In Vitro Identification of Cultivar Responses to Rice Sheath Blight ...

PEST MANAGEMENT: DISEASES

In Vitro Identification of Cultivar Responses to Rice Sheath Blight Pathogen Rhizoctonia solani Y. Jia, P. Singh, G.C. Eizenga, F.N. Lee, and R.D. Cartwright ABSTRACT The goal of this project is to identify critical genes for the control of sheath blight disease. To this end, an in vitro method to examine interactions of rice with the rice sheath blight pathogen Rhizocotnia solani was developed. The initial analysis of R. solani isolates from Arkansas rice fields revealed three classes of most virulent, moderately virulent, and weakly virulent isolates. Differential responses of rice cultivar leaves toward these pathogen isolates were detected using a novel detached-leaf method. Characterized R. solani isolates are being used to identify differentially expressed genes. INTRODUCTION Rice sheath blight caused by Rhizoctonia solani Kühn [(teleomorph: Thanatephorus cucumeris (A.B. Frank) Donk.] is one of the most serious rice diseases worldwide (Rush and Lee, 1992). The lack of host specificity for R. solani has hindered the establishment of a defined genetic system for studying interactions between host and parasite (Bandara and Navaraja, 1979; Mew et al., 1980). In recent years, the incidence and severity of sheath blight have increased following the adoption of new semi-dwarf rice cultivars and the increased application of nitrogen fertilizers (Xie et al., 1990). In order to exploit rice-genome sequence information being made available (Goff et al., 2002; Yu et al., 2002) to develop better control of sheath blight, Jia et al. (2002) characterized a set of R. solani isolates from Arkansas rice fields. It appears these isolates possess minor differences in the internal transcribed spaces of ribosomal DNA

229

AAES Research Series 504

and all belong to the anastomosis groups AG1-IA. The objective of this study was to develop an in vitro method to detect cultivar responses to the pathogen isolates. PROCEDURES Methods for plant growth, purification, and conservation of the pathogen isolates were previously described (Jia et al., 2002). Six rice cultivars: ‘Cypress’, ‘Jasmine 85’, ‘Katy’, ‘Labelle’, ‘Lemont’, and ‘M-202’ were used to evaluate virulence. Briefly, the second youngest leaves, approximately 23 cm in length, were removed at V5 (Counce et al., 2000) and cut with scissors into 16-cm long segments. Immediately, the detached leaves were placed in 24 X 24 X 1.8 cm petri dishes containing moistened filter papers (23 X 23 cm). A 16-cm-long segment was adequate to support mycelial growth for 72 h. The six cultivars were inoculated with fourteen isolates in three replications. A 0.8-cmdiameter potato dextrose agar (PDA) plug was excised with a 1-ml Eppendorf tip and removed with a sterile toothpick. This plug was placed near the middle of the abaxial surface of leaf segments (approx. 11 cm). As a control, leaf segments from each cultivar were inoculated with a PDA plug without hyphae. Petri dishes with inoculated leaf segments were placed on a laboratory bench and maintained at 21ºC to 24ºC under continuous fluorescent light (10-20 µEm-2s-1) for 72 h. Because the lesion length reflects the rate of invasive mycelial growth and may be an indicator for the extent of crop reduction, the distance from the inoculation site to the leading edge of the lesion was measured with a protractor. Based on the mean value of total lesion length, isolates were rated as most virulent (for lesions >6.5 cm), moderately virulent (4 to 6.5 cm), and weakly virulent (6.5 cm). Data of the disease response of detached leaf segments were analyzed with 8.2 SAS (SAS Institute, Cary, NC). The Fisher T-test was used in Welch's ANOVA with the level of significance at P=0.05 to compare the disease response. RESULTS AND DISCUSSION Fourteen isolates were inoculated on six rice cultivars, Cypress, Jasmine 85, Katy, Labelle, Lemont, and M-202. Lesions produced by each isolate were measured to determine the virulence on each cultivar (Table 1). Fig. 1 shows virulent reaction of isolates from these representative classes on two cultivars, Jasmine 85 and M-202. The isolates were divided into three groups: most virulent (RR0102, RG0102, and RG0103); moderately virulent (RR0101, RR0103, RR0104, RR0113, RR0120, RR0125, RR0128, RR0134, and RR0135); and weakly virulent (RR0107 and RR0129) based on a significant difference in lesion sizes on selected cultivars. Isolates RR0102 and RG0102 caused the largest lesions ranging from 5.5 cm (RR0102) and 7.4 cm (RG0102) on Lemont to 9.1 cm (RR0102) and 9.0 cm (RG0102) on Labelle. The least virulent isolate, RR0129, caused the

230

B.R. Wells Rice Research Studies 2002

smallest lesions on all cultivars and almost no lesion development on Jasmine 85. Fig. 2 shows typical symptoms of cultivar responses to representative isolates of the most virulent (RG0102), moderately virulent (RR0128), and weakly virulent (RR0129) groups. Significant differences were observed in each cultivar's response to the different R. solani isolates (Table 1, Fig. 3). Based on detached leaf lesion development, Jasmine 85 was significantly more tolerant than all other cultivars tested. Katy and Lemont were moderately tolerant whereas Cypress, M-202, and Labelle were susceptible (Table 1, Fig. 3). Field observations of these cultivars over the years classify Cypress, Labelle, and Lemont as very susceptible, Katy and M-202 as moderately susceptible to susceptible, and Jasmine 85 as tolerant in Arkansas rice fields (Table 2). Table 2 shows the differences in lesion length that were observed but these were not significantly different when the individual cultivar or isolate were compared between trials. To examine variation among trials, three independent sets of experiments were performed and analyzed (Table 3). Increased tolerance to R. solani can be achieved either by incorporating resistance sources from Oryza species and selected Gramineae species into cultivated rice, or through genetic engineering of novel resistance genes into elite germplasm (Anuratha et al., 1996; Linthorst, 1991; Velazhahan et al., 1998). Detailed analysis of the molecular response of rice plants to R. solani infection has been initiated (Anuratha et al., 1996; Velazhahan et al., 1998; Datta et al., 1999). Expression of some pathogenesis related genes (PR) was correlated with R. solani infection (Anuratha et al., 1996; Velazhahan et al., 1998) and overexpressions of a few cloned PR genes have been shown to enhance resistance to R. solani (Datta et al., 1999; Lin et al., 1995). Establishment of a defined biological interaction system should eliminate some variability in identification of novel sources of tolerance (Guo et al., 1985). This study describes an improved inoculation system for identifying genes that control sheath blight. SIGNIFICANCE OF FINDINGS The differential host responses to the pathogen isolates using detached rice leaves provides a method of identifying differentially expressed resistance genes after inoculation with R. solani. This method of virulence evaluation should have a broader impact for other crop plants. Characterized isolates will not only be a useful tool for identification of resistance-related genes but also for plant breeders to develop resistant cultivars. ACKNOWLEDGMENTS We thank Ms. C. Ridgell and Ms. C. Flowers for technical support. For critical reading we thank Dr. J.N. Rutger. This work was supported in part by the Arkansas Rice Research and Promotion Board.

231


LITERATURE CITED Anuratha, C.S., K.C. Zen, K.C. Cole, T. Mew, and S. Muthukrishnan. 1996. Induction of chitinase and beta-1,3-glucanases in Rhizoctonia solani-infected rice plants: Isolation of an infection-related chitinase cDNA clone. Physiol. Plant 97:39-46. Bandara, J.M.R.S. and V. Navaraja. 1979. Reaction of some common weeds in Sri Lanka rice fields of Corticium sasakii. Int. Rice Res. Newsl.4(3):15-16. Counce, P.A., T.C. Keisling, and A.J. Mitchell. 2000. A uniform, objective, and adaptive system for expressing rice development. Crop Sci. 40:436-443. Datta, K., R. Velazhahan, N. Oliva, I. Ona, T. Mew, G.S. Khush, S. Muthukrishnan, and S.K. Datta. 1999. Overexpression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease. Theor. Appl.Genet. 98:11381145. Goff, S., D. Ricke, T.-H. Lan, G. Presting, R. Wing, et al. 2002. A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296:92-100. Guo, C.J., Z.Y. Chen, and F.M. Wang. 1985. Pathogenic variability in Thanatephorus cucumeris (Frank) Donk and techniques for identifying varietal resistance. Sci. Agri. Sin. 5:50-57. Jia Y., P. Singh G.C. Eizenga, F.N. Lee, and R. Cartwright. 2002. Molecular and pathological characterization of the rice sheath blight pathogen Rhizoctonia solani in Arkansas. In: R.J. Norman and. J.-F. Meullenet (eds.). B.R. Wells Rice Research Studies 2001. University of Arkansas Agricultural Experiment Station Research Series 495:148-153. Lin, W., C.S. Anuratha, K. Datta, I. Potrykus, S. Muthukrishnan, and S.K. Datta. 1995. Genetic engineering of rice to resistance to sheath blight. Biotechnology 13:686691. Linthorst, H.J.M. 1991. Pathogenesis-related proteins of plants. Crit. Rev. Plant Sci. 10:123-150. Mew, T.W., D. Fabellar, and F.A. Elazequi. 1980. Ecology of rice sheath blight pathogen: parasitic survival. Int. Rice Res. Newsl. 5:15-16. Rush, M.C. and F.N. Lee. 1992. Sheath blight. In: R.K. Webster, and P. S. Gunnell (eds.). Compendium of rice diseases. APS Press, St. Paul, MN, USA. pp. 22-23. Velazhahan, R., K. Chen-Cole, C.S. Anuratha, and S. Muthukrishnan. 1998. Induction of thaumatin-like proteins (TLPs) in Rhizoctonia solani-infected rice and characterization of two new cDNA clones. Physiol. Plant 102:21-28. Xie, Q.J., M.C. Rush, and J. Cao. 1990. Somaclonal variation for disease resistance in rice (Oryza sativa L.). Pest management in rice. In: B.T. Grayson, M.B. Green, and L.G. Copping (eds.). Elsevier Applied Science. London. pp. 491-509. Yu, J., S. Hu, G.K.-S. Wang, S. Wong, B. Li, Y. Liu, L. Deng, Y. Dai, Y. Zhou, X. Zhang, et al. 2002. A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science 296:79-92.

232


Table 1. Lesion length caused by different R. solani isolates in detached leaf inoculations of different rice cultivarsz. Cultivars Isolates

Cypress

Jasmine 85

Katy

Labelle

Lemont

M-202

RR0101 RR0102 RR0103 RR0104 RR0107 RR0113 RR0120 RR0125 RR0128 RR0129 RR0134 RR0135 RG0102 RG0103 LSDx

7.17y 8.63 6.97 3.23 5.97 8.20 4.97 4.90 7.00 5.00 6.33 8.23 8.40 9.93 1.06

3.90 5.97 1.23 1.73 0.97 4.43 1.67 3.33 3.17 0.50 4.17 2.80 4.27 4.43 0.98

3.30 6.13 3.77 5.33 2.30 3.73 3.00 4.33 3.83 3.23 3.83 5.50 6.40 4.43 1.30

4.63 9.07 9.80 5.30 3.77 7.43 2.93 7.27 7.47 5.00 8.47 7.33 8.97 8.20 1.05

6.13 5.50 1.63 4.20 1.60 4.97 3.43 3.33 4.57 1.20 5.23 4.50 7.37 4.67 1.22

7.33 7.27 8.17 6.17 9.37 7.53 5.50 5.07 6.30 5.93 7.57 7.20 7.13 8.50 0.87

z y x

Data represent total lesion length (cm) around the inoculation site. Means of three replications. Least significant difference (LSD) calculated based on Welch's ANOVA disease ratings for individual cultivar at P6.5 cm. Data were field observations.

233


Table 3. Interaction among three independent sets of experimentsz. Mean Variables Cultivars Cypress Jasmine 85 Katy Labelle Lemont M-202 Isolates RR0101 RR0102 RR0103 RR0104 RR0107 RR0113 RR0120 RR0125 RR0128 RR0129 RR0134 RR0135 RG0102 RG0103 z y

Trial 1y

Trial 2

Trial 3

6.32 a 2.83 a 3.94 a 6.37 a 3.88 a 6.60 a

6.30 2.98 4.22 6.32 4.14 6.46

a a a a a a

6.40 3.14 4.37 6.41 4.40 6.59

a a a a a a

5.01 7.12 5.62 4.31 4.03 6.32 3.68 4.78 5.41 3.55 6.25 5.75 7.44 6.43

5.27 7.27 5.24 4.92 4.20 6.24 4.19 4.76 5.54 3.76 6.41 5.80 7.29 6.80

a a a a a a a a a a a a a a

5.46 7.04 5.44 4.56 3.99 5.98 3.74 4.97 5.44 3.77 5.98 5.80 7.10 6.27



Subcultures of same original culture plate were used for inoculation in each trial. Trial 1 represents mean of data presented in Table 1, trial 2 and 3 are independent sets of experiments (data not shown). Means compared in each trial with the same letter are not significantly different.

234


Jasmine 85 M202 1

2 3

4

1

2

3

4

Fig. 1. Disease lesions on detached leaf assays for isolates on two rice cultivars. (1) Most virulent (RG0102), (2) Moderately virulent (RR0128), (3) Least virulent (RR0129), and (4) the control (PDA plugs) were inoculated onto indicated cultivars and incubated for 72 h,

1 2 34 5 6

RR0129

12 3 4 5 6

123 45 6

Control

1 23 4 5 6

Fig. 2. Disease lesions on detached leaf assays for isolates on (1) Cypress, (2) Jasmine 85, (3) Katy, (4) Labelle, (5) Lemont, and (6) M-202 after inoculation with indicated isolates and the control (PDA plugs) and incubation for 72 h.

235

10 8 6 4

a

a c

b

a b

2 0

Cy pr es Ja s sm in e 85 Ka ty La be lle Le m on t M 20 2

Mean lesion length (cm)


Fig. 3. Overall cultivar response to all 14 isolates as determined by mean lesion length. Letters above each bar indicate significant difference at P=0.05 ± standard error.

236