(±)-Catechins inhibit prehaustorium formation in the parasitic weed Phelipanche ramosa and reduce tomato infestation
Abstract
BACKGROUND
Phelipanche ramosa L. (Pomel) is a noxious parasitic weed in field and vegetable crops in Mediterranean countries. Control of this pest is complex and far from being achieved, and new environmentally-friendly strategies are being sought. The present study evaluates the possibility of using (±)-catechins as a natural herbicide against broomrapes.
RESULTS
The results show that (±)-catechins have no effect on GR24-induced germination over a wide concentration range (10−4 to 10−10 m), nor on radicle elongation after germination, but strongly inhibit, at 10−4 and 10−5 m, prehaustorium formation in response to the haustorium-inducing factor, cis/trans-zeatin. Accordingly, pot experiments involving the supplies of 10−5 m of (±)-catechins to tomato plants infested or not with P. ramosa demonstrate that (±)-catechins do not influence growth of non-parasitized tomato plants and prevent heavy infestation by strongly reducing parasite attachments and inducing parasite necrosis once they are attached.
CONCLUSION
This study points the potential use of (±)-catechins for parasitic weed control. It raises also the question of the mechanisms involved in the inhibition of prehaustorium formation and the necrosis of parasite attachments in response to (±)-catechins application. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
1 INTRODUCTION
Broomrapes (Orobanche and Phelipanche spp.) are root parasitic plants, and some of them are noxious weeds in economically important crops, such as Phelipanche ramosa L. (Pomel) and the closely related Phelipanche aegyptiaca in field and vegetable crops around the Mediterranean Sea.1 Integrated pest management programmes (IPMPs) exist to control infestations and the soil seed bank, preserving yields: solarization, fumigation and controlled use of herbicides; use of resistant varieties or, in the absence of them, tolerant varieties; integration into rotations of false host crops, and/or cross-cultural trap crops.2 IPMP efficiency depends on crop and infestation level. In addition, several studies point the value of herbivorous insects, fungal pathogens or toxins, or natural compounds (amino acids) in reducing parasitism under controlled conditions, with the hope of including them in the IPMPs in the near future.1, 2 To preserve yields, it is recommended to diversify crops and use tolerant varieties in the absence of effective control.3-5
One of the biological specificities of parasitic plants is the development of a specific organ, the haustorium, necessary for attachment to host's roots and spoliation of water and nutrient. Papillae at the radicle apex (prehaustorium) form after germination, which promote adhesion to the host root surface. Invading the cortex and endodermis of the host root, the haustorial cells connect to vascular tissues, strengthening their attachment to the host.6, 7 Early interaction between host and parasite occurs as allelochemical compounds released by host roots cause germination and haustoriogenesis of the parasitic weed.8, 9
Catechins are natural flavonoids present in many food and medical plants, such as tea, legumes and Rubiaceae. Non-esterified catechins include catechin, gallocatechin, epicatechin, and epigallocatechin, while esterified catechins include epigallocatechin gallate, epicatechin gallate, gallocatechin gallate, and catechin gallate, with various biological activities depending on their chemical structure. Potent benefits for human health through antioxidant and pro-oxidative properties are well documented and accordingly they are increasingly used for medical, pharmaceutical and cosmetic applications.10 Accumulation of catechins is also associated with the protection of several plant species against bioaggressors.11, 12 Interestingly, treatment with gallate catechins induces salicylic acid (SA) signalling and plant defence against fungal pathogens.13, 14 In addition, literature reports the combination of antimicrobial activity and phytotoxicity for (±)-catechins. While their anti-oxidant property is not a determining factor,15 the lipophilicity and membrane-disrupting capacity of (±)-catechins derivatives contribute to antimicrobial activity.16 As evidence for phytotoxicity, the root exudation of catechins by Centaurea stoebe, a noxious invasive weed in North America, is a mechanism to gain advantage over native plants.17 According to Kaushik et al.,18 the activation of cell death genes by catechins triggers reactive oxygen species (ROS)-mediated rhizotoxicity in Festuca idahoensis and Arabidopsis thaliana, the toxicity being entirely due to the (−)-catechin enantiomer.15 (±)-Catechins could then be used as natural antimicrobial and herbicides for plant protection. In this context, parasitic plants are particular weeds due to attachment and vascular connection to host plants. Interestingly, Lewis et al.19 demonstrated that catechins from tea extract were effective in reducing the parasitism of Castilleja indivisa, a facultative root parasitic plant, and of Cuscuta pentagona, an obligate stem parasite, and suggested that catechins specifically trigger inhibition of parasite attachment to the host. Allelopathic activities of other natural flavonoids in relation to weedy broomrapes has been recently reported.20, 21 Two flavonoids purified from Conyza bonariensis, (4Z)-lachnophyllum lactone and (4Z,8Z)-matricaria lactone, strongly inhibit the radicle growth of Orobanche cumana, Orobanche minor, Orobanche crenata, and P. ramosa. Some flavonoids structurally close to catechins, hispidulin purified from Conyza bonariensis,20 quercetin purified from Fagopyrum esculentum and 3-O-acetylpadmatin purified from Dittrichia viscosa21 inhibit also the radicle growth of P. ramosa. In addition, quercetin and 3-O-acetylpadmatin, but not hispidulin, like haustorium inducing factors (HIFs), induce the formation of haustorial papillae (prehaustorium) in this species. Experiments of structure–activity relationships using methyl ether derivates of quercetin suggest that the presence of two ortho-free hydroxyl groups of C ring, like catechins, could be an important feature to impact activity.21 However, assays using catechins are missing in these studies. In addition, the efficiency of such flavonoids to prevent parasite attachment to the host plant should be assessed.
Consequently, this study questions the activity of catechins on the radicle growth and the prehaustorium formation of parasitic plants, and about their potential use for weed control in infested crops. The branched broomrape, P. ramosa, is an appropriate model plant to address this issue. Indeed, since both germination and prehaustorium formation can be induced in vitro, in the absence of the host plant, by using synthetic germination stimulants (here, GR24, a synthetic strigolactone) and HIFs (here, cytokinins, cis/trans-zeatin, and c/tZ), it was quite simple in the present study to assess the inhibitory effect of compounds such as (±)-catechins over a wide range of concentrations. Then, the efficiency of (±)-catechins to prevent infestation of host plants (here, tomato) could be evaluated in pot experiments under controlled conditions.
2 MATERIALS AND METHODS
2.1 Plant materials
Phelipanche ramosa (L.) Pomel seeds (genetic group 122) were collected in 2021 from mature broomrape flowering spikes in an infested oilseed rape field at Saint Martin de Fraigneau (France) and stored at 25 °C in the dark before use. Surface-sterilization of seeds (200 mg) consisted in 5 min incubation in 40 mL of 12% (v/v) sodium hypochlorite and thorough rinsing three times in sterile water.23 Tomato seeds (Solanum lycopersicum L., cv. Saint Pierre, Vilmorin) were purchased from a market.
2.2 Germination and radicle growth assays
Sterilized P. ramosa seeds were suspended (10 mg mL−1) in incubation medium containing 1 mM HEPES-KOH (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-potassium hydroxide, pH 7.5) and 0.1% (w/v) plant preservative mixture (PPM, Sigma-Aldrich, St Louis, MO, USA). Seeds were conditioned by distributing 25 μL of seed suspension (around 100 sterilized seeds) per well in a 96-well plate (Cell Culture Multiwell Plate Cellstar; Greiner Bio-One, Frickenhausen, Germany), and then stored for 7 days at 21 °C in the dark (Fig. 1(A)). Rac-GR24 (synthetic germination stimulant, kindly provided by Dr B. Zwanenburg, with a final concentration of 10−9 m in 0.02% (v/v) acetone) and/or (±)-catechins (Sigma-Aldrich, 290.27 g mol−1, purity ≥ 96%, final concentration of 10−4 to 10−10 m in 0.02% (v/v) acetone) were added to each well and volumes were adjusted to 100 μL with sterile distilled water. (+)-Catechin and its isomer (−)-epicatechin are in a 50:50 mixture. A 0.02% (v/v) acetone solution was used as negative control. Subsequently, plates were incubated for 3 days at 21 °C in the dark and germinated seeds were counted under a stereo microscope (Olympus SZX10; Olympus Europa GmbH, Hamburg, Germany). Seeds were considered as germinated when the radicle protruded out of the seed coat (Fig. 1(B)). Data are mean ± standard deviation (SD) (n = 15, Tukey's HSD (honestly significant difference) for comparative analysis of treatments, P < 0.05).
In addition, radicle growth was monitored for germinated seeds treated or not with (±)-catechins at various final concentrations (10−4, 10−5, and 10−6 m in 0.02% (v/v) acetone). A 0.02% (v/v) acetone solution was used as negative control. Data are mean ± SD (n = 25, Tukey's HSD for comparative analysis of treatments, P < 0.05).
2.3 Prehaustorium formation assays
Conditioned P. ramosa seeds (Fig. 1(A)) were rinsed three times with sterile distilled water and suspended (2.5 mg mL−1) in germination medium (0.5 mm HEPES, pH 7.5, 0.05% (w/v) PPM, rac-GR24 10−9 m). A 100 μL aliquot of GR24-treated P. ramosa seeds (~25 seeds) was then distributed in each well in 96-well plates and placed to germinate at 21 °C in the dark for 4 days. After liquid removal from the wells, germinated seeds were suspended in 100 μL of 10−8 m of c/tZ (cytokinin HIF, in 0.02% (v/v) acetone) and/or various concentration (10−4 to 10−10 m in 0.02% (v/v) acetone) of (±)-catechins.9 Controls corresponded to treatment of germinated P. ramosa seeds with 0.02% (v/v) acetone alone. Papillae formation at the radicle apex of germinated seeds was monitored 3 days after treatments under a stereo microscope (Olympus SZX10; Olympus Europa GmbH, Hamburg, Germany) (Fig. 1(C)). Data are mean ± SD (n = 15, Tukey's HSD for comparative analysis of treatments, P < 0.05).
2.4 Tomato infestation assays
Broomrape seeds (50 mg per pot) were mixed with a sand–vermiculite mixture (50–50% v/v) in a 1.3 L pot. Two tomato seeds were sown into each pot. Two weeks after emergence, tomato seedlings were thinned to one per pot. Pots containing broomrape-free soil were used as controls. Cultures were maintained in a growth chamber at 21 °C/18 °C (day/night temperature) and a 16 h photoperiod (300 μmol m−2 s−1 photosynthetic active radiation), and fertilized weekly with 50 mL of nutritive solution (50% Coïc solution24). Controls and treatments consisted in supplying each pot with 50 mL of 0.02% acetone and 10−5 m (±)-catechins (in 0.02% acetone), respectively, at 14-, 28-, and 35-days post-infection (DPI). Tomato plants were uprooted at 63 DPI before flowering and fruiting. At this date, broomrape attachments were easily detectable on host roots. The total number of broomrape attachments per host plant was determined as well as the fresh weight (FW) and dry weight (DW) of tomato roots and aerial parts. DW was determined following incubation of fresh plant material in an oven at 80 °C for 48 h. The data are mean ± SD (n = 15, Tukey's HSD test for comparative analysis of treatments, P < 0.05 and P < 0.01).
3 RESULTS
3.1 (±)-Catechins have no effect on P. ramosa germination and radicle elongation
In vitro assays show that (±)-catechins used at different concentrations (10−4 to 10−10 m) have no effect on seed germination when stimulated by GR24 (Table 1), since the germination rate is constantly high around 80% whatever the treatment. In addition, as expected and as shown with the control without GR24, (±)-catechins do not induce germination.
| Treatment | Germination rate (%) |
|---|---|
| Negative control (0.02% acetone) | 0a |
| Positive control (10−9 m GR24) | 82.3 ± 1.7b |
| 10−4 m (±)-catechins | 0a |
| 10−4 m (±)-catechins + 10−9 m GR24 | 82.4 ± 3.6b |
| 10−5 m (±)-catechins | 0a |
| 10−5 m (±)-catechins + 10−9 m GR24 | 84.0 ± 1.5b |
| 10−6 m (±)-catechins | 0a |
| 10−6 m (±)-catechins + 10−9 m GR24 | 83.7 ± 4.1b |
| 10−7 m (±)-catechins | 0a |
| 10−7 m (±)-catechins + 10−9 m GR24 | 80.5 ± 5.2b |
| 10−8 m (±)-catechins | 0a |
| 10−8 m (±)-catechins + 10−9 m GR24 | 84.3 ± 4.0b |
| 10−9 m (±)-catechins | 0a |
| 10−9 m (±)-catechins +10−9 m GR24 | 79.4 ± 3.1b |
| 10−10 m (±)-catechins | 0a |
| 10−10 m (±)-catechins +10−9 m GR24 | 82.3 ± 1.6b |
- Note: GR24 and (±)-catechins were dissolved in 0.02% acetone. Means are values ± standard deviation (n = 15). Means with the same letter are not significantly different from each other (Tukey test, P < 0.05).
- Abbreviation: GR24, synthetic stimulant of germination.
In addition, (±)-catechins (10−4 to 10−6 m) were assessed on radicle elongation following germination. Neither phenotype nor growth was affected by the treatments (Figs 2 and 3).
3.2 (±)-Catechins strongly inhibit cytokinin-mediated prehaustorium formation
(±)-Catechins at 10−4 and 10−5 m strongly inhibit cytokinin-mediated papillae formation (Fig. 4), whereas lower concentrations (10−6 to 10−10 m) are inactive. Furthermore, as expected and confirmed by the controls without GR24 and c/tZ, (±)-catechins did not induce prehaustorium formation.
3.3 (±)-Catechin applications reduce tomato infestation and promote parasite necrosis
Infestation was assessed following the supplies of 10−5 m (±)-catechins, a concentration strongly active in the previous in vitro assays (Fig. 5). A two-fold decrease in viable parasite attachments is observed at 63 DPI following the treatments. In addition, catechin applications promoted necrosis of young attachments (Fig. 5), the rate of which was five-fold higher. Applications of the solvent alone (0.02% acetone, controls) did not impact host infestation nor induced parasite necrosis.
3.4 (±)-Catechins have no toxicity on tomato development
Neither 0.02% acetone nor 10−5 m (±)-catechins in acetone 0.02% affect tomato development (Table 2). No rhizotoxicity of catechins is observed, as demonstrated by non-significant changes in root FW and DW of treated and non-parasitized plants. In addition, treatments did not impact FW and DW of aerial parts.
| Treatment | Roots | Aerial parts | ||
|---|---|---|---|---|
| FW (g) | DW (g) | FW (g) | DW (g) | |
| Non-infested tomato | ||||
| Control (0.02% acetone) | 11.81a ± 1.43 | 1.08b ± 0.10 | 51.62c ± 3.77 | 6.16d ± 0.91 |
| 10−5 m (±)-Catechins | 12.02a ± 1.42 | 1.04b ± 0.12 | 51.06c ± 5.45 | 6.09d ± 1.73 |
| Infested tomato | ||||
| Control (0.02% acetone) | 11.48a ± 1.88 | 1.15b ± 0.33 | 51.72c ± 6.89 | 5.94d ± 1.28 |
| 10−5 m (±)-Catechins | 12.96a ± 1.94 | 0.91b ± 0.26 | 50.27c ± 6.69 | 5.52d ± 1.35 |
- Note: (±)-Catechins were dissolved in 0.02% acetone. Tomato plants were uprooted at 63 days post-infection. Means are values ± standard deviation (n = 15). Means with the same letter are not significantly different from each other (Tukey test, P < 0.05).
- Abbreviations: FW, fresh weight; DW, dry weight.
4 DISCUSSION
(±)-Catechins are a potent natural herbicide due to the specific rhizotoxicity of (−)-catechin enantiomer.15 In the present study, (±)-catechins were used and no phytotoxicity was observed on tomato plants when supplied with 10−5 m (±)-catechins. The effect of (±)-catechin treatments to improve host plant protection against P. ramosa could then be assessed.
According to Lewis et al.,19 a single application of mixed catechin tea extract (10 g L−1 PP60, i.e., 34 mm catechins, mainly epigallocatechin gallate) on A. thaliana shoots before parasite inoculation reduced further infestation by the stem parasitic plant Cuscuta pentagona (field dodder). Treatment did not affect host plant. On the contrary, while the parasite's prehaustorial development, consisting in germination and shoot growth prior attachment, did not fail with the preventive treatment, the success of the parasite's shoot attachment to host shoot decreased. This suggests that catechins specifically disrupt the haustorium formation in Cuscuta pentagona. The present study confirms the inhibitory activity of (±)-catechins on the prehaustorium formation in the root parasitic plant P. ramosa. The findings demonstrate that low concentrations of (±)-catechins (10−4 and 10−5 m) do not affect GR24-induced germination and radicle elongation, but prevent cytokinin-induced prehaustorium (adhesive papillae) formation in this species. In addition, pot experiments clearly show that applications of (±)-catechins at 10−5 mm in the rhizophere of tomato plants challenging with P. ramosa do not affect the host plant but strongly decrease parasite attachment. Interestingly, treatments promote also parasite necrosis when attachments succeed. Those findings suggest that (±)-catechins are efficient in lowering host infestation by inhibiting pre- and post-attachment stages of parasite development. When applied in the soil, (±)-catechins may act directly on the parasitic plant and prevent prehaustorium formation. Regarding attachment necrosis, the hypothesis of a direct necrogenic activity of catechins on tubercles cannot be ruled out while (±)-catechins did not induce necrosis of treated radicles (Fig. 2), nor that of induced resistance in tomato following applications of catechins. Further studies are needed to test these hypotheses and to decipher the involved mechanisms. Our findings are consistent with previous studies20, 21 suggesting that catechin-like compounds sharing the presence of two ortho-free hydroxyl groups of C ring, display allelopathic activities in relation to parasitic weeds, notably P. ramosa. However, our findings suggest that these activities in P. ramosa differ according to the catechin-like compound since, at similar concentrations (10−5 and 10−4 mm), (±)-catechins are inactive on the radicle growth and inhibit the prehaustorium formation while quercetin and 3-O-acetylpadmatin inhibit the radicle growth and induce the prehaustorium formation.21
Mechanisms involved in the failure of the cytokinin-induced prehaustorium formation and parasite attachment to host roots by catechins need future investigations, notably on the potent disruption of cytokinin signalling. In the light of the strong inhibition (at the μm level) of specific protein kinases and related signalling pathways by epigallocatechin 3-gallate in human cells,25-27 the cytokinin reception by hybrid histidine protein kinases (cytokinin receptors) could be affected by catechins. In addition, future investigations are required on the effects of catechins on the enzymes involved in cell wall modification during papillae formation,9 and host root invasion by intrusive haustorial cells, such as pectin methyl esterases (PMEs). Indeed, Lewis et al.28 showed that catechins inhibit fungal and plant PMEs.
To conclude, this research is a complement to earlier studies on the benefits of catechin-like compounds for plant protection, particularly against parasitic weeds. As low concentrations are efficient in reducing parasite attachment without affecting the host plant and the parasite seed germination, (±)-catechins could help with crop protection and decrease in P. ramosa seed banks in soils due to attachment failure, resulting in suicidal germination.29 Pure catechin compounds can be chemically synthesized,30 but they are mostly isolated from plant sources using various techniques including solvent extraction (water, ethanol), chromatography (such as high-performance liquid chromatography) and membrane filtration. (±)-Catechins are prevalent in (green) tea leaves and cocoa, and, in smaller amounts, in fruits (berries, grape seeds) and bark of few trees (acacia for example).31, 32 Hence, applying (±)-catechins to soils, notably in vegetables crops as it is done with more or less effectiveness with herbicides to control broomrape in processing tomatoes33, 34 may contribute to control more efficiently the parasitic weed. Screening wild tomato species for catechin production, which can be used as rootstocks, could be a better solution than using soil application.35 Depending on the country's legislation regarding the use of genetically modified organisms (GMOs) in agriculture, other solutions may be considered, such as transgenic rootstocks producing catechins and tomato rhizosphere microbes engineered to produce catechins and added to transplant plugs.36 Finally, future studies should be also addressed to provide clarification about the most active catechin epimer in the balanced mixture used in the present study and their potential interactivity.
AUTHOR CONTRIBUTIONS
CV, EB, and PS conceived and designed the experiments. CV and EB carried out the experiments. CV, PD, and PS wrote the manuscript.
ACKNOWLEDGEMENTS
The authors would like to thank Johannes Schmidt from US2B for his technical expertise in plant cultivation.
CONFLICT OF INTEREST STATEMENT
The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this article.
Open Research
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
