Resistance of Oryza nivara and Oryza eichingeri derived lines to brown planthopper , Nilaparvata lugens ( Stal )

This study evaluates the basis of brown planthopper (BPH) resistance in Oryza nivara (9864, WRAC 04), Oryza eichingeri (TC 01, TC 02), IR 54751-2-34-10-6-2 (an introgression line of Oryza officinalis), Ptb 33, Bg 380 and Bg 379/2 accessions by screening for resistance, and characterizes the genes by molecular techniques. Four BPH populations reared on Ptb33, Bg 380, Bg 379/2, IR 54751-2-34-10-6-2 and a virulent BPH population collected from rice fields in Kegalle area Sri Lanka were used for screening tests. Screening for resistance was conducted following the standard honeydew test and molecular screening was carried out using specific molecular markers for bph2, Bph10 and Bph13 genes. Results of the screening tests for resistance showed that the level of resistance of the tested wild rice accessions (O. nivara and O. eichingeri) was not significantly different from that of Ptb 33. These accessions also showed high level of resistance to all the BPH populations used in this study. Molecular screening revealed positive results with O. nivara (WRAC 04) accession for bph 2 and Bph 13 markers and O. eichingeri derived lines (TC 01 & TC 02) for Bph 13 marker. Results showed that the emergence of virulent biotype in field rice cultivations have the potential to overcome the BPH resistance in Bg 379/2.


INTRODUCTION
The brown planthopper (BPH), Nilaparvata lugens (Stal) (Homoptera: Delphacidae) is one of the most serious rice pests in Sri Lanka.Annually BPH damage leads to a loss of about 5-10% of national rice production.At present, farmers depend mostly on chemical pesticides for the control of this pest (Kudagamage & Nugaliyadda, 1995).This dependence leads to many adverse effects.The most effective way of BPH management is considered as host plant resistance.Varietal resistance is the most economic, least complicated and environmentally friendly approach for the control of insect pest damage (Pathak & Kush, 1979).Rice is an annual grass that belongs to genus Oryza, which includes 22 wild species.These wild species of rice contain genes, which can be used for improvement of rice.Identification and characterization of these are important for their future transfer from wild rice to cultivated rice varieties.Researchers at the Rice Research and Development Institute (RRDI) at Bathalagoda use some BPH resistant lines developed from O. officinalis and O. australiensis in their breeding programmes.Fundamental studies have been undertaken to evalute the use of O. nivara and O. eichingeri in rice breeding programmes in Sri Lanka.BPH resistance has been detected in O. nivara and introgression lines derived from O. eichingeri and O. sativa at the RRDI (Kumari et al., 2007;Hemachandra et al., 2008).Therefore, understanding the basis of BPH resistance in these species will be very useful for varietal improvement programmes of rice in Sri Lanka.Bioassay of honeydew extraction has been effectively used for screening resistant lines (Heinrichs et al., 1985).
Molecular markers have become efficient tools for screening of BPH resistance and marker assisted selection (MAS) in rice.They have been helpful for the identification of donors of BPH resistance and the genetic study of BPH resistance (Su et al., 2006).Over 18 major BPH resistant genes have already been identified and characterized (Jena et al., 2006).Of these resistant genes Bph1, bph2, Bph9 and Bph10 were located on chromosome 12, Bph3 and bph12 were located on chromosome 4, bph4 was located on chromosome 6, Bph6 was located on chromosome11 and bph11 and Bph13 were located on chromosome 3 (Ishii et al., 1994;Hirabayashi et al., 1999;Renganayaki et al., 2002;Jena et al., 2003;Sharma et al. 2003).Primers specific to above resistant genes can be used to identify BPH resistant genes in O. nivara and O. eichingeri derived lines.The present study screened the above lines for BPH resistance using honeydew extraction bioassay and polymerase chain reaction (PCR) analysis using the sequence tagged site (STS) markers such as KAM4 (bph2), RG457 (Bph10) and AJ 096 (Bph13).
Four BPH populations developed from BPH culture maintained at the RRDI, Batalagoda and virulent BPH population collected from fields in Kegalle and reared on Bg 379/2 at Bathalagoda in separate cages (volume; 1.215 m 3 ) were cultured as described in Tables 1 & 2 in a plant house at the RRDI, Bathalagoda for 3 months were used in this study.
The rice lines and varieties (9864, WRAC 04, TC 01, TC 02, IR 54751-2-34-10-6-2, Ptb 33, Bg 379/2 and Bg 380) were grown singly in 15 cm clay pots and managed free from BPH infestations.Screening for resistance was done when they reached 2 months in age.Screening was done inside feeding chambers.Whatman No.2 filter paper stained with bromocresol green pigment (2 mg/1 mL of 70% ethanol) was placed around the base of the test plant before infesting BPH for screening.A pair of BPH females previously starved for 4 to 5 h were placed in the feeding chamber and allowed to feed for 24 h.Honeydew excreted by BPH was absorbed to filter paper, which appeared as a blue spot.The area of the spot was assumed to be proportional to the honeydew production and amount of BPH feeding (Heinrichs et al., 1985).Reaction of BPH to host plants and plant resistance to BPH were determined based on honeydew production.Screening of rice lines and varieties for resistance was done following the randomized complete block design with five replicates.DNA was extracted following the CTAB "Miniprep" protocol, (Anonymous, 2003).DNA extracted from the rice lines and varieties were screened by PCR amplification using RG 457 FL/RL (Bph 10), KAM 4 (bph 2) and AJ096 (F/R) (Bph 13) STS primers.
The PCR mixture contained 20 -100 ng template DNA, 1 pmol of each primers, 0.25 mM dNTP's each, 1X buffer (20 mM Tris pH 8.0 including 20 mM MgCl 2 ) and 0.5 units of Taq polymerase in a total volume of 10 -15 µL.PCR was performed by using a Master Cycler Gradient.PCR mixtures were initially denatured at 94 0 C at 5 min.This was followed by 40 cycles of PCR amplification using following parameters: denaturation of 94 0 C (30 s), primer annealing at 66 0 C (1 min) and primer extension at 72 0 C (1 min).After PCR, products of RG 457 primer were subjected to digestion by restriction enzyme Hinf1 incubating at 37 0 C for 1.5 h.PCR products were electrophorated in 1.4% agarose gel to separate the amplified DNA fragments.The gel was subsequently stained by ethidium bromide and amplified DNA fragments were visualized under UV light as bands.PCR products of AJ096 primer were electrophorated in 10% Silver stained urea PAGE to confirm the separated bands in 1.4% agarose gel.

RESULTS
PCR amplifications with KAM 4 primer gave rise products of 300bp, (Murai et al., 2001) with Ptb 33 and WRAC 04 (Figure 1-A).This indicates the presence of bph2 gene in the genome of Ptb 33 and WRAC 04 accessions.Gene bph2 is one of the 12 BPH resistant genes so far identified in several indica cultivars.Ptb 33 shows high level of BPH resistance.Khush (1979) reported that it was due to combination of two different resistant genes, bph2 and Bph3.PCR analysis with Bg 379/2, the variety developed by RRDI, Bathalagoda using Ptb 33 as the source of resistance, did not indicate bph2 gene in its genome (Khush, 1979).PCR analysis with forward and reverse primers of RG 457 showed amplification of 750bp DNA fragment with DNA of all the tested lines and varieties (Figure 1-C) (Nguyen et al., 1999).These amplified products were detected only after digestion by Hinf1 restriction enzyme.Further analysis of them showed DNA fragments of 300bp, 250bp and 200bp in the amplified products of Bg 357 and DNA fragments of 550bp and 200bp in the amplified products of all the other entries (Figure 1-D).This indicated the presence of Bph10 gene only in the genome of Bg 357 (Nguyen et al., 1999).
Analysis of honeydew productions showed a significant difference among the tested rice lines and varieties (α = 0.05) (Figure 2-A).O. nivara (WRAC 04,9864), O. eichingeri (TC 01, TC 02) and Ptb 33 showed significantly lower honeydew productions.This indicated their resistance to BPH.Bg 380 showed significantly higher honeydew production indicating no resistance to BPH.Moderate but significantly different honeydew production was observed in Bg 379/2 and IR line (Figure 2-B) indicating moderate resistance to BPH (CV= 42%).
Figure 3A shows the mean areas of honeydew excretions in relation to tested BPH populations on all the tested lines and varieties.Analysis of mean areas of honeydew excretions showed a significant difference among populations (α = 0.05).The significantly high honeydew excretions were produced by populations reared on Bg 379/2 and the virulent population of Kegalle.The significantly low honeydew excretions were produced by populations reared on IR 54751-2-34-10-6-2 and Bg 380.The population reared on Ptb 33 showed no significant difference of honeydew excretions to any populations.Thus, virulence of the Kegalle population and the population reared on Bg 379/2 were significantly higher than that of populations reared on IR 54751-2-34-10-6-2 and Bg 380.This also supports the report of the emergence of new biotype in cultivated fields of Bg 379/2 variety in Kegalle (Fernando et al., 2007).
Data of honeydew excretions also indicated a significant interaction between different populations and tested varieties (α= 0.05) (Figure 3-B).Bg 380 variety was highly susceptible to BPH populations reared on Ptb 33 and Bg 379/2 and the virulent population of Kegalle.

DISCUSSION AND CONCLUSION
BPH resistance has been identified in a wide range of traditional rice varieties and wild rice species (Pathak & Khush, 1979;Heinrichs et al. 1985).Scientists have already characterized and exploited some of the genes responsible for resistance and upgraded cultivated rice varieties.(Wu et al., 1986).Further research in other wild relatives is suggested as they may be useful as donors of BPH resistance in variety improvement programmes in the future.
Screening with specific molecular markers in this study was revealed the presence or absence of three BPH resistant genes (bph2, Bph10 and Bph13) in O. nivara and O. eichingeri derived lines.It has identified the presence of Bph10 gene in O. australiensis (Nguyen et al., 1999) Results of the honeydew test indicated resistance in Ptb 33 and all the tested wild rice accessions to all BPH populations.However their degree of resistance was not significantly different among each other.Therefore, the BPH resistance identified in O. nivara accession (9864), O. nivara accession (WRAC 04) and O. eichingeri derived lines (TC 01, TC 02) may be due to the presence of other resistant genes or new genes.Identification of additional BPH resistant genes and exploiting them to widen the genetic base of cultivated rice varieties need to be continued in view of overcoming future BPH outbreaks by emergence of new biotypes.Results of this study highlight the possibility of using genetic resources of wild rice, O. nivara and O. eichingeri in order to develop broad based and durable resistance for BPH in cultivated rice varieties in the future.

Figure 1 :Figure 2 :
Figure1: PCR amplification of DNA of tested rice entries by KAM 4 primer followed by electrophoration in 1.4% agarose gel (A), by AJ096 primer followed by electrophoration in 1.4% agarose gel (B), by RG 457 FL/RL primers followed by electrophoration in 1.4% agarose gel (C) and digested products of (C) by restriction enzyme Hinf1 followed by electrophoration in 1.4% agarose gel (D)

Figure 3 :
Figure 3: Mean area (mm 2 ) representing honeydew excretion by 5 BPH populations when fed on tested rice lines and varieties (A) and interaction between BPH populations and tested rice lines and varieties (B); Means with the same letter are not significantly different at 5% level (DMRT).

Table 1 :
Description of BPH populations used for BPH Bioassay

Table 2 :
Status of BPH resistance in host plants used in BPH cultures To date these varieties play a significant role in controlling BPH outbreaks in rice cultivations.However development of virulent BPH populations is becoming a serious threat for rice cultivation at present.Thus, constant search for resistant genes is very important.Wild relatives of rice, O. australiensis and O. officinalis have already become donors of BPH resistance (Renganayaki et al., 2002).Several reports have highlighted wild rice species of O. latifolia, O. nivara, O. punctata and O. minuta as important sources of BPH resistance

Area of honeydew spots (mm 2 ) Varieties of
. This gene is absent in O. nivara and O. eichinigeri derived lines.The analysis showed the presence of bph 2 gene in the genome of O. nivara (WRAC 04) accession.However, analysis did not indicate any evidence for the presence of bph 2 gene in O. nivara (9864) accession.Molecular analysis with markers indicated the presence Bph 13 gene in Bg 380, Bg 357, Bg 379/2, IR 54751-2-34-10-6-2 line, O. nivara (WRAC 04) and O. eichingeri derived lines (TC 01 and TC 02).Honeydew test results showed that Bg 380 is susceptible to the BPH, Bg 379/2 and IR 54751-2-34-10-6-2 line moderately resistant to the BPH while, O.nivara (WRAC 04) and O.eichingeri (TC 01 and TC 02) resistant to the BPH and Bg 357 known to be moderately resistant to the BPH.Although Bg 380 is susceptible to the BPH it showed positive results for Bph 13 gene indicating Sri Lankan BPH is virulent to the Bph 13 gene.Hence resistance observed in Bg 379/2, IR 54751-2-34-10-6-2 line, O. nivara (WRAC 04), O. eichingeri (TC 01 and TC 02) and Bg 357 due to the presence of other resistant genes but not due to the presence of Bph 13 gene.The lower level of resistance in Bg 379/2 in comparison to Ptb 33 may be related to the absence of bph2 gene in BG 379/2, which is present in Ptb33.