Abstract: RYMV transmission by insect vectors is considered to fully represent how RYMV disease is spread under natural field conditions. The present study aimed to use Oxya hyla, Locris rubra and Chnootriba similes vectors after acquisition of the virus to determine RYMV movement and distribution in insect body and transmission to rice cultivars. RYMV susceptible BG 90-2 was sown in 5 L plastic pots each at 0.5, 1 and 1.5 m distance from test entries and seedlings were mechanically inoculated with a highly virulent RYMV Nigerian isolate 14 days after sowing. Seven days after inoculation of BG 90-2, test entries were sown in 5-litre plastic pots and same day Oxya hyla, Locris rubra and Chnootriba similes vectors were introduced into the screen house to feed on RYMV infected BG 90-2. RYMV content in Oxya hyla, Locris rubra and Chnootriba similes whole body was 71.8, 44.1 and 50 and head part was 42, 44.6 and 10.1%. RYMV incidence at 0.5, 1.0 and 1.5 m vector migration distance was 14.6, 16.0 and 19.0% for Oxya hyla, 31.3, 35.2 and 39.6% for Locris rubra and 13.7, 16.2 and 19.9% for Chnootriba similes. Cluster dendrogram revealed three groups (GrpA, GrpB, GrpC) of RYMV cultivar screening methods. GrpA was typical of Locris rubra, GrpB has mechanical and Oxya hyla while Chnootriba similes formed GrpC. The information reported in this study would help to better understand RYMV disease epidemic in farmers’ fields and to develop durable resistant rice varieties against the disease.
INTRODUCTION
Rice production in Africa is seriously affected by diseases. Rice Yellow Mottle Virus (RYMV) remains a major constraint to rice production in Africa (Sere et al., 2008a; Ochola and Tusiime, 2011b), especially in the lowland and irrigated rice ecologies (Banwo et al., 2004). The virus is widely spread, indigenous to Africa and very infectious to rice with varying mottle and yellowing symptoms (Banwo et al., 2004; Onasanya et al., 2006; Gnanamanickam, 2009). Different serotypes (Sere et al., 2007) and pathotypes (Onasanya et al., 2004; Ochola and Tusiime, 2011a) of RYMV isolates are known to exist. Yield losses of between 4-90% have been reported which depend on genotype and infection time (Onwughalu et al., 2010, 2011). There is need to replace existing susceptible rice varieties with new varieties that are better adapted to lowland and irrigated conditions (Sie et al., 2008).
RYMV transmission and spread under field condition is usually by insect vectors and mechanical inoculations (Nwilene et al., 2009). Research study has revealed that cows, donkeys and grass rats transmit the virus in irrigated rice fields (Sarra and Peters, 2003) while the disease is not transmitted by seed or nematode (Abo et al., 2004). Abiotic transmission of RYMV through soil and contact between plants now revealed (Traore et al., 2005, 2008). The virus has also been observed on some grasses, cultivated rice and wild rice (Abo et al., 2000a). Previous studies on insect vectors have led to the identification of Chaetocnema pulla, Dicladispa gestroi, Trichispa sericea, Sessilia pusilla, Chaetocnema abyssinica, C. kenyensis and C. pallidipes Oxya hyla, Locris rubra and Chnootriba similis as important vectors of RYMV (Abo et al., 1998; Sere et al., 2008b; Nwilene et al., 2009). In natural condition, the insect vectors played a significant role in disease epidemic as they are able to transfer RYMV from surrounding infected fields and contaminated weeds to the new rice fields.
The use of resistant varieties has been considered as most reliable control measure to RYMV disease. In order to identify resistant material to be released or to be used as donor in breeding programs, rice varieties are usually screened under artificial conditions by mechanical inoculation of RYMV isolates into the rice plants (Onasanya et al., 2004, 2006). Such screening method does not demonstrate how RYMV disease is spread or transmitted under natural or field environment (Sere et al., 2008b). Most importantly, there is need to know the vector that acquired or retailed more RYMV in their body parts (head, thorax and abdomen) before transmission to rice plants. Therefore, the objective of the current study was to identify among Oxya hyla, Locris rubra and Chnootriba similes insect vectors RYMV contents after acquiring the virus, determine the virus movement and distribution across the insect head, thorax and abdomen and finally investigate their RYMV transmission potential to rice cultivars as compared to mechanical transmission approach.
MATERIALS AND METHODS
Research location: The study was conducted between April to October 2009 at Africa Rice Center, Nigeria Station, Ibadan, Oyo State, Nigeria.
Rice genotypes: Eight differential rice genotypes (Table 1) used in this study were obtained from the Africa Rice Center (AfricaRice), Plant Pathology Unit, Cotonou, Benin Republic.
| Table 1: | Identity of differential rice genotypes used for the study |
RYMV isolate: The highly virulent Nigerian isolate of RYMV (Sere et al., 2008b) used for this study was first propagated in the susceptible rice variety BG 90-2 following mechanical inoculation of 21-day-old rice seedlings in the screen house. Four weeks after inoculation, leaves bearing typical yellow mottle symptoms were harvested and used to prepare the viral inoculum. The viral inoculum was prepared by grinding the RYMV-infected leaf samples in 0.01 M phosphate buffer pH 7.0 at the ratio of 1:10 (w/v) and the resulting homogenate filtered through cheesecloth. Carborundum powder (600 mesh) was added to the inoculum to aid the penetration of the virus into leaf tissues during mechanical inoculation.
Insect vector RYMV inoculation: For the infected row, the susceptible variety BG 90-2, was first sown in 5 litre plastic pots each at 0.5, 1 and 1.5 m distance respectively from test entries. The rice seedlings in the infected row were mechanically inoculated with a highly virulent RYMV Nigerian isolate 14 days after sowing and 7 days later, the test entries (Table 1) were sown in 5 L plastic pots. Insect vectors were introduced into the screen house on the development of the first symptoms at 14 days after mechanical inoculation to allow them to feed on the infected row of rice plants. Five days after feeding some insect vectors were removed for RYMV serological diagnosis. One species of insect vector per experiment was used and a total of three species (Oxya hyla, Locris rubra and Chnootriba similis) were tested. In another experiments 14-day-old test entries of rice seedlings were mechanically inoculated with the same RYMV isolate while controls were not inoculated. Each of the experiments was laid down on a Randomized Complete Block Design (RCBD) with three replications, each in a separate insect-proofed screenhouse.
Insect vectors RYMV serological diagnosis: Indirect-antigen coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) was performed on head, thorax, abdomen and whole insect as described by Sere et al. (2007).
Data collection: At 42 days after the introduction of the insect vector, chlorophyll content was measured using a SPAD 502 Chlorophyll Meter (Martines and Guiamet, 2004) and Disease Incidence (DI) was evaluated according to Onasanya et al. (2004). Insect vector viral (head, thorax, abdomen and whole insect) content (VC) was determined using enzyme linked immunosorbent assay (Sere et al., 2007). SPAD measurement was obtained both for test and control genotypes.
Data analysis: Based on the SPAD readings and viral content, the percentages of chlorophyll reduction and viral content were calculated. IRRISTAT statistical software was used for all the analyses (Zhu and Kuljaca, 2005). Variance of percentage disease incidence and chlorophyll reductions were performed. RYMV cultivar transmission fitness of the three insect vectors (Oxya hyla, Locris rubra and Chnootriba similes) and migration distance (0.5, 1 and 1.5 m) was plotted using regression analysis (Sere et al., 2008b). Additive Main Effect and Multiplicative Interaction (AMMI) analysis was performed to identify the insect vector that screened rice cultivars like the mechanical method (Ebdon and Gauch, 2002).
RESULTS
Insect vector RYMV serological detection and distribution: After acquired RYMV by feeding on leaves of RYMV inoculated BG 90-2 seedlings, RYMV was detected on selected Oxya hyla, Locris rubra and Chnootriba similes after serological test. RYMV content in Oxya hyla, Locris rubra and Chnootriba similes whole body was 71.8, 44.1 and 50%, respectively (Fig. 1).
| Table 2: | Analysis of variance for cultivar percentage chlorophyll reduction (%SPADR42) and percentage disease incidence (%DI) due to RYMV transmitted by Oxya hyla, Locris rubra and Chnootriba similes |
| **: Significant at 1% level, *: Significant at 5% level, NS: Not significant | |
| Fig. 1: | RYMV content acquired by Oxya hyla, Locris rubra and Chnootriba similes and distribution within the insect vector body |
On the head part, RYMV content in Oxya hyla, Locris rubra and Chnootriba similes was 42, 44.6 and 10.1%, respectively (Fig. 1). The thorax part has RYMV content of 39.7, 57.5 and 80% for Oxya hyla, Locris rubra and Chnootriba similes, respectively while abdomen part has 66.4, 45.9 and 13.4% RYMV content for Oxya hyla, Locris rubra and Chnootriba similes, respectively (Fig. 1). Oxya hyla and Locris rubra have more RYMV in their body parts (head and abdomen) than Chnootriba similes which has more viral content only in thorax part.
Insect vector RYMV transmission potential to rice cultivars: Analysis of variance for percentage chlorophyll reduction (%SPADR42) and percentage disease incidence (%DI) revealed significant (p = 0.01; p = 0.05) RYMV transmission by Oxya hyla, Locris rubra and Chnootriba similes into the 8 rice cultivars (Table 2).
| Table 3: | Analysis of variance for cultivar percentage chlorophyll reduction (%SPADR42) and percentage disease incidence (%DI) due to combined effect of RYMV transmitted by Oxya hyla, Locris rubra, Chnootriba similes and mechanical method |
| **: Significant at 1% level, *: Significant at 5% level, NS: Not significant | |
| Table 4: | Cultivar age mean comparison effect on percentage chlorophyll reduction (%SPADR42) and percentage disease incidence (%DI) due to RYMV infection by Oxya hyla, Locris rubra and Chnootriba similes |
| In a column, means followed by a common letter are not significantly different at the 5% level by Duncan’s Multiple Range Test | |
| Fig. 2: | Oxya hyla, Locris rubra, Chnootriba similes migration distance relative to rice cultivar % disease incidence |
The insect vector viral transmission was significantly different (p = 0.01; p = 0.05) at different migration distance (0.5, 1.0 and 1.5 m) (Table 2). Rice cultivar mean percentage disease incidence (%DI) increased as insect vector migration distance increased (Fig. 2). Rice cultivar mean %DI at 0.5, 1.0 and 1.5 m vector migration distance was between 14.6-19.0% for Oxya hyla, 31.3-39.6% for Locris rubra and 13.7-19.9% for Chnootriba similes (Fig. 2). The highest %DI was caused by Locris rubra (39.6%), followed by Chnootriba similes (19.9%) and the least was Oxya hyla (19.0%) (Fig. 2). On the cultivar age, RYMV infection on young and old leaves caused by Oxya hyla and Locris rubra was significant (p = 0.01) and not significant with Chnootriba similes (Table 2). Percentage DI of young and old leaves was between 11.2-21.9% for Oxya hyla, 30.1-40.6% for Locris rubra and 16.5-16.7% for Chnootriba similes Table 4.
| Table 5: | Rice cultivar and viral transmission method mean comparison on percentage chlorophyll reduction (%SPADR42) |
| In a column, means followed by a common letter are not significantly different at the 5% level by Duncan’s multiple range test. OH: Oxya hyla, LR: Locris rubra, CS: Chnootriba similes, M: Mechanical | |
| Table 6: | Rice cultivar and viral transmission method mean comparison on percentage disease incidence (%DI) |
| In a column, means followed by a common letter are not significantly different at the 5% level by duncan’s multiple range test. OH: Oxya hyla, LR: Locris rubra, CS: Chnootriba similes, M: Mechanical | |
This revealed that Oxya hyla and Locris rubra RYMV transmission to rice cultivar is dependent on cultivar age while Chnootriba similes viral transmission is independent of cultivar age. Besides, analysis of variance also revealed significant interaction (p = 0.01) between rice cultivars and viral transmission methods (Oxya hyla, Locris rubra, Chnootriba similes and mechanical) (Table 3). Cultivar mean chlorophyll reduction (%SPADR) caused by viral transmission methods was 42.1 a for mechanical, 36.6 ab for Locris rubra, 31.1b for Oxya hyla and 25.9 c for Chnootriba similes (Table 5). Besides, cultivar mean %DI caused by viral transmission methods was 35.3 a for Locris rubra, 25.4 b for mechanical, 16.6 c for Chnootriba simile and 16.5 c for Oxya hyla (Table 6). Indicating that Oxya hyla, Locris rubra, Chnootriba similes and mechanical method screened the 8 rice cultivars differently.
Classification of RYMV cultivar screening methods: Cluster dendrogram revealed three main groups (GrpA, GrpB, GrpC) of RYMV cultivar screening methods. GrpA was typical of Locris rubra cultivar screening method. GrpB was made up of mechanical and Oxya hyla cultivar screening methods while Chnootriba similes cultivar screening methods dominated GrpC (Fig. 3).
| Fig. 3: | Cluster dendrogram showing diversity among the four screening methods using additive main effects and multiplicative interaction (AMMI) analysis. V: Rice variety, CS: Chnootriba similes, OH: Oxya hyla, LR: Locris rubra, M: Mechanical |
DISCUSSION
In terms of epidemiology, insects are the most important factors in plant virus disease. Approximately 80% of the plant viruses depend on insect vectors for transmission (other vectors can be nematodes and fungi) and the plant virus vector interactions are very specific (Hohn, 2007; Uzest et al., 2007; Nwilene et al., 2009; Sere et al., 2008b). Thus, the recent spreading of RYMV throughout Africa might be caused by the known and unknown RYMV insect vectors (Nwilene et al., 2009; Sere et al., 2008b). This spreading by insect vectors provides the opportunity for the virus to be transmitted to a variety of rice cultivars in Africa (Nwilene et al., 2009). The present study did not only support these previous findings but also provides new knowledge into how vectors could transmit RYMV under rice field conditions irrespective of their distance from the rice fields. It revealed RYMV transmitted by Oxya hyla, Locris rubra and Chnootriba similes into the 8 rice cultivars has led to significant chlorophyll reduction and increased disease incidence among rice cultivars. Our study has demonstrated the importance of insect vector migration distance and effective viral transmission to rice cultivars. Rice cultivar RYMV disease incidence increases as insect vector migration distance increases. Short migration distance by insect vector reduced infection while long migration distance increase infection in the rice cultivars. There is evidence that Oxya hyla and Locris rubra transmit RYMV to young and old rice plants discriminately and cause different infection rates similar to mechanical transmission method. This evidence revealed that Oxya hyla and Locris rubra RYMV transmission rate was different in young and old rice cultivar while viral transmission rate was the same as with Chnootriba similes. It was very cleared in our study that Oxya hyla, Locris rubra, Chnootriba similes and mechanical method screened the 8 rice cultivars differently.
Non-circulative transmission has been considered the most adopted strategy for virus vector interaction. Having fed on infected plant, the vector acquired the virus and moved to inoculate a new host plant. The viral components involved in this interaction have been established in Cucumovirus, Potyvirus and Caulimovirus where domains of the viral coat protein directly recognize unknown retention sites in the vector mouthparts (Hohn, 2007; Uzest et al., 2007; Ziegler-Graff and Brault, 2008). In the present study, after acquired RYMV by feeding on leaves of RYMV inoculated BG 90-2 seedlings, RYMV was detected on selected Oxya hyla, Locris rubra and Chnootriba similes after serological test. This gave the evidence that the three vectors actually acquired the virus which confirmed what was obtained in previous studies with Cucumovirus, Potyvirus and Caulimovirus vectors (Hohn, 2007; Uzest et al., 2007; Ziegler-Graff and Brault, 2008). Oxya hyla and Locris rubra have more RYMV in their body parts (head and abdomen) than Chnootriba similes which has more viral content only in the thorax part. The presence of more RYMV content in Oxya hyla and Locris rubra mouthparts might possibly explained the reason for their efficient rice cultivar viral transmission (Uzest et al., 2007). This efficient viral transmission from Oxya hyla mouthparts could possibly explained why it was similar to mechanical viral transmission method in their cultivar screening potential as revealed in our study. The implication of this finding is that Oxya hyla can be used in place of mechanical method for routine RYMV cultivar screening for durable resistance in order to demonstrate natural field RYMV infection pattern.
In past RYMV vector studies researchers focused on vectors feeding on infected plants, acquired the virus and moved to inoculate a new host plants (Abo et al., 2000b; Sere et al., 2008b; Nwilene et al., 2009). Besides, high concentrations have been given also to researches on the population structure of the virus and cultivar resistance (N’Guessan et al., 2000; Fargette et al., 2004; Traore et al., 2005; Sorho et al., 2005; Fargette et al., 2008; Ndjiondjop et al., 2001; Albar et al., 2003; Ioannidou et al., 2003; Onasanya et al., 2004, 2006). However, no investigation was carried out on RYMV content acquired by vector species and vector migration distance as demonstrated in the present study.
CONCLUSION
RYMV content in insect vectors, viral distribution within the insect vectors and vector migration distance were reported for the first time in the present study. The study linked insect vector viral transmission efficiency to higher RYMV content in the vector mouthparts and wider migration distance. RYMV movement and distribution in vector body now understood. There is need to investigate the population structure and genetic diversity of RYMV vectors species and biotypes with specific linkage to RYMV strains carried by different vectors. This would help to better understand RYMV disease epidemic in farmers’ fields and develop durable resistant rice varieties against the disease.
ACKNOWLEDGMENTS
We are very grateful to the Government of Japan (Ministry of Foreign Affairs) for providing funds for this research. The authors would also like to acknowledge Mr. Bayo Kehinde and Mr. Soton Georges for technical support.