments. With the magnification set in the mid-range, follow the main root and its branches to look for *Striga* that have attached. Circle and mark each with a unique number on the bottom of the plate and take a picture of each at higher magnification. The position of each on the root system (whether they are on the main root or a branch, for instance) can be recorded from a whole plate image taken with a smart phone at each observation day. If you can get two or three images over time of particular attached *Striga* on a live host, you can have a measure of its ability to support early *Striga* growth. Imaging individual attachments marked at 10 days and reimaging at 15 and 20 days (or there about) after planting the sorghum allows one to get an idea of whether and how well *Striga* grow on a particular sorghum accession. Simple data collected from tracked individual attachments as "successful" if the parasite grows over the course of observation or "unsuccessful" if an attached *Striga* does not grow allows one to compare sorghum accessions both in terms of the average number of attached parasites per seedling and percentage of those that

**Fig. 14** Example data sheet for keeping track of *Striga* attachments and quantifying postattachment resistance in sorghum in agar plates

succeed (Fig. 14). Such measures can indicate differences in their compatibilities as *Striga* hosts. With the proper checks, the extended agar assay in 25 mm plates can be used as an initial screen for post-attachment *Striga* resistance.

It is very important that you keep track of the fitness of the sorghum on the plate with each set of images and make sure the agar hasn't dried out if you go to the trouble to follow the growth of individual parasites over time (Fig. 15). Whole plate images are useful for this purpose.  Don't bother looking at plates with a sorghum whose shoot is brown or on which the agar layer is drying up. Also your record keeping needs to be such that you know which image corresponds to which *Striga* on which plate so that you can put together a string of images at the end of the observation period (perhaps 3 images over 2 weeks, Fig. 16). This is, of course, laborious and probably not worth it if your attachment frequency is low. Attachment frequency tends to be higher in thicker agar layers (60 mL) in deep (25 mm) plates. Resistance at this early post-attachment stage, generally called incompatibility, may be observed if you have several good plates of a given accession. If a sorghum accession consistently shows fewer attachments than a susceptible check in the same set, this may indicate some post-germination, pre-attachment resistance. If the *Striga*  attaching to plants of one accession don't grow, turn purple or grows at a slower rate than those on a susceptible check, you might be observing incompatible resistance (Fig. 17).

The agar assay is not ideal for measuring these post-germination resistance mechanisms because the sorghum seedling is so confined and attachment frequency tends to be lower in agar than on other media like that described in Chapter "Phenotyping for Resistance to *Striga Asiatica* in Rice and Maize Mutant Populations In Madagascar". With several sets of good plates with multiple *Striga* attachments and careful

**Fig. 15** If given proper light and humidity and a thick enough layer of agar, it's possible to keep plates for three weeks or even longer. Even in this set of standard depth (15 mm) plates, young *Striga* can be seen growing on 20-day-old sorghum seedlings. The sorghum eventually succumbs to the cramped conditions and lack of nutrients

**Fig. 16** Images of the same *Striga* attached to the root of a susceptible sorghum over time in agar. Images taken at 7, 10 and 14 days after planting the sorghum seedling in agar embedded with conditioned *Striga* seed. Plate was treated with GR24 after reading MGD in the Agar Gel Assay. In this case, images at the latter days had to be taken at lower magnifications than the original image to capture the larger *Striga* shoot

**Fig. 17** Images of the same *Striga* attached to the root of a resistant sorghum over time in agar. Images taken at 7, 10 and 14 days after planting the sorghum seedling in agar embedded with conditioned *Striga* seed. Plate was treated with GR24 after reading MGD in the Agar Gel Assay. In contrast to the series of images collected on the susceptible sorghum, this attached *Striga* does not grow

observations, such reactions can be indicated from the same plates used to determine if pre-attachment *Striga* resistance mechanisms are at play. Post-attachment *Striga*  resistance can then be confirmed in other co-culture media.

#### **Conclusions**

The most important information (and what the assay was originally designed for) one gets from the agar gel assay is the MGD, which is an established measure of *Striga* germination stimulant activity (Hess et al. 1992). As long as one has the proper amount of *Striga* seed embedded in the agar, and the germinability of that seed is adequate (> 30%, as measured on the blank plates), one can determine whether a sorghum accession has the resistance mechanism of low *Striga* germination stimulant activity. To date, the only *Striga* resistance gene identified in sorghum is *LGS1* which controls germination stimulant activity (Gobena et al. 2017). Recessive alleles at this locus changes the type of strigolactone exuded by sorghum roots. Mutagenesis could result in new *lgs1* alleles which could be confirmed in mutant lines by chemical analysis of root exudates (This chapter) and sequencing of PCR amplicons targeting *LGS1*. More useful would be gained resistance expressed as germination inhibition, lower haustorial inducing capacity or at the post-germination stages of the *Striga*-host interaction. Then *lgs1* could be introgressed into these mutant lines through marker assisted backcrossing to offer more durable *Striga* resistance through pyramiding. The extended agar assay may detect such novel resistance mechanisms and ultimately, with further investigation, lead to gene identification underpinning the gained *Striga*  resistance.

**Acknowledgments** This work was supported though a grant provided by the Joint FAO/IAEA Centre for Nuclear Techniques in Food and Agriculture (CRP D25005, #20698) and the Bill and Melinda Gates Foundation (OPP1174896).

#### **References**

The opinions expressed in this chapter are those of the author(s) and do not necessarily reflect the views of the IAEA: International Atomic Energy Agency, its Board of Directors, or the countries they represent.

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 3.0 IGO license (http://creativecommons.org/licenses/by/3.0/igo/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the IAEA: International Atomic Energy Agency, provide a link to the Creative Commons license and indicate if changes were made.

Any dispute related to the use of the works of the IAEA: International Atomic Energy Agency that cannot be settled amicably shall be submitted to arbitration pursuant to the UNCITRAL rules. The use of the IAEA: International Atomic Energy Agency's name for any purpose other than for attribution, and the use of the IAEA: International Atomic Energy Agency's logo, shall be subject to a separate written license agreement between the IAEA: International Atomic Energy Agency and the user and is not authorized as part of this CC-IGO license. Note that the link provided above includes additional terms and conditions of the license.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Histological Analysis of** *Striga* **Infected Plants**

**Mafrikhul Muttaqin, Songkui Cui, and Satoko Yoshida**

**Abstract** Post-attachment resistance can be verified by observing the infection and growth rates of *Striga* attached to host roots. These protocols explain how to infect *Striga hermonthica* on host roots using rhizotron chambers, a semi-in vitro system that allows one to observe *Striga* infection without disturbing the host's roots. Sectioning parasitic attachments and subsequent histological methods are also described, which allow closer investigations of the extent to which the *Striga* haustoria penetrate host tissues. Such observations can reveal whether any mechanical resistance exists in the host. These histological methods help further characterize resistance mechanisms in rice or sorghum.

**Keywords** *Striga* · Root parasitic plants · Rhizotron · Safranin · Xylem staining · Histology

#### **Introduction**

*Striga* resistance can be classified into two types, pre-attachment and postattachment. As *Striga* parasitism advances on its cereal hosts, pre-attachment processes include germination and haustorium formation, whereas post-attachment processes include penetration, reaching the host stele, xylem connection, and nutrient transfer. To characterize any gained *Striga*-resistance in mutagenized hosts, it is essential to quantify and qualify the resistance. For qualification of *Striga* parasitic success, developing a system in which multiple *Striga* synchronously infect host roots with minimal influence from environmental conditions are key. For this purpose, we

M. Muttaqin · S. Cui · S. Yoshida (B)

Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma, Nara, Japan e-mail<EMAIL_ADDRESS>
M. Muttaqin

© IAEA: International Atomic Energy Agency 2024

Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University (IPB University), Bogor 16680, Indonesia

A. M. A. Ghanim et al. (eds.), *Mutation Breeding and Efficiency Enhancing Technologies for Resistance to Striga in Cereals*, https://doi.org/10.1007/978-3-662-68181-7\_7

use a rhizotron system, in which host roots infected by *Striga* are easily observed without disturbing root architecture (Gurney et al. 2006; Yoshida and Shirasu 2009). The rates of infection are quantified with low-power stereo microscopy or magnification lenses at different days of co-culture. When a *Striga* seedling has more than three pairs of leaves, the vascular connection generally is established (Yoshida and Shirasu 2009). To characterize the resistance mechanisms in detail, histological analysis is useful. The xylem bridge can be observed with simple Safranin O staining, and the cellular level interaction can be analyzed by making thin sections from Technovit 7100-embedded samples. The staining process may then reveal some post-attachment resistance mechanisms (Yoshida and Shirasu 2009), including incompatibility after vascular connection, endodermis blockage, or mechanical barriers to penetration at the root cortex.

#### **Protocols**

*Rhizotron system for observing* Striga *infection*. Although the rhizotron method is described for rice, it is also suitable for use with a sorghum host. If using sorghum, follow the surface sterilization and germination protocol described in Chapter "An Agar-Based Method for Determining Mechanisms of Striga Resistance in Sorghum".

Surface sterilize rice seeds by soaking in a tube with 10% commercial bleach (0.525% NaOCl) for 15 min, gently vortexing periodically.  Germinate the seeds and grow for one week in a growth chamber at 26 °C with the 16 h light/8 h dark photoperiod.

Assemble the rhizotron by removing a 2 cm square notch on the shorter side of a square petri dish and its lid using a pair of pliers or a soldering iron. If using a soldering iron, work under a fume hood to remove fumes from melting plastic (Fig. 1). Make two drain holes on the opposite short side of the rectangular petri dish using the soldering iron (Fig. 1). Cut the rockwool and nylon mesh to the same dimensions of the bottom plate of the petri dish. Immerse the rockwool in a tray filled with tap water. Place the rockwool into the petri dish, and cover with nylon mesh. Place the rice seedling on the nylon mesh. Close the petri dish with its lid and secure it with two rubber bands. Place the petri dish vertically on a tray with water, cover the root parts with aluminum foil, and give half-strength MS for rice plants. Grow them in a glasshouse or growth chamber (28 °C light for 12-h/20 °C dark for 12-h).

*Preparation of* Striga *seeds (preconditioning)*. At 1–2 weeks before infection day, surface-sterilize *Striga* seeds by immersing them in a 20% commercial bleach solution diluted to contain approximately 1.2% sodium hypochlorite (NaOCl) for 5 min. Replace the solution at least once, wait for 5 min, and wash with sterile water more than five times. Place the sterilized seeds on the glass microfiber filters GF/A paper with 8–10 ml water in a petri dish (90 mm diameter), cover it with aluminum foil, and keep it in a 25 °C plant growth chamber for 1–2 weeks.

**Fig. 1** Materials and structure of a rhizotron

Striga *infection*. Before *Striga* infection, add germination stimulants (final concentration of 10 nM strigol, or 1 µM GR24) onto the filter paper with preconditioned *Striga.* Return the petri dish at 25 °C and incubate for 2–24 h in the dark. Open the rhizotron and place the *Striga* seeds carefully along the rice roots using forceps (5–10 mm distance between seeds, Fig. 2). Put the rhizotron lid back and secure the rhizotron with two rubber bands. Cover the root parts with aluminum foil. Place it back to the glasshouse or plant growth chamber. In the case of two hours exposure to germination stimulants, *Striga* germination can be observed at one day, penetration at two days, and xylem bridge formation at 3–5 days after infection. In the case of 24 h germination stimulant treatment, *Striga* germination can be readily observed before rice infection, and penetration and xylem bridge formation can be observed at one day and 3–4 days after infection, respectively.

*Quantification of* Striga *infection*. After a few weeks of infection, *Striga* infection rates can be quantified depending on which stage you want to observe. The later stage can be analyzed at two and four weeks after infection. Carefully remove the rubber bands and the lid from a rhizotron without disturbing host roots. Observe the infection stage of each *Striga* seedling under a stereo microscope or magnification

**Fig. 2** Rhizotron infection system; P: parasite (*Striga hermonthica*), H: host (rice)

lenses. Count the number of *Striga* in each stage. *Striga* infection can be classified into the following seven groups, (a) not germinated, (b) germinated but not attached, (c) penetrated, but the seed coat is still covered, (d) penetrated but dead, (e) 1–2 pairs of open leaves, (f) 3–5 pairs of open leaves or (g) more than six pairs of open leaves (Yoshida and Shirasu 2009) (Fig. 3).

*Safranin O staining for the observation of xylem bridges.* Prepare a fixation solution by combining 75% ethanol, 25% acetic acid, and 0.1% Safranin O. Prepare a clearing solution by dissolving 1 g chloral hydrate (C2H3Cl3O2) in 1 mL glycerol and 2 mL water.

Excise 1–2 cm of *Striga*-infected root segments with scissors. Place root samples in the fixation solution and vacuum infiltrate for 15 min. Wash with sterile water three times. Add enough 0.1% Safranin O stain to cover the sample and heat at 90 °C for 5 min. Wash with sterile water three times. Soak in the clearing solution overnight. Observe the xylem bridge formation under microscopy. Xylem cells are stained in red with Safranin O (Fig. 4). The staining can also be observed under fluorescent microscopy with the filter setting for red fluorescent protein (RFP).

*Making Technovit 7100-embedded thin sections.* Prepare an FAA fixative solution by combining 1 mL formaldehyde, 0.5 mL glacial acetic acid, 5 mL ethanol, and 3.5 mL water. Prepare the following steps at room temperature. Cut the samples and put them into the FAA fixative solution, vacuum infiltrate for 15 min and leave at room temperature for more than two hours (place at 4 °C if you do it overnight). Dehydrate using an ethanol series. Replace fixative solution with 70% ethanol and incubate for 30 min with gentle agitation at room temperature. Next, replace 70% ethanol with 100% ethanol and incubate for 30 min with gentle agitation at room temperature. Repeat 100% ethanol incubation one more time.

**Fig. 3** Classification of *Striga hermontica* infection on a sorghum host. **a** Not germinated, **b** germinated but not attached, **c** penetrated, but the seed coat is still covered, **d** penetrated but dead, **e** 1–2 pairs of open leaves, **f** 3–5 pairs of open leaves, and **g** more than six pairs of open leaves. Scale bars: 1000 µm

**Fig. 4** Safranin-O staining of *Striga hermonthica* infecting a sorghum root. Scale bars: left 500 µm, right 100 µm

Add 1/4 volume (250 µL for 1 mL ethanol) of Technovit 7100 liquid (Heraeus Kulzer GmbH; liquid, Hardener I, Hardener II), and incubate for 30 min with gentle agitation. Repeat until the solution contains an equal volume of Technovit 7100 liquid and ethanol (four times). Incubate for more than one hour (or overnight) with gentle agitation. Remove half the volume of Technovit 7100 liquid: ethanol solution. Add the same volume of Technovit 7100 liquid as you removed. Incubate for 30 min with gentle agitation. Prepare Technovit infiltration solution (add 1 g of Hardener I to 100 mL Technovit 7100 liquid). Replace the solution containing the sample with the Technovit infiltration solution. Incubate it for more than one hour with gentle agitation. Replace the solution with a new Technovit infiltration solution. Incubate it overnight with gentle agitation. Replace the solution with a new Technovit infiltration solution. Incubate it for 1–2 h(s) with gentle agitation. Prepare the Technovit embedding solution by mixing 1 mL Technovit infiltration solution and 66 µL Hardener II (this will start solidifying soon).

To embed the infiltrated *Striga*/root sample, cut it under a microscope to the appropriate size and shape. Prepare a proper mold (e.g., cap of 1.5 ml Eppendorf tube, etc.). Put the sample into the mold and add the Technovit embedding solution. Adjust the angle of samples. Leave it at room temperature (RT) until the sample becomes solid (1 h at RT or 37 °C, or overnight at 4 °C or RT).

Prepare a Technovit 3040 (Heraeus Kulzer GmbH; powder, liquid) mounting solution by mixing well Technovit 3040 powder and liquid at a 2:1 ratio under a fume hood. Pour the Technovit 3040 solution onto the embedded sample's surface and attach a small wood block to the solution (Optional: a quick-drying glue can be used). Leave the samples until the Technovit 3040 solution becomes solid.

For preparing thin sections of the sample for microscopic observation, preheat the slide warmer to 42 °C. Place water droplets onto a MAS-coated slide glasses to the approximate size of the sections. Insert the wood block with sample into the sample holder of a Manual Rotary Microtome (Leica RM2235 or equivalent) and a TC-65 Tungsten blade to the blade holder of the microtome. Carefully slice the sample with manual rotation to 2–4 µm thickness. Hold the section with thin forceps and place each section onto the water droplet on the slide glass. Check the section under a light microscope. Place the slide glass onto the preheated slide warmer until the water dries up.

*Double staining with Safranin-O and Fast Green.* Preheat a hot plate (Corning 6795-420D hot plate stir or equivalent) to about 80 °C. Drop 0.1% Safranin solution on the slide containing the sample sections to cover them. Place the slide on the heat block until the Safranin solution is almost dried up. Briefly wash the slide glass with water. Set the slide(s) in a staining basket.  Wash with water until no more red pigment comes off from the slide. Dip the slide glass in 0.5% picric acid in 95% ethanol for a few seconds. Next, dip the slide glass in 95% EtOH with ammonium solution for a few seconds. Dip the slide glass in 95% EtOH with a few drops of HCl. Wash with 100% ethanol. Stain with 0.1% Fast Green FCF for 30 s to 1 min (ideal duration may vary). Wash with 100% ethanol. Place the slide glasses on the slide glass holder and dry them in the air, and observe with a bright field microscope (Fig. 5a).

*Simple staining with toluidine blue.* The section on the slide glass can be stained with toluidine blue solution (Fig. 5b) instead of the double stainng listed above

**Fig. 5** Thin-sections *Striga hermonthica* infecting a rice root stained by different methods: **a** Safranin O and Fast Green double-stained cross-section of *Striga hermonthica* infecting a rice root. Scale bar, 50 µm. **b** toluidine blue-stained longitudinal section of *Striga hermonthica* infecting a rice root. S: *Striga*, H: rice

(Fig. 5a). Drop 0.01% toluidine blue solution (100 mg Toluidine Blue [Sigma-Aldrich] in 10 mL water) on the top of slide glass to cover the specimen. Wait for 2 min. Wash the slide glass by immersing in water. Observe with a bright field microscope.

#### **Conclusion**

The protocols herein described are useful for characterizing post-attachment *Striga*  resistance in rice and sorghum. They may also be applied to maize with some modification (e.g., using larger plates to construct the rhizotrons). Although they can indicate pre-attachment resistance mechanisms, for instance by applying ungerminated preconditioned *Striga* seeds to host roots in the rhizotron, such observations are perhaps more suitable in the agar system described in Chapter "An Agar-Based Method for Determining Mechanisms of *Striga* Resistance in Sorghum". The application of both co-culture methods can pinpoint the point at which resistance gained through mutagenesis counters *Striga* parasitism in cereal mutant lineages.

#### **References**

The opinions expressed in this chapter are those of the author(s) and do not necessarily reflect the views of the IAEA: International Atomic Energy Agency, its Board of Directors, or the countries they represent.

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 3.0 IGO license (http://creativecommons.org/licenses/by/3.0/igo/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the IAEA: International Atomic Energy Agency, provide a link to the Creative Commons license and indicate if changes were made.

Any dispute related to the use of the works of the IAEA: International Atomic Energy Agency that cannot be settled amicably shall be submitted to arbitration pursuant to the UNCITRAL rules. The use of the IAEA: International Atomic Energy Agency's name for any purpose other than for attribution, and the use of the IAEA: International Atomic Energy Agency's logo, shall be subject to a separate written license agreement between the IAEA: International Atomic Energy Agency and the user and is not authorized as part of this CC-IGO license. Note that the link provided above includes additional terms and conditions of the license.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# *Striga* **Germination Stimulant Analysis**

**Benjamin Thiombiano, Kristýna Floková, Aimee Walmsley, and Harro J. Bouwmeester**

**Abstract** *Striga* is a flowering parasitic plant that can infect a broad range of crops including sorghum, maize and rice. The life cycle of *Striga* is intricately linked to its host, using host derived signals to initiate germination and attach to its host. Host susceptibility to *Striga* is thus directly correlated to the production and exudation of these signals. In this chapter we discuss the strigolactones, the most important class of *Striga* germination stimulants. We review the structural diversity of the strigolactones, their transport and biological relevance in plant and rhizosphere, and their importance for host specificity. In addition, we describe methods to analyse germination stimulant production by a *Striga* host: how to, reproducibly, collect the root exudate of a host, how to process these exudates, partially purify them and analyse them using analytical chemistry or a high-throughput image analysis-based germination bioassay.

**Keywords** Parasitic plant · Strigolactone analysis · Root exudate collection · *Striga* germination

#### **Introduction**

Selected *Striga* resistant cereal mutants derived from gamma irradiated stocks (Chapters "Screening for Resistance to *Striga* Hermonthica in Mutagenized Sorghum and Upland Rice in Burkina Faso"–"Phenotyping for Resistance to *Striga* Asiatica in Rice and Maize Mutant Populations in Madagascar") determined through laboratory assays to have lower *Striga* germination stimulant activity (Chapter "An Agar-based Method for Determining Mechanisms of *Striga* Resistance in Sorghum") than their unmutagenized progenitors may be further characterized by examining the strigolactones in their root exudates. This chapter provides further details of the parasitic

B. Thiombiano (B) · K. Floková · A. Walmsley · H. J. Bouwmeester

Plant Hormone Biology Group, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands e-mail<EMAIL_ADDRESS>
<sup>©</sup> IAEA: International Atomic Energy Agency 2024 A. M. A. Ghanim et al. (eds.), *Mutation Breeding and Efficiency Enhancing Technologies for Resistance to Striga in Cereals*, https://doi.org/10.1007/978-3-662-68181-7\_8

association of *Striga* with its cereal hosts with particular emphasis on strigolactones, the germination stimulants in host root exudates. Protocols for collecting and chemical and biological characterization of root exudates are also described.

Striga *life cycle*. The life cycle of *Striga* commences with the germination of its seed, followed by its attachment to the host, marking the start of the parasitic phase. Before the seed can respond to germination stimuli, various metabolic processes occur in what is known as the "conditioning" phase. Full imbibition, which occurs within a day, must be followed by several days (optimally 10–14) of sustained moisture and warmth to become responsive to germination stimulants. If conditioned seed does not encounter a germination stimulant, it eventually returns to an unresponsive state of secondary dormancy (Matusova et al. 2004). With germination, the *Striga*  radicle emerges from the seed coat and grows up to a centimeter. Once the radicle encounters a potential host root, its apex forms an haustorium that aids attachment and penetration of the epidermis, cortex and ultimately stele where xylem connection is established with the host (LeBlanc et al. 2012). A *Striga* shoot will only emerge with successful vascular connection. Germination without attachment and vascular contact with a suitable host will kill *Striga* since it cannot survive on its own seed reserves. An established parasite eventually emerges from the ground, flowers and sets up to 200,000 seeds/plant. This leads to the formation of vast seed banks, which pose a significant challenge in the management of parasitic weeds, as these seeds can remain dormant and viable in the soil for many years until they encounter a host.

Scientists had long ago observed that the root exudates of clover, maize, sorghum, and linseed contain compounds that induce the germination of parasitic plant seeds. (Saunders, 1933; Brown et al. 1949, 1951). The first witchweed (*Striga lutea* Lour.) germination stimulant was isolated from the root exudate of cotton (*Gossypium hirsutum* L.) and named strigol (Cook et al. 1966, 1972). Over 30 different molecules with similar properties to strigol, collectively referred to as strigolactones (SLs), have been discovered in the plant kingdom since then.

*Germination stimulants*. After its discovery in cotton, strigol was subsequently identified in the root exudates of *Striga* hosts such as maize (*Zea mays* L.), sorghum (*Sorghum bicolor* (L.) Moench), and proso millet (*Panicum miliaceum* L.) (Siame et al. 1993). Two additional germination stimulants were subsequently identified in the root exudates of sorghum, sorgolactone (Hauck et al. 1992), and from cowpea [*Vigna unguiculata* (Walp)], alectrol (which was later identified as orobanchyl acetate; Ueno et al. 2015; Xie et al. 2008). Both share basic chemical structure to strigol. In 1995, Butler coined the name strigolactones for these strigol-related compounds (Butler 1995). Some years later, orobanchol, a germination stimulant for the broomrape, *Orobanche minor* (Smith), was isolated from the root exudate of red clover (*Trifolium pratense* L.) (Yokota et al. 1998). This showed that SLs are used by both *Striga* and *Orobanche* spp. as germination cues to ensure germination in the presence of a host root. Subsequently, sorgomol (Fig. 1; Table 1) was identified in the root exudate of *Sorghum bicolor* and shown to induce germination in *Striga hermonthica* and *O. minor* (Xie et al. 2008). Fabacol, fabacyl acetate (germination stimulants of *O. minor*) and strigone (a highly active germination stimulant of *S. hermonthica*) were isolated from the root exudates of pea (*Pisum sativum* L.) (Xie et al. 2009) and from the non-host, *Houttuynia cordata* (Kisugi et al. 2013), respectively.

The discovery of new, non-canonical SLs that do not possess the consistent ABCDring structure (as shown in Fig. 1) was a fascinating and unexpected development. One such example is the compound methyl carlactonoate (MeCLA), which has been found in *Arabidopsis* (van Ha et al. 2014; Seto and Yamaguchi 2014). Other examples are heliolactone, identified in the root exudate of sunflower (Ueno et al. 2014) and zealactone, identified in maize root exudate (Xie et al. 2017) that induces *S. hermonthica* germination (Charnikhova et al. 2017). Avenaol was identified in the root exudate of black oats (*Avena strigosa* Schreb.) and is a strong germination stimulant for *Phelipanche ramosa*, while it is virtually inactive in *S. hermonthica* and *O. minor* (Kim et al. 2014). To date, all examined plant species have been found to release a combination of multiple SLs. The SL profiles can vary among different species, and sometimes, even within different cultivars of the same species. Moreover, the quantities and proportions of SLs can fluctuate under varying growth conditions and developmental stages (Yoneyama et al. 2008).

*Other roles of strigolactones.* For many years, plant scientists were puzzled by the question of why plants exude SLs into the rhizosphere, thereby inviting parasitic weed attacks. In 2005, Akiyama et al. finally provided an answer to this quandary by demonstrating that SLs play a crucial role in establishing beneficial arbuscular mycorrhizal (AM) fungi symbiosis (Akiyama et al. 2005). Strigolactones stimulate hyphal branching in AM fungi, enabling them to extend towards and colonize the host root. This symbiotic relationship is a vital factor in the growth and survival of terrestrial plants, as demonstrated by its prevalence in approximately 80% of all plant species (Bonfante and Genre 2015; Gutjahr and Parniske 2013).

This mutualistic relationship plays a crucial role in providing the host plant with minerals that are absorbed by the fungal hyphae, effectively expanding the plant's root surface area and allowing it to exploit a much larger volume of soil. In exchange, the heterotrophic fungal partner receives reduced carbon compounds from the plant's photosynthesis to support its own growth (Gutjahr and Parniske 2013; Bonfante and Genre 2015). The benefits of AM symbiosis explain why plants, particularly under phosphorus deficiency, release SLs into the soil (Gutjahr 2014; Khosla and

**Fig. 1** Chemical structures of sorghum strigolactones and the synthetic strigolactone, GR24

**Table 1** Optimized UPLC-MS/MS conditions for analysis of selected strigolactones

MS settings are following: capillary voltage 1.2 kV; ion source/desolvation temperature 120/550 °C; desolvation/cone gas flow 1000/150 L/h; collision gas flow 0.15 ml/min. Individual compounds were analyzed using characteristic quantifying (a) and confirming (b) MRM transitions. Internal standard (c) used for analyte quantification

Nelson 2016). It is plausible to assume that root parasitic weeds, which evolved much later than AM symbiosis, have co-opted the SL signal as a reliable indicator of host presence. This was achieved through the evolution of a highly sensitive SL detection system coupled with the induction of seed germination. It is worth noting that the function of SLs as a plant hormone was reported three years after their role in AM symbiosis was revealed (Gomez-Roldan et al. 2008; Umehara et al. 2008). The identification of SLs as a plant hormone regulating shoot branching and tillering was made possible by the availability of mutants with increased shoot branching and tillering from various plant species. Through genetic analysis and grafting studies, these mutants were classified into two groups: those deficient in the synthesis of a presumed mobile shoot branching-inhibitory signal and those affected in the perception of this signal. The biosynthetic mutants lack SLs, and application of the SL analog GR24 restores their branching and tillering phenotypes. This finding showed that SLs are the elusive shoot branching inhibitory signal. The supposed perception mutants, on the other hand, did not respond to GR24, which further supports the conclusion that SLs are the signal in question. (Gomez-Roldan et al. 2008; Umehara et al. 2008). Thus, SLs are a new plant hormone that plays an important role in the adaptation of plants to nutrient deficiency, regulating senescence, root development, and stem secondary growth (Koltai 2011; Ruyter-Spira et al. 2013; Brewer et al. 2013; Al-Babili and Bouwmeester 2015; Waters et al. 2017; Jia et al. 2018). Moreover, SLs seem to play a role in the plant response to biotic and (other) abiotic stresses (van Ha et al. 2014; Torres-Vera et al. 2014; Wang et al. 2015; Decker et al. 2017).

*Strigolactone biosynthesis*. The discovery in 2008 that strigolactones act as the long-sought branching-inhibiting hormone (Gomez-Roldan et al. 2008; Umehara et al. 2008) facilitated the rapid elucidation of the core strigolactone biosynthesis pathway. This pathway involves a ß-carotene isomerase (DWARF27) and two carotenoid cleavage dioxygenases (CCD7 and CCD8), which produce carlactone, the precursor for all known strigolactones. However, biosynthesis of strigolactones diverges strongly after carlactone, leading to the production of different types of strigolactones, including orobanchol-type and non-canonical strigolactones that lack the classic ABC-ring structure of canonical strigolactones. Our understanding of the details of strigolactone biosynthesis after carlactone is limited, with only a few enzymatic steps identified in *Arabidopsis* towards non-canonical strigolactones and in rice towards orobanchol-type strigolactones (Bouwmeester et al. 2021). Notably, maize strigolactones with unique structures have been identified, but their biosynthesis remains unknown (Charnikhova et al. 2017).

*Strigolactone transport*. Grafting is a common technique used to study hormone and metabolite transport between plant shoots and roots. Mutants lacking strigolactones (SLs) exhibit a distinct high-branching phenotype. However, when these mutants were grafted onto wildtype rootstocks, the bushy phenotype was fully rescued, indicating that SLs are transported from roots to shoots (Kohlen et al. 2011). Conversely, grafting wildtype scions onto SL-deficient rootstocks did not result in increased branching, suggesting that SLs can also be synthesized in aerial tissues (Kohlen et al. 2011). Grafting studies have also shown that SL biosynthesis intermediates can be transported from roots to shoots. For example, grafting of MORE AXILLARY BRANCHING (MAX) mutants *max3* or *max4* onto *max1* rootstocks restored wildtype branching to the mutant shoots, suggesting that carlactone or its derivatives, which accumulate in *max1* rootstocks, are transported into the mutant shoots where they are further metabolized into SLs that inhibit branching (Booker et al. 2005).

Transport of SLs from roots to shoots has been suggested to occur through the xylem (Kohlen et al. 2011). However, labeled orobanchol and 4-deoxyorobanchol, that were applied to the roots, were detected in the shoot, but not in the xylem (Xie et al. 2015). Moreover, transport of the labeled SLs was slower than expected for xylem-mediated movement (Xie et al. 2015). In addition to transport from root to shoot, SLs are exuded into the rhizosphere by specific transporters, such as the Petunia PLEIOTROPIC DRUG RESISTANCE 1 (PhPDR1), an ATP-binding cassette (ABC) transporter (Kretzschmar et al. 2012). PhPDR1 is located in the plasma membrane of sub-epidermal cells in lateral roots, the hypodermal passage cells where AM fungi penetrate the roots. The activity of PhPDR1 may indicate the presence of a local maximum of SL, which could guide AM fungal hyphae towards the hypodermal passage cells. PaPDR1, an ortholog of PhPDR1 found in *P. axillaris*, exhibits asymmetric localization in different root tissues, with its transporter localized at the apical membrane of root hypodermal cells in root tips, potentially facilitating the transport of SLs towards the shoot (Sasse et al. 2015). In hypodermal passage cells above the root tips, PaPDR1 is localized in the outer-lateral membrane, consistent with its postulated role in transporting SLs from roots into the rhizosphere.

*SLs and host specificity*. Many parasitic plant species display host specificity, often with a narrow host range, such as *O. cumana* that mostly only parasitizes sunflower. For other species, the host range is wider, such as *P. ramosa* that can infect several members of both Solanaceae and Brassicaceae (Gibot-Leclerc et al. 2016; Perronne et al. 2017). Within the broad host range *P. ramosa*, however, there are ecotypes that seem to exhibit a narrower host specificty. For example, the germination of *P. ramosa*  seeds harvested from different hosts, is differentially stimulated by the exudates of these different hosts (Perronne et al. 2017). Also in *S. hermonthica*, although it can infect several cereals (maize, sorghum, millet, rice), we find ecotypes that better parasitize millet than sorghum and maize and vice-versa (Kim, 1994; Mohemed et al. 2018). Whether this specificity is based on the recognition of germination stimulants is an intriguing question. Indeed, the sunflower parasite, *O. cumana*, germinates in response to dehydrocostuslactone, a molecule present in the exudate of sunflower, but not to strigolactones (Auger et al. 2012). In *S. hermonthica*, strigolactone composition of the host root exudate seems to play a role in host specificity. For example, a sorghum ecotype of *S. hermonthica* germinates much less well with a millet exudate and vice-versa (Mohemed et al. 2018). In sorghum, maize and rice, *S. hermonthica*  susceptibility seems to correlate with 5-deoxystrigol production (Jamil et al. 2012; Xie et al. 2015; Mohemed et al. 2018). In contrast, orobanchol seems to correlate with reduced susceptibility to *S. hermonthica* (Gobena et al. 2017; Mohemed et al. 2018).

Similar to auxin and other plant hormones, strigolactones also need a receptor to exert their effects on biological processes. The branched phenotype of mutants has been instrumental in identifying components involved in strigolactone perception and downstream signaling. This includes the discovery of two canonical α/β hydrolase fold proteins: the strigolactone receptor, D14, and the homologous receptor, D14like1/KAI2/HTL (abbreviated as HTL) (Waters et al. 2017). D14 is a receptor with hydrolase activity and the strigolactone ligand is hydrolyzed upon binding (Seto et al. 2019). Although the crystallographic studies indicate that the D-ring is essential for the interaction between D14 and MAX2, it remains uncertain whether hydrolysis is a prerequisite for signaling or merely a result (Yao et al. 2016; Seto et al. 2019). When a strigolactone or the D-ring binds to D14, it causes a conformational change that allows D14 to recruit the F-box protein MAX2/D3. MAX2/D3 then targets repressor proteins such as DWARF53 and SMXLs for ubiquitination, leading to their proteasomal degradation. As a result, gene expression is induced and plant development is altered, including the inhibition of bud outgrowth (Al-Babili and Bouwmeester 2015). While D14 has been confirmed as the receptor responsible for regulating plant development through strigolactones, several impressive studies have demonstrated that HTL has evolved new ligand binding specificity in root parasitic broomrapes and witchweeds through duplication and adaptation (Conn et al. 2015; Toh et al. 2015). This facilitated germination of the parasites upon perception of strigolactones secreted by their host. Intriguingly, the function and ligand of HTL in non-parasitic plants is unknown (Waters et al. 2017). With the strigolactone receptors in hand we can now screen for ligands. Uraguchi et al. (2018), for example, used a *S. hermonthica*, HTL to find new synthetic suicidal germination stimulants (Uraguchi et al. 2018).

Testing individual SLs for their ability to induce parasitic plant seed germination has confirmed that seeds of different species differentially respond to specific SLs (Wang and Bouwmeester 2018). For example, germination of *O. minor* is induced with about 200-fold lower *ent-*2'*-epi*-orobanchol concentration than *S. hermonthica*  (Ueno et al. 2011). Similarly, in the same concentration, 5-deoxystrigol only induced germination in *S. hermonthica* but not in of *S. gesnerioides* (Ueno et al. 2011).

*Role of SLs in other belowground interactions of the host*. Plants utilize multiple adaptation strategies when exposed to stress such as phosphate or nitrogen deficiency. The most crucial ones include altering the architecture of roots and shoots, establishing beneficial interactions with microorganisms, and modifying the pH of the rhizosphere (Bouwmeester et al. 2007; Péret et al. 2011; Auger et al. 2012; Kumar et al. 2015). All of these mechanisms aim to enhance the availability of nutrients for plants. It is interesting to note that when plants are grown in media deficient in nitrate, and particularly phosphate, there is a stimulated increase in the production of SLs (López-Ráez et al. 2008; Yoneyama et al. 2012; Marzec et al. 2013; Torres-Vera et al. 2014). There are various indications that the up-regulation of SL production is involved in the plant adaptation to low-nutrient conditions. For instance, in the absence of phosphate, plants tend to prioritize the production of lateral roots (Péret et al. 2011), which increases the surface area in contact with the soil. This adaptation is facilitated by the important role of auxin in initiating lateral root primordia and promoting the emergence of lateral roots (Chiou and Lin 2011; Sun et al. 2014), but SLs also play an important role in the adaptation of root architecture to phosphate deficiency (Ruyter-Spira et al. 2011; Sun et al. 2014; Kumar et al. 2015) and, in cross talk with auxin, SLs induce an increase in lateral root density (Ruyter-Spira et al. 2011). The alteration in root architecture that occurs in response to low-nutrient conditions may also impact the susceptibility of host plants to parasitic infections. This modification increases the likelihood of host roots encountering parasitic plant seeds. Furthermore, the increased production and release of SLs in response to nutrient deficiency not only promotes the colonization of plant roots by beneficial microorganisms but also stimulates the germination of parasitic plant seeds, thereby increasing the risk of infection (Jamil et al. 2012, 2014).

In addition to the interaction with AM fungi (Akiyama et al. 2005; Besserer et al. 2006), SLs were also shown to regulate nodulation. The SL deficient increased branching (*ramosus*) pea *rms1* mutant displayed a reduced nodule number (Foo et al. 2013). Also in soybean, nodulation was decreased in the SL deficient *GmMAX3b*  mutant line while overexpression resulted in an increased nodule number (Haq et al. 2017). The precise mechanism behind this impact of SLs on nodulation is not fully understood, including whether it is due to their signaling function or hormonal effect.

In addition to SLs, plants also exude a wide range of other chemicals into the rhizosphere. These molecules provide carbon for microorganisms and serve as signaling molecules that play a critical role in recruiting and selecting specific microorganisms. Among the most extensively studied examples are phenylpropanoids, which are involved in both symbiotic and pathogenic interactions in the rhizosphere (Abdel-Lateif et al. 2012; Liu and Murray 2016) and allelopathy (Bais et al. 2006).

Recent work on sorghum showed that the *Striga* resistant genotype SRN39, which exudes mostly the SL orobanchol (Gobena et al. 2017) recruited a different microbiome than other genotypes that mostly exude 5-deoxystrigol (Schlemper et al. 2017). Also in rice a significant correlation was detected between orobanchol and a number of micro-organisms, including AM fungi and *Burkholderia*–*Caballeronia*– *Paraburkholderia* and *Acidobacteria* that potentially solubilize phosphate (Kim et al. 2022). Observations suggest that these changes at the microbiome level can mitigate infection of the host by parasitic plants. From a *Striga* suppressive Kenyan soil, bacteria were isolated that induced up to 45% of decay in *Striga* seeds (Neondo et al. 2017). Other mechanisms by which soil micro-organisms could suppress parasitic plants include the production of germination inhibiting factors, inhibitors of radicle growth and haustorium formation, strengthening the vigor of the host plant by activating plant defense mechanisms or competitive utilization of signaling molecules inducing parasitic plant seed germination.

*SLs and parasitic weed management*. Controlling parasitic Orobanchaceae weeds poses several challenges. On the one hand, their parasitic nature makes it difficult to intervene without harming the host plant, due to the physical and physiological connection between the two. On the other hand, the properties of their seeds, such as their small size, fecundity, and easy dispersal, make it challenging to prevent the spread of the weed. These factors lead to a rapid increase in the soil seed bank. Therefore, preventing the distribution of seeds and containing infested areas should be a significant focus of any parasitic weed management strategy, in addition to direct control measures against the parasites (Rubiales et al. 2009; Rubiales and Fernández-Aparicio 2012). In this Chapter, we will not review all possible control and management strategies of parasitic weeds, but focus on methods—that are already used or can potentially be developed—that are based on the importance of SLs in the lifecycle of these parasites. Indeed, there are several strategies of weed management focusing on the SLs, trying to avoid the stimulation of germination, or conversely to favor it, in the absence of a host. These practices are briefly considered in the next sections.

*Host resistance through low strigolactone exudation*. As discussed above SLs are the main germination stimulants for root parasitic plants. In studies that evaluated the induction of parasitic plant seed germination by exudates from different genotypes and cultivars of several crop species, a positive correlation was demonstrated between the SL concentration in the root exudate and the germination rate (Jamil et al. 2011a, b; Fernández-Aparicio et al. 2014; Yoneyama et al. 2015; Mohemed et al. 2018).

An approach for the management of parasitic plants in agricultural crops could thus be to reduce germination of the parasitic plant seeds by reducing the exudation of the germination stimulants. Several studies explored natural variation in germination stimulant production, for example in the New Rice for Africa (NERICA) rice cultivars. This work showed that several NERICA cultivars (1, 2, 5, 10 and 17) displayed post-germination resistance to *S. hermonthica* and *S. asiatica* unlike NERICA 7, 8 and 11, which were susceptible (Rodenburg et al. 2015). In parallel, variation in SL production in the NERICA genotypes was demonstrated, resulting in differences in *Striga* germination induction (Jamil et al. 2011b). The combination, by breeding, of germination-related resistance with post-germination resistance could result in better durable *Striga* resistance (Cissoko et al. 2011; Jamil et al. 2011b).

A pea genotype with reduced SL exudation displayed partial field resistance to *O. crenata* infection (Pavan et al. 2016). Further in sorghum, the *Striga* resistant genotype SRN39 produced much less 5-deoxystrigol than the susceptible Tabat (Yoneyama et al. 2010). Later, SRN39 was shown to produce more orobanchol, instead of 5-deoxystrigol, due to a tentative modification in the SL biosynthetic pathway (Gobena et al. 2017). This mechanism was also observed in a number of other *Striga* resistant sorghum genotypes (Mohemed et al. 2018). Interestingly, the differences in SL composition in maize and sorghum did not affect the level of AM colonization (Gobena et al. 2017).

Besides exploiting natural variation, biotechnological approaches aiming to generate low SL exuding plants could be a strategy to reduce infestation by parasitic plants (Torres-Vera et al. 2014). Indeed, it was demonstrated in tomato that by knocking down SL biosynthesis through genetic modification, the mutant was more resistant to *O. ramosa* infection (Kohlen et al. 2012). A reduction in SL production to obtain parasitic weed resistance was also achieved unintentionally. The broomrape resistant tomato mutant Sl-ORT1 developed by Dor and coworkers was later found out to be SL-deficient and the resistance thus associated to the low amount of exuded SLs (Dor et al. 2010). Breeding, through conventional or biotechnological approaches, for a reduction in exuded SLs potentially also has negative consequences given their importance for the control of shoot and root architecture and the acquisition of nutrients through AM fungi (López-Ráez et al. 2008). This could possibly be prevented by approaches that reduce transport of SLs into the rhizosphere, which is facilitated by an ABC transporter, PDR1 (Borghi et al. 2015). However, under certain abiotic stress conditions, this could still negatively affect the adaptive capacity of plants by hampering mycorrhization. Particularly the example of sorghum shows that solutions in which the composition rather than the level of the SLs is changed may be the best solution (Gobena et al. 2017). Nevertheless, several examples show that a reduction in SL production results in an acceptable level of resistance without large consequences for the plant phenotype (Jamil et al. 2011a, b; Pavan et al. 2016). In order to prevent that this partial germination-based resistance is overcome by parasitic plant populations, a combination of pre-and post-attachment resistance mechanisms is necessary.

In this chapter we describe how to analyse the level of germination stimulant produced by a *Striga* host, with an emphasis on the SLs as the most important class of *Striga* germination stimulants. With the methods described here it is possible to screen collections of germplasm and/or mutants suspected to induce lower *Striga*  germination, to find genotypes that display (partial) *Striga* resistance through a lower (or no) induction of *Striga* germination. We describe how to reproducibly collect the root exudate of hosts, how to process these exudates, partially purify them and analyse them using a *Striga* germination bioassay, for which we will also describe a method to analyse the results through high-throughput image analysis, or using advanced Multiple Reaction Monitoring—Liquid Chromatography Coupled to Mass Spectrometry (MRM-LC-MS).

#### **Protocols**

*Root exudate collection*. One of the of plant adaptive characteristic features is their capacity to constantly communicate with their surrounding environment through metabolites. These chemical compounds are widely diverse and involved in a broad range of responses: attraction of beneficial or harmful micro-organisms, iron chelation, rhizosphere pH modulation, nutrient uptake, etc. It has been shown that to face a specific biotic or abiotic stress plants modulate the composition of their root exudates to attract the right beneficial micro-organisms. Due to coevolution, harmful organisms are often able to hijack these signals. A well-studied dual effect compounds example are the strigolactones (SLs) that the plant uses to attract arbuscular mycorrhizal fungi and that also induce parasitic plant seed germination. In their attempt to mitigate phosphate deficiency through mycorrhization, SL levels in roots and root exudates increase under phosphate stress. Therefore, characterizing the SL profiles of *Striga* hosts is often done under phosphate starvation. In addition to manipulating nutrition, temperature and light to optimize SL exudation, other aspects such as SL stability need to be considered while collecting root exudates. The following steps have been successfully used in our lab to collect root exudates from cereal *Striga*  hosts like maize, millet, rice and sorghum.

Place the desired number of deglumed seeds in a 15 ml Falcon tube. Add 10 mL of 4% sodium hypochlorite with 0.2% Tween-20 and shake the tube for 45 min on a rotating mixer. After removing the bleach, soak the seeds for 30 s in 10 mL of 70% ethanol and wash with 10 mL of sterile MilliQ water (repeat this step three times). Wash the seeds an additional four times with 10 mL of sterile MilliQ water.

Moisten autoclaved 90 mm Whatman® glass microfiber filters (GF/A Grade/ qualitative filter paper) with 3 mL sterile MilliQ water in a sterile 90 mm petri dish, then place 10–15 seeds on each filter paper. Seal the plates with parafilm and wrap them with aluminum foil, then incubate them for two days at 30 °C. Select the best seedlings and transfer them to 50 mL Falcon tubes filled with 45 mL of washed sand. Sand can be prewashed by placing a bulk quantity in a sieve and placing under running water. Move the planted tubes to a greenhouse or growth chamber set at 28 °C/25 °C, 70% relative humidity, 16 h photoperiod, 450 μmol. photons m−2 s−1. This light intensity is ideal but a range of 200–450 μmol. photons m−2 s−1 could be used. Select the better growing plants after two days. Water the plants every two days with 5 mL of modified half strength Hoagland's solution, containing 10% of the normal amount of phosphate. The use of sand for plant growth gives a relatively clean exudate and facilitates the low nutrient treatment. However, it has the disadvantage of drying very quickly. Daily watering is therefore very important for the first three days after the transfer of the seedlings. Grow the plants under these conditions for two weeks.

Elute the exudates with 30 mL of 5% ethanol–water and collect 20 mL. The analyte enrichment can be improved by addition of a suitable organic modifier at 5% to the root exudate collection solvent; also see below (Floková et al. 2020). Keep the exudates on ice for the whole exudate collection process. During the root exudate collection, the stability of SLs can be an issue. Keep the exudates therefore refrigerated until they are combined with the solid-phase extraction (SPE) sorbent. Centrifuge the exudates (4 °C, 3000 rpm for 20 min) and transfer supernatant to a second Falcon tube.

*Processing the exudates, preserving and storing samples*. The process of sample extraction and further purification significantly influences the overall sensitivity of final UHPLC-MS/MS analysis. The universal solid phase polymer-based sorbent for neutral compounds Oasis HLB® (Waters), combining both hydrophilic and lipophilic retention characteristics, was successfully applied in pre-concentration of broad spectrum of strigolactones (Floková et al. 2020). The additional selective purification step using polar interactions with silica sorbent eliminates the co-extraction of lipophilic contaminants, greatly improves the sample purity and decreases detection limits of mass analysis on the sample background. The complete sample preparation procedure is shown in Fig. 2.

Filter ~ 100 mL of collected exudates using a Büchner vacuum funnel1 and transfer 20 mL of filtered exudates to a 50 mL Falcon tube. Add the mixture of internal standards (authentic or stable deuterium-labelled counterparts of strigolactones) to the sample and vortex.2

Before sample concentration with SPE column, activate the sorbent of Oasis HLB® with 100% acetonitrile (3 mL) and subsequently equilibrate with 3 mL of 5% ethanol/water (v/v). Solvents and sample extracts are passed through the SPE cartridge Oasis HLB® at a consistent flow rate 1–2 drops per second.

Load the extract of root exudates onto conditioned SPE sorbent (~ 50 mL). Discard the flow-through fraction. Wash sorbent with deionized water (3 mL) to remove

<sup>1</sup> The complete set up for pressure assisted filtration consists of a Büchner vacuum filter funnel (100 mL volume), side-arm flask (250 mL volume), adapters, silicone pump tubing, portable diaphragm vacuum pump (max. vacuum 160 mbar), grade 1 filter paper with pore size 11 μm.

<sup>2</sup> Internal standard stock solutions are generally prepared beforehand by dissolving each standard in 50% acetonitrile/water (v/v) to final concentration 10−5 M, and stored at <sup>−</sup> 20 °C. Working solution of internal standards is a mixture of stock solutions, diluted to final concentration 5 <sup>×</sup> <sup>10</sup>−7 M. The mixture of internal standards used in this example was GR24 and [2H6]-5-deoxystrigol prepared in 50% acetonitrile/water (v/v) mixed to final concentration 5 <sup>×</sup> <sup>10</sup>−7 M (5 pmol of each compound in 10μL of solution).

#### 126 B. Thiombiano et al.

**Fig. 2** Analysis of *Striga* germination stimulants. Root exudate collection and concentration for strigolactones measurement using UPLC-MRM-MS/MS (left panel) and image analysis pipeline for analysis of *Striga* germination rate (right panel)

salts and unretained compounds. Discard the flow-through fraction. Place 4 mL borosilicate glass vials in the rack of a solid-phase extraction (SPE) glass block vacuum manifold (e.g., 24-port manifold lid) and perform the elution with 80% acetone (v/v, 3 mL). Evaporate samples to dryness using vacuum sample concentrator. Keep dry samples at − 20 °C until the following purification step.

To purify the sample, dissolve dry sample in 150 μL of 100% ethyl acetate and vortex. Fill the reconstituted sample up to 3 mL with 100% hexane (2.85 mL). Before sample purification with SPE column, activate the sorbent of Strata® SI-1 Silica with 100% ethyl acetate (3 mL) and subsequently equilibrate with 100% hexane (3 mL). Solvents and sample extracts are passed through the SPE cartridge Strata® SI-1 Silica at a consistent flow rate 1–2 drops per second. Avoid drying out the silica-based SPE sorbent bed and loss of column capacity. Load dissolved sample onto conditioned SPE sorbent (3 mL). Discard the flow-through fraction. Wash the sorbent with 100% hexane (3 mL). Discard the flow-through fraction. Place 4 mL borosilicate glass vials in the rack of vacuum manifold and perform the elution with 80% ethyl acetate/ hexane (v/v, 3 mL). Evaporate samples to dryness using vacuum sample concentrator. Keep dry samples at − 20 °C until the final LC–MS analysis.

*Germination stimulant analysis using UPLC-ESI–MS/MS: strigolactones*. Ultra-high performance liquid chromatography (UHPLC) coupled to fast scanning tandem mass spectrometry (MS/MS) is routinely applied for simultaneous profiling and quantitation of strigolactones, isolated from different sample matrices (Boutet-Mercey et al. 2018; Floková et al. 2020; Rial et al. 2019, p. 20; Sun et al. 2014). After analyte enrichment and additional purification using SPE-based sorbents, special attention has to be paid to suitable chromatography system and mobile phase composition. The sufficient retention of strigolactones is achieved on reversed-phase ethylenebridged hybrid polymer-based columns (BEH) packed with sub-2-micron particles, improving both separation efficiency and speed of analysis (Fig. 3). The presence of eluent additive formic acid in dual-component mobile phase (water/acetonitrile) facilitates the chromatographic resolution between strigolactone stereoisomers with the same MS-fragmentation pattern, such as 5-deoxystrigol and 4-deoxyorobanchol (Floková et al. 2020). However, the main reason of mobile phase acidification is to support protonation of the strigolactone molecules [M + H]+ in the electrospray (ESI) source, operating in positive mode, and thus improve analyte extraction by the mass analyzer. Compounds are analysed in multiple reaction monitoring mode (MRM) using two specific precursor-to-product ion transitions, listed in Table 1.

To analyse SLs, reconstitute the dried sample with 100 μL of 25% acetonitrile/ water (v/v) and vortex. Transfer the dissolved sample onto the membrane of a 750 μL micro-centrifugal nylon filter with pore size 0.2 μm and spin in a pre-cooled (4 °C)

**Fig. 3** The chromatographic separation of five authentic strigolactones coupled to internal standards with similar retention time: strigol, orobanchol and sorgomol to GR24 (red peak line); sorgolactone and 5-deoxystrigol (5-DS) to [2H6]-5-deoxystrigol ([2H6]-5-DS, blue peak line). Separation is performed on Acquity UPLC™ BEH C18 column

centrifuge at 10,000 g for one minute. Transfer the eluent to a 250 μL glass insert in a 1.5 mL LC/MS autosampler vial. Place the vials in the autosampler of the UPLC System,3 maintained at 10 °C. Inject 5 μL of the sample onto an analytical Acquity UPLC™ BEH C18 column (2.1 × 100 mm, 1.7 μm particle size). Separate analytes with a 12 min binary gradient elution, consisting of 15 mM formic acid in both water (eluent A) and acetonitrile (eluent B), at flow rate 0.45 mL/min and constant temperature of 45 °C. Set the elution profile as follows: 0–0.4 min initial isocratic elution at 15% B; linear gradient increase to 27, 40 and 65% B in 0.65, 5 and 8 min, respectively; isocratic elution at 65% until 8.7 min. Finally wash the column with 95% B for 1.6 min and equilibrate back to initial conditions (15% B) for 1.7 min. The retention time of individual compounds is listed in Table 1.