New Rice for Africa (NERICA) cultivars exhibit different levels of post-attachment resistance against the parasitic weeds Striga hermonthica and Striga asiatica

Authors


Author for correspondence:
Julie D. Scholes
Tel: +44 (0)114 222 4780
Email: j.scholes@sheffield.ac.uk

Summary

  • Striga hermonthica and S. asiatica are root parasitic weeds that infect the major cereal crops of sub-Saharan Africa causing severe losses in yield. The interspecific upland NEw RICe for Africa (NERICA) cultivars are popular amongst subsistence farmers, but little is known about their post-attachment resistance against Striga.
  • Here, we evaluate the post-attachment resistance levels of the NERICA cultivars and their parents against ecotypes of S. hermonthica and S. asiatica, characterize the phenotype of the resistance mechanisms and determine the effect of Striga on host biomass.
  • Some NERICA cultivars showed good broad-spectrum resistance against several Striga ecotypes, whereas others showed intermediate resistance or were very susceptible. The phenotype of a resistant interaction was often characterized by an inability of the parasite to penetrate the endodermis. Moreover, some parasites formed only a few connections to the host xylem, grew slowly and remained small.
  • The most resistant NERICA cultivars were least damaged by Striga, although even a small number of parasites caused a reduction in above-ground host biomass. The elucidation of the molecular genetic basis of the resistance mechanisms and tolerance would allow the development of cultivars with multiple, durable resistance for use in farmers’ fields.

Introduction

Rice is an economically important cereal crop in sub-Saharan Africa (Balasubramanian et al., 2007) and is mostly cultivated by resource-poor farmers (Nwanze et al., 2006). Both cultivated rice species, Oryza sativa (L.) and Oryza glaberrima (Steud), are grown in Africa. Oryza glaberrima germplasm, domesticated in West Africa, possesses useful genetic traits, such as weed competitiveness and good levels of resilience against abiotic and biotic stresses (Sarla & Swamy, 2005), but generally has a low yield potential and unfavourable agronomic characteristics, such as grain shattering and lodging (Koffi, 1980). Oryza sativa (L.), originating from Asia, is most appreciated for its high yield and grain quality. To take advantages of the superior traits from both species, the Africa Rice Center and partners developed interspecific rice cultivars for rain-fed upland ecosystems, called NERICA (NEw RICe for Africa), using backcross coupled with double haploid breeding (Jones et al., 1997a,b,c). These cultivars are currently distributed across Africa and are popular among subsistence rice farmers (Diagne, 2006; Kijima et al., 2006). Despite the introduction of improved cultivars, such as the NERICAs, the average yield obtained in rain-fed upland rice in sub-Saharan Africa is only c. 1 ton per hectare because of biophysical production constraints, including biotic stresses and the low capacity of resource-poor rice farmers to use inputs (Balasubramanian et al., 2007). Weed-inflicted yield losses in rain-fed upland rice (despite control efforts) are estimated to be 16%, equivalent to an estimated annual loss of US$ 418 M for African economies (Rodenburg & Johnson, 2009). Increasingly important weeds in rice are the parasitic weeds, with Striga spp. being the most prominent (Rodenburg et al., 2010).

Striga hermonthica (Del.) Benth. and Striga asiatica (L.) Kuntze are the most economically important root parasitic weeds in Africa (e.g. Mohamed et al., 2006; Parker, 2009). Infection with Striga leads to stunting and loss of grain yield in upland rice (Johnson et al., 1997). As obligate hemiparasites, they only germinate in the presence of host-derived stimulants, called strigolactones (Bouwmeester et al., 2003; Yoneyama et al., 2010). Each germinated Striga seedling forms a radicle which, in response to host-derived haustorial initiation factors, forms an organ called the haustorium. On contact with a host root, the haustorium develops a wedge-shaped group of cells that penetrates the host root cortex and endodermis to establish parasite–host xylem–xylem connections. This allows the transfer of water, carbohydrates and nutrients from host to parasite (Parker & Riches, 1993; Press & Graves, 1995). Once attached to the host root, the parasite grows towards the soil surface, emerges above the ground and flowers to produce many tiny seeds which can remain viable in the soil for many years (Parker & Riches, 1993). Striga infection distorts host plant growth and development very early after parasite attachment, although the severity of these effects depends on many factors, such as nitrogen availability and host genotype (tolerance), Striga species and ecotype (determining virulence), and infection time and level (Cechin & Press, 1993; Gurney et al., 1999). Because Striga has an impact on the host so soon after attachment, effective control methods should either prevent Striga attachment or its development beyond attachment (Scholes & Press, 2008). Both can be achieved through the use of improved cultivars. Host genotypes producing small amounts or less effective types of germination stimulants (as shown by Jamil et al., 2011) prevent parasite attachment, whereas genotypes with post-attachment resistance prevent parasite development (e.g. Gurney et al., 2006). The use of such resistant cultivars is commonly considered to be an effective and affordable component of an integrated Striga control strategy (e.g. Haussmann et al., 2000; Rodenburg et al., 2005; Yoder & Scholes, 2010).

Previous studies on rice have shown that cultivars exhibiting resistance (i.e. with only a few emerged parasites) may be an effective Striga control method (Harahap et al., 1993; Johnson et al., 1997). Gurney et al. (2006) demonstrated that rice germplasm contains sources of post-attachment resistance to S. hermonthica. For instance, Nipponbare, an O. sativa japonica lowland cultivar, exhibits a high resistance response, particularly to S. hermonthica, with parasites failing to make xylem–xylem connections after attachment to the host. Apart from the cultivars and resistance mechanisms identified in these studies, few resistant rice cultivars have been found that are well adapted to upland ecosystems (Rodenburg et al., 2010), and a good understanding of the molecular genetic basis of host post-attachment resistance to Striga is still lacking (Yoder & Scholes, 2010).

Despite the wide distribution of NERICA cultivars in Striga-infested regions in Africa, very little is known about their susceptibility to different species and ecotypes of Striga, information which is critical to the selection of the most appropriate cultivar for particular regions or agro-ecological zones. The objectives of the current study are to determine the post-attachment resistance levels of all 18 upland NERICA cultivars and their parents to ecotypes of S. hermonthica and S. asiatica, to characterize the phenotype of the resistance mechanisms and to evaluate the effect of Striga on the biomass of the NERICA cultivars.

Materials and Methods

Plant materials

Eighteen upland NERICA cultivars were developed by AfricaRice from three series of crosses between the O. glaberrima cultivar CG14 and one of three O. sativa ssp. japonica cultivars WAB56-104 (NERICA cultivars 1–11), WAB56-50 (NERICA cultivars 12–14) or WAB181-18 (NERICA cultivars 15–18) (Jones et al., 1997a). The NERICA cultivars 1–18 and their parents were all evaluated in the present study. Rice seeds were provided by the Genetic Resources Unit of AfricaRice. The rice cultivars Nipponbare, Kasalath, Koshihikari and IAC165 (Gurney et al., 2006) were used as reference cultivars in this study. The origin and description of the different S. hermonthica and S. asiatica seed populations (ecotypes) used in this study are shown in Table 1.

Table 1. Striga species and ecotypes used to infect the NEw RICe for Africa (NERICA) cultivars
Striga speciesEcotype nameDetailsLatitude : longitude
S. hermonthicaSh-KibosCollected from maize (H511) growing at the Kenyan Agricultural Research Institute (KARI), Kibos, Kisumu, Kenya in 19970°02′20″S : 34°47′57″E
S. hermonthicaSh-BusiaCollected from maize growing in a farmer’s field near Busia, Kenya in 20090°28′N : 34°05′E
S. hermonthicaSh-MedaniCollected from sorghum growing near Wad Medani, Sudan in 200614°24′N : 33°31′E
S. hermonthicaSh-KoutoCollected from rice (NERICA 1) growing in Kouto near Korhogo, Ivory Coast in 200909°24′23′′N : 05°31′37′′W
S. asiaticaSa-USACollected from maize growing in North Carolina, USA in 1989. Seeds have bulked on maize (WH 505) at the University of SheffieldNorth/South Carolina
S. asiaticaSa-KyelaCollected from rice (cv Supa India aka Kilombero) growing in Kyela near Mbeya, Tanzania in 20099°37′30″S : 33°52′30″E

Growth and infection of rice plants with Striga

Rice seeds were germinated between blocks of moistened horticultural rockwool (growdan®Vital, Growdan, Roermond, The Netherlands) for 6 d, after which a single rice seedling was transferred to a root observation chamber (rhizotron), as described previously by Gurney et al. (2006). Each rhizotron consisted of a 25 × 25 × 2 cm3 Perspex container packed with vermiculite (Sinclair, Gainsborough, UK) onto which a 100 μm polyester mesh was placed (Plastok Group, Birkenhead, Merseyside, UK). Roots of the rice seedling grew down the mesh, and openings at the top and bottom of the rhizotron allowed for shoot growth and water drainage, respectively. Rhizotrons were covered with foil to prevent light from reaching the roots. Rhizotrons were supplied with 25 ml of 40% Long Ashton nutrient solution (Hewitt, 1966) containing 2 mM ammonium nitrate four times each day via an automatic watering system.

Striga seeds were surfaced sterilized in 10% bleach, thoroughly washed and then incubated (pre-conditioned) on moistened glass-fibre filter paper (Whatman®, Maidstone, Kent, UK) in Petri dishes for 12–15 d at 30°C before use (Gurney et al., 2006). Eighteen hours before infection of the rice seedlings, 1 ml of 0.1 ppm solution of GR24 (an artificial germination stimulant) was added to Petri dishes containing pre-conditioned Striga seeds to trigger germination. The germination percentage of each Striga ecotype was checked before infection. Sixteen days after sowing, rice plants were infected with 12 mg of pre-germinated Striga seeds by aligning them along the roots using a paint brush (Gurney et al., 2006). Infection of rice roots with pre-germinated Striga seeds is essential when quantifying post-attachment resistance, as it ensures synchronous attachment of the parasites to the roots and eliminates any differences that may occur as a result of variation in the production of germination stimulants by the different rice cultivars (see Jamil et al., 2011). Control, uninfected plants were treated in a similar manner, but without the infection step. In every experiment, a minimum of four replicates was evaluated for each cultivar × treatment combination.

The 18 NERICA cultivars, their parents and IAC 165, Nipponbare, Koshihikari and Kasalath were assessed for their resistance against S. hermonthica (ecotype Sh-Kibos) and S. asiatica (ecotype Sa-USA) (Table 1). Plants were grown in a temperature-controlled glasshouse environment with day : night temperatures of 28°C : 24°C and 60% relative humidity. Plants were screened during the summer months (June–August 2009) when irradiance averages were > 400 μmol m−2 s−1 at plant height. If the irradiance fell below 200 μmol m−2 s−1, supplementary lighting was switched on automatically. In order to determine whether resistance was specific to a particular ecotype or species of Striga, selected cultivars (WAB56-104, CG14, NERICA 1, 7, 9 and 10) were also infected with four different ecotypes of S. hermonthica and two ecotypes of S. asiatica (Table 1).

Quantification of post-attachment resistance and the effect of Striga on host biomass

Post-attachment resistance was quantified 21 d after infection (DAI) of the roots. Before harvest, the root system of each rhizotron was photographed using a Canon EOS 300D (Canon (UK), Reigate, Surrey) digital camera. Striga seedlings growing on the roots of each infected plant were then harvested, placed in Petri dishes and photographed using a Canon EOS 300D digital camera. The number and length of Striga seedlings from each rice plant were determined from the Petri dish photographs using Image-Pro® (Media Cybernetics, Bethesda, USA). Striga plants were then dried at 48°C for 2 d and the amount of dry biomass per host plant was determined. In order to assess the effect of Striga on the growth and partitioning of host biomass, the numbers of tillers on control and infected plants were recorded at 21 DAI and the plants were harvested and separated into roots, stem (culm and leaf sheath) and leaves. Plant material was dried for 1 wk at 48°C and dry biomass was determined thereafter. The effect of Striga on the host was quantified by expressing the above-ground biomass of infected plants as a percentage of the uninfected control biomass.

The phenotype of resistance

The phenotype of resistance was investigated by photographing parasites developing on the root systems of each cultivar at different stages after infection using a Leica MZFLIII (Leica Microsystems Ltd, Heerbrugg, Switzerland) stereo microscope and Diagnostic Instruments camera (Model 7.4), or by cutting small sections of root (with attached parasite) and mounting on a glass slide in water. The root tissue was then observed using an Olympus BX51 microscope (Olympus Optical Ltd., London, UK) employing differential interference contrast microscopy (Nomarski) and photographed using a digital camera (Olympus DP11; Olympus Optical Ltd.). In order to examine the stage of parasite development on the host root, small sections of tissue were taken at intervals after infection, and fixed and embedded in Technovit solution according to the manufacturer’s instructions. Small sections of root tissue plus parasite were placed into an Eppendorf tube containing Carnoy’s fixative (4 : 1, 100% ethanol : acetic acid) and vacuum infiltrated for 20 min. Samples were incubated in Carnoy’s fixative overnight and then washed with 2 × 100% ethanol for 2 h each. Samples were transferred to 100% Technovit solution for 15 min and then transferred into fresh solution for 3 d. Samples were transferred into moulds and Technovit solution and hardener (1 : 2) were added. As the Technovit resin became viscous, samples were positioned in the correct orientation for sectioning. The resin blocks were covered with foil and baked in an oven at 37°C for 30–60 min. The resin blocks were mounted onto histoblocs, trimmed and sectioned using a microtome (Leica RM 2145). Sections (3–5 μm thick) were transferred to microscope slides (poly-lysine slides; SLS, Nottingham, UK). Sections were stained with toluidine blue O in 100 mM phosphate buffer, pH 7.0, dried on a hot plate at 65°C for 30 min and mounted with Depex (BDH, Poole, UK). Sections were observed and photographed using the Olympus BX51 microscope and camera.

Statistical analyses

The statistical package R (version 2.8.1) was used for ANOVA and Pearson’s product-moment correlation analyses. Data for Striga dry biomass and number of Striga seedlings were log-transformed to meet the assumptions of ANOVA. Tukey’s honestly significant difference (HSD) test was then performed to calculate the corresponding critical HSD and to establish the different groups.

Results

How resistant are the NERICA rice cultivars to S. hermonthica and S. asiatica?

Fig. 1 shows the mean biomass of S. hermonthica (Sh-Kibos) and S. asiatica (Sa-USA) attached to the roots of all 18 NERICA cultivars, their parents and two susceptible (IAC165 and Koshihikari) and two resistant (Nipponbare and Kasalath) cultivars at 21 DAI. The NERICA cultivars and their parents exhibited a range of susceptibility to the two Striga species and were ranked from the most susceptible to the most resistant to the S. hermonthica ecotype from Kibos, Kenya. Interestingly, the cultivar ranking of resistance against this ecotype of S. hermonthica was quite similar to that of the resistance against the ecotype of S. asiatica tested in this experiment (Fig. 1). Some NERICA cultivars, e.g. NERICA 7, 8, 9, 11 and 14, were very susceptible to S. hermonthica, supporting between 100 and 150 well-developed parasites per host root system (Figs 1, 2a). NERICA 6, 15, 16, 18 and WAB56-104 were also susceptible, supporting between 40 and 100 parasites, although the parasites were, on average, smaller (Fig. 2a). The remaining NERICA cultivars and CG14, WAB56-50 and WAB181-18 exhibited good levels of post-attachment resistance to the ecotypes of the two Striga species (Figs 1, 2). NERICA 1 and 10 exhibited the greatest resistance to both S. hermonthica and S. asiatica, and were more resistant than their O. glaberrima parent (CG14) and Nipponbare (Figs 1, 2, 3d–f). These cultivars had very few successful attachments at 21 DAI and the parasites were very small: < 3 mm (Sh-Kibos) and 1 mm (Sa-USA) in size (Fig. 2a,b). By contrast, NERICA 9 and 7 (Fig. 3a,b) were more susceptible than their O. sativa parent WAB56-104 (Fig. 3c), and were as susceptible as IAC165 (Fig. 1). Parasites on the most susceptible cultivars grew rapidly and were relatively large. In general, the biomass and size of S. asiatica seedlings attached to the rice cultivars were smaller than those of S. hermonthica seedlings, although, on the susceptible cultivars, there were more S. asiatica than S. hermonthica attachments.

Figure 1.

Evaluation of post-attachment resistance of the NEw RICe for Africa (NERICA) cultivars to Striga hermonthica (Sh-Kibos, grey bars) and S. asiatica (Sa-USA, black bars) ecotypes. One-way ANOVA showed that the genotype effect is highly significant (< 0.0001) for both Striga ecotypes. Tukey’s honestly significant differences (HSDs, < 0.05) are represented for each Striga ecotype. Data are presented as means ± SE.

Figure 2.

The relationship between the number and length of Striga hermonthica (Sh-Kibos) (a) and S. asiatica (Sa-USA) (b) seedlings attached to the roots of the NEw RICe for Africa (NERICA) cultivars. Data are presented as means ± SE. The genotypes used for further analyses of Striga resistance are indicated as white symbols and are named. Pearson’s product-moment correlation probability and coefficient are indicated for each Striga ecotype.

Figure 3.

Striga hermonthica (Sh-Kibos) growing on the roots of selected NEw RICe for Africa (NERICA) cultivars (in rhizotrons) 21 d after infection. NERICA 7 and 9 are very susceptible, showing many S. hermonthica attachments (a, b). Fewer parasites were attached to WAB56-104 (c), and CG14, NERICA 1 and 10 exhibited good levels of resistance to the parasite with only a few viable attachments (d–f).

Although the number of parasites undergoing a resistance response varied on the different NERICA cultivars, the visible phenotype of the resistance reaction to S. hermonthica and S. asiatica was similar (Fig. 4a,b). Striga hermonthica and S. asiatica attached to the root systems of the NERICA cultivars within 2–3 d of inoculation in both susceptible and resistant interactions. However, by day 7, parasites that had elicited a host resistance response were clearly dying, as the haustoria failed to increase in size and the host root exhibited intense necrosis at the site of attachment (Fig. 4a,b). In susceptible interactions (e.g. NERICA 7 and 9), S. hermonthica parasites penetrated through the cortex and endodermis and, by day 6–7, had begun to form connections with the host xylem resulting in the differentiation of the haustorium (Fig. 4c) and emergence of the shoot. In resistant interactions (e.g. CG14, NERICA 1 and NERICA 10), transverse sections through the rice root at the site of attachment revealed two phenotypes. In the most frequently observed phenotype, the parasite penetrated the cortex, but was unable to traverse the endodermis to form host–parasite xylem continuity. The parasites grew around the vascular cylinder and sometimes exited the root again (Fig. 4d,e). In some cases, the parasite was able to penetrate the endodermis, although this took longer than in the more susceptible interactions. A few connections to the vascular system were visible, but they were often associated with the deposition of dense staining material and the resulting parasites remained small and grew slowly (Fig. 4e).

Figure 4.

The phenotype of resistance in the NEw RICe for Africa (NERICA) cultivars to Striga hermonthica (Sh-Kibos). (a, b) The phenotype of resistance against both S. hermonthica (Sh-Kibos) and S. asiatica (Sa-USA) is associated with intense necrosis at the site of attachment. (c–f) Transverse sections through the root and parasite attachment in compatible and incompatible interactions 6 d after infection with pre-germinated S. hermonthica (Sh-Kibos) seeds. (c) In the compatible interaction (NERICA 7), the parasite has penetrated the cortex and endodermis, and is beginning to form parasite–host xylem connections. (d, e) In many incompatible interactions (e.g. CG14, NERICA 1 and 10), the parasite penetrates the cortex, but is unable to traverse the endodermis and form a connection with the xylem vessels of the host. (f) In some interactions, the parasite is able to penetrate the endodermis and establish a few connections to the vascular system. However, this is associated with the deposition of dense staining material and the parasites remain small (e.g. NERICA 10).

How broad spectrum is the resistance found in the NERICA cultivars?

The NERICA cultivars showed a wide range of resistance levels to an ecotype of S. hermonthica (Sh-Kibos) and an ecotype of S. asiatica (Sa-USA) (Fig. 1). In order to determine whether the resistance exhibited by some of the NERICA cultivars was broad spectrum or specific to the ecotype of Striga used, the two most resistant (NERICA 1 and NERICA 10) and two most susceptible (NERICA 7 and NERICA 9) cultivars (together with their parent cultivars CG14 and WAB56-104) were infected with four different ecotypes of S. hermonthica and two ecotypes of S. asiatica (Table 1).

Fig. 5 shows the amount of Striga biomass, number of Striga seedlings and the average length of Striga seedlings of each Striga ecotype on the different rice cultivars. The rice cultivars showed the same pattern of resistance to three of the four S. hermonthica ecotypes (Sh-Kibos, Sh-Busia and Sh-Medani) and to the two S. asiatica ecotypes (Sa-USA and Sa-Kyela), despite the fact that they were collected from different host species and from different regions of Africa (Table 1). Essentially, NERICA 7 and 9 were very susceptible with in excess of 100 well-developed Striga seedlings per root system in the case of the S. hermonthica ecotypes (Fig. 5b,c). WAB56-104 was also susceptible and supported between 50 and 70 well-developed parasites. CG14, NERICA 1 and NERICA 10 exhibited high levels of resistance to both S. hermonthica and S. asiatica ecotypes (Fig. 5a–c). As observed in the previous screen of all 18 NERICA cultivars, the parasites of the S. asiatica ecotypes were smaller than those of S. hermonthica, in both weight and length. However, although the pattern of resistance of the rice cultivars to the S. asiatica ecotype from Kyela was similar to that observed for all other Striga ecotypes, the total number of attachments was significantly smaller when compared with the ecotype from the USA (Fig. 5b), suggesting that the Kyela population was less virulent on the cultivars used in this study.

Figure 5.

Evaluation of post-attachment resistance of NEw RICe for Africa (NERICA) 1, 7, 9 and 10 and their parents to a range of Striga hermonthica and S. asiatica ecotypes. Striga dry biomass (a), number of Striga seedlings (b) and average length of Striga seedlings (c) were measured after harvesting at 21 d after infection. Data shown for the Sh-Kibos and Sh-USA ecotypes are also shown in Figs 1, 2. Data presented are means ± SE. For the three traits, genotype, ecotype and genotype × ecotype effects were highly significant (two-way ANOVA, < 0.001). The significance of a genotype effect calculated by a one-way ANOVA for each Striga ecotype and trait combination is shown as: *, < 0.05; **, < 0.01; ***, < 0.001. The letters above each bar indicate the different significance groups after Tukey’s pairwise comparisons.

The interaction between the rice cultivars and S. hermonthica collected from NERICA 1 growing in Kouto, Ivory Coast (Sh-Kouto) differed in several respects from the interaction observed with the other S. hermonthica ecotypes. NERICA 7, NERICA 9 and WAB56-104 were susceptible to Sh-Kouto. The total number of attachments on the root systems was similar to that observed for the other S. hermonthica ecotypes (Fig. 5b), but the average size of the parasites was smaller (Fig. 5c). NERICA 10 was slightly more susceptible to this ecotype than to the other S. hermonthica ecotypes, but CG14 remained very resistant to this ecotype. NERICA 1, which exhibited high levels of resistance to all other Striga ecotypes, was as susceptible to the Sh-Kouto ecotype as NERICA 7, NERICA 9 and WAB56-104.

The impact of Striga on the biomass of susceptible and resistant NERICA cultivars

The tiller number and dry biomass of the infected NERICA cultivars were compared with those of the uninfected control plants to evaluate the impact of Striga infection on the growth of the host. A large variation in the biomass dry weights of the uninfected control plants was observed among the cultivars (Table 2). The dry biomass of the control plants ranged from 1.57 g for NERICA 4 to 3.44 g for NERICA 9. Infection of the NERICA cultivars with either S. hermonthica (Sh-Kibos) or S. asiatica (Sa-USA) altered the partitioning of biomass to the roots, stems and leaves in comparison with the uninfected controls (Table 2). The most noticeable difference was in the above-ground biomass, where the partitioning of dry matter to the stems and leaves was severely reduced by Striga infection (Table 2). In the two most susceptible cultivars, NERICA 7 and NERICA 9, the stem biomass of infected plants was 74% and 81% less than that of the control plants, respectively (when infected with either S. hermonthica or S. asiatica) and the leaf biomass was between 60% and 66% (NERICA 7) and 65% and 75% (NERICA 9) less than that of control plants (Table 2). In the two most resistant cultivars, NERICA 1 and NERICA 10, the effect of the parasite on the stem and leaf biomass was less severe, but still significant (Table 2).

Table 2.   The effect of Striga on biomass and tiller production of Striga-infected NEw RICe for Africa (NERICA) cultivars compared with uninfected controls
   Biomass of Striga-infected plants as percentage of the control  
CultivarEcotypeTotal dry biomass (g)RootStemLeafNo. of tillers
  1. Data presented for total dry biomass and numbers of tillers are means ± SE. For the two traits, genotype, ecotype and genotype × ecotype effects were highly significant (two-way ANOVA, < 0.001). The significance of a treatment effect was calculated by a one-way ANOVA for each genotype, and the letters next to each mean value indicate the different significance groups after Tukey’s pairwise comparisons (< 0.05).

NERICA 1Control1.77 ± 0.21a4.0 ± 0.00a
Sh-Kibos1.30 ± 0.07ab−3−36−263.0 ± 0.45a
Sa-USA0.94 ± 0.08b−43−50−453.2 ± 0.37a
NERICA 2Control1.67 ± 0.10a4.0 ± 0.00a
Sh-Kibos1.12 ± 0.20ab2−43−373.0 ± 0.32b
Sa-USA0.71 ± 0.14b−55−61−562.5 ± 0.29b
NERICA 3Control1.88 ± 0.26a4.3 ± 0.33a
Sh-Kibos1.11 ± 0.15b−20−46−452.8 ± 0.37b
Sa-USA0.97 ± 0.06b−41−50−503.2 ± 0.20ab
NERICA 4Control1.57 ± 0.27a4.3 ± 0.25a
Sh-Kibos1.09 ± 0.08ab10−42−342.5 ± 0.29b
Sa-USA0.84 ± 0.11b−37−52−453.0 ± 0.45ab
NERICA 5Control1.95 ± 0.06a4.8 ± 0.25a
Sh-Kibos1.02 ± 0.20b−29−53−492.8 ± 0.25b
Sa-USA1.00 ± 0.07b−41−52−493.6 ± 0.24b
NERICA 6Control2.16 ± 0.11a3.8 ± 0.25a
Sh-Kibos0.72 ± 0.10b−34−74−722.4 ± 0.40b
Sa-USA0.70 ± 0.09b−53−70−701.3 ± 0.25b
NERICA 7Control2.41 ± 0.45a3.3 ± 0.33a
Sh-Kibos0.91 ± 0.10b−38−74−602.3 ± 0.25a
Sa-USA0.81 ± 0.26b−52−73−662.0 ± 0.00a
NERICA 8Control3.20 ± 0.16a6.5 ± 0.50a
Sh-Kibos1.03 ± 0.11b−36−79−683.4 ± 0.24b
Sa-USA1.15 ± 0.21b−45−70−653.3 ± 0.33b
NERICA 9Control3.44 ± 0.27a7.0 ± 0.41a
Sh-Kibos1.12 ± 0.06b−37−81−654.0 ± 0.00b
Sa-USA0.82 ± 0.08b−66−81−752.6 ± 0.24c
NERICA 10Control1.76 ± 0.17a4.0 ± 0.00a
Sh-Kibos1.26 ± 0.12ab−10−36−283.0 ± 0.00b
Sa-USA1.08 ± 0.11b−30−42−393.2 ± 0.37ab
NERICA 11Control3.43 ± 0.31a7.0 ± 0.32a
Sh-Kibos1.25 ± 0.18b−38−75−633.6 ± 0.24b
Sa-USA1.06 ± 0.08b−62−74−683.2 ± 0.20b
NERICA 12Control2.45 ± 0.12a5.6 ± 0.40a
Sh-Kibos1.43 ± 0.14b−25−55−353.2 ± 0.20b
Sa-USA0.78 ± 0.12c−60−73−663.2 ± 0.37b
NERICA 13Control2.66 ± 0.15a5.5 ± 0.29a
Sh-Kibos1.12 ± 0.04b−35−71−532.6 ± 0.24c
Sa-USA1.12 ± 0.09b−46−63−574.2 ± 0.20b
NERICA 14Control2.63 ± 0.14a4.6 ± 0.24a
Sh-Kibos1.08 ± 0.13b−34−71−593.0 ± 0.00b
Sa-USA1.04 ± 0.22b−46−68−602.6 ± 0.24b
NERICA 15Control1.68 ± 0.16a3.3 ± 0.25a
Sh-Kibos0.48 ± 0.06b−37−85−711.6 ± 0.24b
Sa-USA0.59 ± 0.01b−44−72−651.0 ± 0.00b
NERICA 16Control1.89 ± 0.21a3.5 ± 0.29a
Sh-Kibos0.54 ± 0.09b−50−82−701.5 ± 0.29b
Sa-USA0.58 ± 0.03b−60−75−681.0 ± 0.00b
NERICA 17Control2.93 ± 0.29a6.4 ± 0.60a
Sh-Kibos1.66 ± 0.18b−20−56−413.2 ± 0.20b
Sa-USA1.62 ± 0.28b−30−53−443.6 ± 0.24b
NERICA 18Control1.78 ± 0.20a3.5 ± 0.29a
Sh-Kibos0.52 ± 0.05b−47−86−671.6 ± 0.24b
Sa-USA0.62 ± 0.04b−48−72−651.0 ± 0.00b
CG14Control2.69 ± 0.20a8.0 ± 0.77a
Sh-Kibos1.83 ± 0.18b−6−46−315.4 ± 0.75a
Sa-USA2.37 ± 0.22ab17−24−145.6 ± 0.75a
WAB181-18Control1.96 ± 0.10a5.8 ± 0.37a
Sh-Kibos1.17 ± 0.05b4−58−413.8 ± 0.20b
Sa-USA1.58 ± 0.17ab27−32−234.2 ± 0.20b
WAB56-104Control1.98 ± 0.17a4.0 ± 0.00a
Sh-Kibos0.91 ± 0.13b−33−64−532.8 ± 0.20b
Sa-USA1.00 ± 0.21b−43−53−492.5 ± 0.50b
WAB56-50Control2.37 ± 0.15a4.5 ± 0.29a
Sh-Kibos1.31 ± 0.10b−12−62−393.0 ± 0.00b
Sa-USA1.11 ± 0.14b−40−62−492.4 ± 0.40b

Figure 6 shows the relationship between the percentage reduction in total host biomass of infected plants (compared with control plants) and the amount of parasite biomass on the roots. There was a significant linear relationship between the effect of S. hermonthica (Sh-Kibos) on host biomass and the amount of parasite biomass on the roots, that is the most susceptible NERICA cultivars were generally the most badly affected, whereas the most resistant NERICA cultivars were the least affected (Fig. 6, Table 2). This negative relationship between host and parasite biomass was less obvious when plants were infected with S. asiatica (Sa-USA), as different cultivars were affected to different extents for a similar amount of parasite biomass on the roots. For example, CG14 and NERICA 12 showed similar levels of infection with 1.52 mg and 1.34 mg of Striga biomass, respectively, yet the percentage reduction in host biomass (relative to the controls) was 31% and 82%, respectively, suggesting a cultivar difference in tolerance to the deleterious effects of infection by S. asiatica. Infection of the NERICA cultivars with either S. hermonthica or S. asiatica also suppressed tillering significantly when compared with the uninfected controls (Table 2).

Figure 6.

Relationship between the percentage loss in total host biomass of infected plants compared with control plants and the amount of parasite biomass on the roots of Striga hermonthica (Sh-Kibos) (a) and S. asiatica (Sa-USA) (b). Data are presented as means ± SE.

Discussion

This study has shown, for the first time, that the 18 upland NERICA cultivars exhibit different levels of post-attachment resistance against different Striga species and ecotypes, ranging from extremely susceptible (e.g. NERICA 7, 8, 9, 11 and 14) to highly resistant (e.g. CG14, NERICA 1, 2, 3, 5 and 10). Interestingly, the resistance of the different NERICA cultivars against the ecotype of S. hermonthica (Sh-Kibos) was similar to the resistance observed against the ecotype of S. asiatica (Sa-USA), although the biomass and size of S. asiatica seedlings attached to the NERICA cultivars were lower than those of S. hermonthica seedlings. This probably reflects the different morphology, structure and rate of development of the two Striga species, rather than an inherent difference in virulence. In addition, there were more S. asiatica parasites on the susceptible NERICA cultivars when compared with S. hermonthica, which may have led to a greater level of intraspecific competition for host nutrients, contributing to the smaller size of S. asiatica parasites. Although none of the NERICA cultivars (or their parents) exhibited complete resistance to the ecotypes of Striga used in this study, the strongest resistance was associated with very few, small Striga seedlings. The most resistant cultivars, for example CG14 and NERICA 1 and 10, were more resistant than Nipponbare, which is known to exhibit very strong post-attachment resistance to S. hermonthica (Gurney et al., 2006; Kaewchumnong & Price, 2008).

The different groups of NERICA cultivars share a large part of their genomes between themselves and their respective O. sativa recurrent parent because of their breeding history (Jones et al., 1997a). For example, the genetic background of NERICAs 1–7 is derived, predominantly, from WAB56-104, with between 3.4% and 12.1% of loci coming from their donor parent CG14 (Semagn et al., 2007). Thus, it might be expected that only a few genetic regions would explain the difference in susceptibility to Striga observed in our screen. However, we observed a wide range of susceptibility of the NERICA cultivars to Striga infection, from those that were much more susceptible than the O. sativa parent to those that exhibited greater resistance than found in CG14. The strong resistance in some of the NERICA cultivars is probably controlled by a few loci, with the resistant allele coming from CG14. However, the larger than expected transgressive segregation towards susceptibility may be a result of the introduction of susceptibility alleles from CG14 at different loci to those present in the O. sativa parent, or to genetic introgressions (nonparental alleles) from unknown rice parent(s) during the breeding process. The presence of nonparental alleles has been detected for c. 3% of loci analysed in NERICA cultivars 1–7 (Semagn et al., 2007).

Some of the upland NERICA cultivars exhibit strong, broad-spectrum post-attachment resistance against Striga ecotypes

Our study indicates that the resistance in some of the NERICA cultivars is relatively broad spectrum and therefore potentially of great interest to farmers in the short term, as well as for longer term studies of the genetic basis of resistance for breeding programmes. The resistance ranking of the 18 upland NERICA cultivars was similar when challenged with S. hermonthica (Sh-Kibos) and S. asiatica (Sa-USA). In addition, the pattern of susceptibility/resistance of NERICA 7 and 9 (susceptible), 1 and 10 (resistant), CG14 (resistant) and WAB56-104 (intermediate) was largely maintained when challenged with another three ecotypes of S. hermonthica and another ecotype of S. asiatica collected from different host species and from different regions of Africa (Table 1).

However, there was one exception to this general pattern of resistance and susceptibility. The S. hermonthica ecotype Sh-Kouto was very virulent on NERICA 1, despite the fact that this cultivar exhibited strong post-attachment resistance to all other ecotypes tested and also produced small amounts of germination stimulants (Jamil et al., 2011). This S. hermonthica ecotype was collected in 2008 from NERICA 1 growing in fields in the Kouto area of northern Ivory Coast. NERICA 1 had been cultivated in this area for several years in succession and was highly infested with S. hermonthica at the time at which the seeds were collected (J. Rodenburg, pers. comm.). This suggests that either the S. hermonthica population in this area was already highly virulent against NERICA 1 before its cultivation or, more probably, that the high genetic diversity of the Striga seedbank led to rapid evolution/build-up of virulent genotypes from a subset of the natural seed bank population over a period of time (Huang et al., 2011).

The deployment of resistant cultivars is considered to be an important and cost-effective component of integrated Striga management programmes, and the fact that NERICA 10 and, particularly, CG14 still showed good resistance to the Sh-Kouto ecotype suggests that the genetic basis of resistance in the NERICA cultivars is multilayered and controlled by different loci. However, the results of this study clearly demonstrate the need to understand the genetic basis of both host resistance and the adaptation of Striga populations (parasite virulence) to new host resistance phenotypes. Such insights would facilitate the stacking of appropriate resistance loci in farmer-preferred and Striga-tolerant cultivars to enhance the durability and stability of defence in the long term (Scholes & Press, 2008; Rodenburg & Bastiaans, 2011).

The phenotype of Striga resistance in the NERICA cultivars

Different numbers of attached parasites developed successfully (compatible) or died (incompatible) on the different NERICA cultivars and their parents. The failure of parasites to develop after attachment was often associated with the appearance of host necrosis at the site of attachment within 4–6 d of placing pre-germinated seeds on the roots (Fig. 4a). A similar resistance phenotype was observed in the O. sativa cultivar Nipponbare infected by S. hermonthica (Gurney et al., 2006), in some sorghum cultivars following infection by S. asiatica (Mohamed et al., 2003) and in resistant cowpea cultivars infected by S. gesnerioides (Li & Timko, 2009). In the case of cowpea resistance, the necrosis reaction was associated with a gene-for-gene resistance mechanism (Li & Timko, 2009). In the NERICA cultivars, their parents and Nipponbare infected with S. hermonthica, it is not clear whether the underlying resistance mechanisms involve gene-for-gene interactions, as resistance is considered to be controlled by several quantitative trait loci, although there are indications that a proportion of this quantitative resistance is controlled by a few genes of major effect (Gurney et al., 2006). The analysis of changes in gene expression in the roots of Nipponbare undergoing a resistance reaction to S. hermonthica revealed that genes encoding resistance and hypersensitive response (HR) protein homologues, pathogenesis-related (PR) proteins and WRKY transcription factors were all up-regulated (Swarbrick et al., 2008). How the alteration in the expression of defence genes and pathways relates to the genetic basis of resistance awaits the fine mapping of the genes underlying Striga resistance quantitative trait loci.

In this study, transverse sections through incompatible parasite attachments, whether on very resistant or on the more susceptible cultivars, often revealed that the parasite endophyte had penetrated the host root cortex, but not the endodermis. The parasite was therefore unable to form the parasite–host xylem connections necessary to access host nutrients and or developmental signals within the host vascular system. This phenotype was associated with failure of the parasite haustorium to differentiate fully and initiate shoot growth, and it appears to be quite common in many rice cultivars, including Nipponbare (Gurney et al., 2006; Yoshida & Shirasu, 2009), Koshihikari (Yoshida & Shirasu, 2009) and Kasalath (J. D. Scholes, unpublished). On the most resistant NERICA cultivars (e.g. NERICA 1 and 10), some parasites took longer to penetrate the endodermis than in very susceptible interactions. In addition, fewer successful connections to the parasite xylem were visible, and the cells and xylem vessels associated with parasite ingress into the vascular cylinder had become occluded by dense staining material (Fig. 4f). It is not clear whether this represents lignifications of the cell walls or the deposition of materials, such as callose, as observed in some resistance responses to Orobanche species (e.g. Fernández-Aparicio et al., 2008; Pérez-de-Luque et al., 2008).

Does resistance and/or tolerance to Striga reduce the damaging effects of the parasite on the host plant?

Resistance reduces the number of successful attachments and, consequently, the reproductive output of the parasite (Rodenburg et al., 2006b). However, even highly resistant cultivars usually have some attachments which will grow successfully, and many studies have shown that the deleterious effects of Striga on plant growth, morphology and yield are complex and are not always related, in a linear manner, to the number of infections or to the amount of parasite biomass on the host roots (Graves et al., 1989; Frost et al., 1997; Gurney et al., 1999; Rodenburg et al., 2006a). Some cultivars appear to perform better than others under similar parasite loads, a phenomenon known as ‘tolerance’. Recent findings regarding the physiological expression of tolerance (Gurney et al., 2002; Rodenburg et al., 2008), coupled to an increased understanding of the genetic mechanisms underlying this trait, could enable plant breeders to incorporate tolerance into largely resistant cultivars to improve yields further (Haussmann et al., 2001; Rodenburg et al., 2005; Rodenburg & Bastiaans, 2011). In this study, partitioning of dry matter to stems and leaves (above-ground biomass) was reduced in the NERICA cultivars infected with S. hermonthica or S. asiatica compared with their uninfected controls, but the most resistant cultivars were also the least affected (Fig. 6). The total biomass of the most resistant cultivars NERICA 1 and 10, when infected by Striga, was c. 30% (Sh-Kibos) or 40% (Sa-USA) lower than the uninfected controls, compared with a 70% (Sh-Kibos) or 80% (Sa-USA) reduction in NERICA 7 and 9. The fact that the percentage reduction in the host biomass of infected plants (compared with controls) was linearly related to the amount of parasite biomass on the host roots (particularly for S. hermonthica) suggests that the most resistant NERICA cultivars may also produce the highest grain yields in Striga-infested fields. Our data also indicate that there is some genetic variation for tolerance to Striga amongst the cultivars. For example, CG14 lost c. 18% of above-ground biomass, Nipponbare 28%, NERICA 4 48% and NERICA 12 70% (compared with uninfected plants) when parasitized by 1.2–1.5 mg of S. asiatica (Fig. 6b), highlighting the need to further elucidate the physiological/molecular mechanisms underlying these differences in tolerance to similar amounts of Striga. At present, it is not clear whether the correlation between resistance/tolerance and percentage loss in above-ground biomass, revealed in this study, is a good predictor of grain yield in the field. In order to test this relationship (and the expression of resistance and tolerance), we are currently evaluating the performance and yield of the NERICA cultivars in field trials under S. hermonthica and S. asiatica infestation in Kenya and Tanzania, respectively.

The way forward

This study has shown that some of the NERICA cultivars exhibit very strong post-attachment resistance and tolerance to a range of S. hermonthica and S. asiatica ecotypes, and that the resistance in some of the cultivars is relatively broad spectrum. Jamil et al. (2011) have also shown that the NERICA cultivars exhibit differences in the amounts and types of germination stimulants produced, resulting in differences in pre-attachment resistance to Striga. We suggest that rice cultivars which combine low germination stimulant production and strong post-attachment resistance will perform and yield well in areas in which Striga infestation is prevalent. However, in order to increase the durability of host defence, when faced with a genetically diverse Striga seed bank, it is essential to use resistant cultivars as part of an integrated control programme and further our understanding of the molecular nature of host–parasite specificity.

Acknowledgements

The authors would like to thank the Biotechnology and Biological Sciences Research Council (BBSRC) and the Department of International Development (DfID) for funding this work through the Sustainable Agriculture for International Development (SARID) Programme. We would also like to thank the Africa Rice Center (AfricaRice) for providing the seeds of the NERICA cultivars used in this study.

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