' USDA-ARS, Sugarcane Field Station, Canal Point, FL Florida Sugar Cane League, Canal Point, FL 33438

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1 J.C. Comstock, et a1 SCREENING FOR RESISTANCE TO RATOON STUNTING DISEASE IN F'LORIDA J.C. Comstock', J.D. Miller1, J.M. Shine, Jr.l& P.Y P. Tail ' USDA-ARS, Sugarcane Field Station, Canal Point, FL Florida Sugar Cane League, Canal Point, FL ABSTRACT A screening program for detecting resistance to ratoon stunhig disease (RSD) at the USDA Sugarcane Field Station was established. Screening starts with Stage I1 when there are approx varieties. All RSD screening is conducted in separate tests parallel to the varietal development program. About 15 % of the Stage I1 varieties were rated as susceptible and most were discarded. They had > 10 colonized vascular bundles per 1 cm stalk core, a value comparable to that of a widely grown susceptible commercial variety. RSD susceptibility alone is not sufficient grounds to merit discarding in subsequent stages because extensive testing for multiple traits has found at least one defect in almost all varieties, making it difficult to discard a variety for a single defect. Correlations for RSD susceptibility between tests varied, but increased as no. of stalks sampled increased. Results indicated that the resistance level of the parental variety influenced the percent of RSD resistantlsusceptible progeny; thus crosses between two parents that produce progeny with high percentages of RSD-susceptible clones are not recommended. Keywords: Sugarcane, ratoon stunting disease, disease screening, disease resistance, Clavibacter xyli subsp. xyli, varietal development, USA. rntr0duction Ratoon stunting disease (RSD) was first recognized in Australia during the summer of by the abnonnal stunting of Q 28 plants in ratoon fields. Since then the disease has been identified in almost all sugarcane-growing areas of the world. The disease is insidious because of the general lack of external symptoms although there is an internal reddish discoloration of the vascular bundles at the basal nodes (Gillaspie & Teakle 1989). It has been documented that RSD causes a reduction in yield that is estimated at a 5 % loss in Florida (Dean & Davis 1989, Irey 1986) and > 30 % in other areas (Gillaspie & Teakle 1989). A bacterium was associated with the disease in 1973 (Glllaspie et a1 1973, Teakle et a1 1973), which was subsequently described as Clavibacter xyli subsp. xyli (Davis et a1 1984). In recent years several assays have been used to diagnose RSD. These include isolation in culture by the dilution plate technique, direct observation of the pathogen using phase contrast microscopy, fluorescent-antibody staining (Davis 1985) (Davis & Dean 1984), alkaline-induced metaxylem autofluorescence (Damann 1988), dot blot (Irvme & Irey 1991) and tissue blot immunoassay (Davis et al 1994, Harrison & Davis 1988). Although these assays are helpful m the detection of RSD, none is foolproof. Traditiona1,control practices have relied on heat therapy to eliminate the pathogen from systematically infected seedcane and on sanitary praotioes to prevent reinfection (Gillaspie & Teakle 1989). Without periodic heat therapy treatment, plants of susceptible varieties in commercial fields become infected within a few years (Comstock et a1 1993). Even with heat therapy, RSD control may not be completely successful (Damann & Benda 1983, Roach 1992, Roach & Jackson 1992). Heat therapy practices require strict adherence to procedures and field sanitation to prevent the RSD pathogen from being reintroduced and spread. It is not uncommon for commercial operations to eliminate the hot-water treating of seedcane for RSD control to expedite planting to meet schedules. Moreover, reports of the 1991, Roach 1992, Roach & Jackson 1992) indicate the difficulty in maintaining disease-free s commercial fields. 520

2 Biology: Pathology variety reported to be immune to the disease because it had no internal Table 1. Summary of RSD screening program for 1993 and vasculardiscolorations~tom (Wismer ). Sitice then the diagnostic assays that have been developed have made it No. CP No. No. CP No. No. possible to screen large numbers of Stage sampled series tested' susceptible series tested1 Susceptible clones for their RSD reaction based on relative pathogen populations rather Seedlings 0 94.OL 0 95 O'(0) 0 than inconspicuous symptoms. I o (o)~ o (0) 10 Screening for RSD resistance was I (65) (81) 82 incorporated into the USDA cane m (56) (91) 9 varietal development program at Canal III inc (92) (97) 3 Point in The purpose was to N (100) (92) 3 assist breeders in developing RSDresistant varieties. Since then others The varietal program has ca. 100,000 seedlings. have suggested the possibility of There are ca. 10,000 clones in Stage I. breeding for RSD resistance (Davis et The number in 0 indicates the percent varieties screened for their a1 1994, Roach 1992, Roach & Jackson RSD reaction. 1992). The objective of this paper is to Three from One plot. describe tile screening program for There were 20 stalks sampled, 5 stalks from 4 replicated plots. Resistant Susceptible Resistant Figure 1. A serologically developed nitrocellulose membrane with stalk samples of 6 varieties (l/column) with 5 different stalks each. controlling RSD at the USDA-ARS Sugarcane Field Station and indicate how it affects the varietal development program. A preliminary report was published by Comstock et a1 (1991). MATERZALS AND METHODS Inoculation Stalks of each variety were inoculated by cutting whole stalks of cane in the furrow into 0.5-m seed pieces. The cane knife was dipped in undiluted cane juice expressed from infected stalks of CP 53-1, a known RSD-susceptible variety that supports high populations of the bacterial pathogen C. xyli, responsible for RSD. Stalk sampling and processing At 10 mo after inoculation, samples were taken from tlie plant cane crop by cutting the basal 2-3 internode portion of 3 stalks from Stage 11 and Stage III plots dnd 5 stalks from the increase plots of Stage 111 and Stage N plots. The samples were processed according to the tissue blot immunoassay method (Davis et a1 1994, Harrison & Davis 1988). Using a 1-cm dim. cork borer, a longitudinal core of tissue was removed from the stalk internode, cut to 1-cm length and placed in a plastic template with the cut vascular bundles resting on a nitrocellulose membrane stacked on a piece of 3-mm Whatman paper and on absorbent paper toweling. The stalk cores were centrifuged to force out the sap from the xylem forming a pellet of bacteria on the nit~ocellulose membrane surface directly below the colonized vascular bundles while the liquid moved through the membrane and was absorbed by the filter paper atid toweliug. The membranes were dried for 1 h at 80 C. The membranes were serologically processed (Harrison & Davis 1988) by first treating them with a protein reagent 52 1

3 J. Comstock, et al to block nonspecific binding sites for antibodies. The membranes were then incubated for 1 h in a rabbit antiserum specific for the RSD pathogen provided by M.J. Davis (Univ. of Florida, Homestead). The membranes were then rinsed with buffer and incubated for 1 h with a second antibody conjugated with alkaline phosphatase. This second antibody was specific for rabbit antibodies and attached to the first antibody. Lastly, Past Blue BB salt substrate solution was added, which reacted with alkaline phosphatase, turning blue. The membranes were rinsed in sodium hypochlorite to remove a general brownish discoloration, after which they were rinsed in water to remove the sodium hypochlorite and dried at room temperature. The membranes showed the impression of the vascular bundles with RSD-infected vascular bundles stained blue. The RSD reaction was determined by the no. of colonized vascular bundles (CVB), which were counted using a stereomicroscope or a video image analysis system (Shine & Comstock 1992). The impressions of the vascular bundles can be seen as "dots," which locate the vascular bundles containing C. xyli, which are stained serologically. RSD rating scale The current rating scale was established to discard varieties that are either as susceptible or more so than commercial var. CP , which usually has 10 CVB11-cm core of stalk tissue in inoculated and naturally infected plants, Varieties with > 10 CVB were.rated as susceptible; those < 10 CVB, resistant. Varietal testing The no. of varieties tested at the various stages in the varietal development program is shown in Table 1. A single stalk of each variety was inoculated and planted in a single plot for the Stage II (approx. 1000) and Stage 111 varietal (131) evaluations. The same inoculation procedure, replicated 4 times, was used for the varieties increased in Stage 111 (41) and in Stage IV (11). Varieties were repeatedly tested in parallel tests separate from the regular selection program, with increasing numbers of samples as they were advanced from one stage to the next. Plant materials from the RSD screening tests were destroyed following evaluation: and none of this material was replanted in the regular varietal development program. RESULTS A serologically developed nitrocellulose membrane that had 6 varieties each in separate columns with 5 stalks sampled per variety is shown in Figure 1. The samples of varieties in columns 3 and 5 from the left have a large number of small "dots" that represent the bacteria from individual CVB collected during centrifugation and were subsequently stained serologically. The large number of CVB indicates that the varieties in columns 3 and 4 from the left are RSD susceptible. The samples of varieties in the columns 3, 5 and 6 have no such stained CVB, indicating that they are RSD resistant. There are a few stained CVB in samples of the variety in column 1, indicating an intermediate reaction. In Stage II a total of 591 and 611 varieties were evaluated in 1993 and 1994, resp., for their reaction to RSD. This represents 65 and 79% of the varieties in Stage I1 for 1993 and 1994, resp. Of the Stage I1 varieties evaluated during these years, only 11 and 13 % were susceptible and only a few of these were advanced. Of the 131 Stage IU varieties 56 and 91 % were evaluated for their RSD reactions in 1993 (CP 91 series) and 1994 (CP 92 series), resp. Of these Stage I11 varieties, 16% were susceptible in 1993 (CP 91 series) and 7% were susceptible in 1994 (CP 92 series). Over 90 % of the varieties in the increase phase of Stage I and in Stage N were evaluated in both years. The percent susceptible varieties in 1994 ranged from a low of 8 % for varieties in increase in Stage III to 27 X in Stage N. In 1993 the levels of susceptible varieties for these stages fell between these values. The correlation of RSD ratings between years varied depending on the no. of stalks sampledlvariety (Table 2). The correlation coefficient was r = 0.22 for the 82 varieties of the 92 series tested that had 3 stalksssampled both years; r = 0.38 for the 21 varieties of the 91 series tested both years with 3 stalks sampled the first year and 20 stalks sampled the second year; and r = 0.84 for the 10 varieties of the 90 series that had 20 stalks sampled both years. The frequency of RSD-susceptible progeny varied according to parental clones (Table 3). US , used either as a female or male parent, produced 33.3 % susceptible progeny; whereas none of the progeny of CO 285 was susceptible. Parental varieties CP and CP also produced a high incidence of susceptible progeny, 20.8 and 18.1 %, resp. In contrast, CP , CL and CP all gave rise to a low frequency of susceptible progeny (i.e., < 6 %). Wide differences in frequency of RSD susceptibility were noted between families (Table 4). The cross CP

4 Biology: Pathology x SP had 26.1 % susceptible progeny; while cross CO 285 x CP produced no susceptible progeny. Another cross (CP x SP ) produced a high proportion of RSD-susceptible progeny. Five crosses involving CP as a female parent crossed with 5 different male parents produced from % susceptible progeny depending on the male parent. The male parents that generally gave rise to less-susceptible progeny did so even when crossed with CP As expected, the average CVB of the family varied, analogous with the percent susceptible progeny. DISCUSSION Table 2. Comparison of RSD ratings of varieties tested in 1993 and The first point where the RSD reaction of varieties can be evaluated is in Stage I1 when there are approx. No. of Stalks sampled r No. RSD susceptible 1m varieties. Up thir point in the Series varieties value Both varietal development program, there is ** not sufficient seedcane to evaluate NS varieties for their RSD reaction in a * separate parallel test to that of the NS = Nonsignificant regular development Program- Even *, ** Significance at 5% or 1 % probability, resp. at this point, only a single nonreplicated plot can be evaluated. Currently, an RSD resistance level of 10 CVB was set as a cutoff between susceptible and resistant varieties. At this level most susceptible varieties can be eliminated from the varietal development program without elirmnating a disproportionate number of varieties. Nevertheless, it should be noted that a near-zero CVB is required to limit the incidence of stalk infection to low levels without hot-water treatments and rigorous field sanitation (Cornstock et al, unpub. data); thus the cutoff will be adjusted downward as the average CVB for the varieties in the development program decreases. In Stage 11, % of the varieties were susceptible to RSD; in 1994, 82 varieties in Stage 11 (CP 93 series) were susceptible. Of these only 23 had acceptable cane and/or sugar yields for advancement; only 3 of the highest yielders were actually advanced to Stage III. At Stage 11 RSD susceptibility is usually a sufticient defect for eliminating a variety. At later stages RSD susceptibility is taken into consideration as one of the factors, but it alone is not sufficient reason to eliminate a variety. Elimination of susceptible varieties in Stage II and the increased use of RSD resistant varieties as parents should lower the percentage of RSD susceptible varieties 111 the later stages of the varietal development program in the future. The r values for tests involving 3 stalks (Table 2) were much lower than the correhon between inoculated and naturally infected tests, reported as ranging from r = 0.68 to r = 0.94 (Roach & Table 3. Influence of parental clone on incidence of progeny RSD susceptibility. Used as female Used as male % Combined Parental No. of % RSD No. of % RSD RSD clone progeny susceptible progeny susceptible susceptible CL CO CP CP CP CP CP CP CP CP SP SP US Jackson 1992). This is probably due to small sample size and to escapes 111 inoculation technique (i.e., cutting st& with caie hives dipped in juice expressed from infected plaits). Only 3 stalks are sampled from a

5 C. Comstock, et a1 single nonreplicated plot. When 4 replications of 5 stalks were used, the correlation was much higher. Starting this year, 2 replications will be evaluated for Stage II varieties with 5 stalk samples assayed per plot. The stalks are cut at 0.5-m lengths so some lateral buds that germinate may be considerable distance from an inoculation site and may escape infection. The plants that Roach & Jackson (1992) sampled were either inoculated as single-bud cutthlgs or naturally infected and had reached RSD equhbriurn. We have obtained a correlation value of r = 0.95 for 8 varieties rated in an RSD-itioculated test (20 stalk samples) and sampled from naturally infected commercial fields (Comstock et al, unpub. data). It is believed that when the CVB no. varies between screening tests and/or between stalks sampled, the higher no. is probably correct. A false rating for RSD resistance is more likely due to a failure in moculation tlm to a false rating for RSD susceptibility. Thus the errors of this system favor the breeder because resistant selections are not falsely discarded. The development of resistant varieties is a practical method of controlling RSD in Florida. Certain cornrnerclal CP-varieties in Florida express RSD resistance as a low no. of CVB- Var. CP , Table 4. Incidence of RSD susceptibility in progeny of released in 1982 (Miller et a1 1984), occupies eight biparental crosses % of the commercial area in Florida and is RSD resistant. A random survey was made No. of % RSD Avg. no. of 28 commercial fields of the variety in which Biparental cross progeny susceptible CVB seedcane lad never been heat treated and little or no phytosanitation had been practiced at ~~~~l~ Male random. Only 1.4 % of the stalks were co 285 x cp infected, with an average of 0.15 CVBIsample. cp cp This is in contrast to RSD-susceptible var. CL CP CL and CP , which are essentially cp x cp % infected under similar conditions. Two ~p x cp other newer CP-varieties (CP & CP cp cp ) appear resistant as their avg stalk cp ~p infections were 8.0% for 28 commercial fields ~p x SP and 1.0 % stalk infection for 7 commercial cp x cp fields, resp. Var. CL , developed in Florida by the US Sugar Corp. also appears to be resistant, with an avg of 2.7% stalk infection for 9 commercial fields. Decisions of what parental varieties are crossed should be made with the knowledge of the frequency of RSDsusceptible progeny that have occurred in the past using individual parental clones. As a general rule, crosses between two parents that produce high frequencies of RSD-susceptible progeny should not be madg*? Families that have higher frequencies of RSD susceptible progeny also average high no. of CVB as would be expected. The breeding program in the 1994 crossing season had a total of 560 crosses made, of which 19.5% of them involved at least one RSD-resistant parent. The 10 varieties identified as having RSD resistance for the commercial breeding program resulted in 32 resistant x resistant crosses and 77 involving one resistant parent in The 36 RSD-resistant parental varieties were planted for use in the 1995 breeding season. As RSD resistance is highly heritable (Miller et a1 1995), the increased use of resistant parental varieties combined with screening for RSD resistance in the middle and later stages of the varietal development program should permit the development of RSDresistant commercial varieties in the near future. ACKNOWLEDGMENTS The authors acknowledge the contributions of M.J. Davis and his associates in developing the tissue blot immunoassay, as well as his and J.L. Dean's research on RSD, which form the basis of the current screening program reported here. REFERENCES Anon. (1991). The RSD situation in the industry. In SASEX ann. rept , Mount Edgecombe, pp Anon. (1992). Ratoon stunting disease. BSES 92nd ann. rept., Indooroopilly, p

6 Biology: Pathology Autrey, L.J.C.; Dookun, A.; Saumtally, S.; Dhayan, S. & Sullivan, S. (1991). Soil transmission of the ratoon stunting disease bacterium Clavibacter xyli subsp. xyli. Sugar Cane Bailey, R.A. & Tough, S.A. (1991). Mechanisms of infection of sugar cane by the ratoon stunting disease bacterium Clavibacterxyli subsp. xyli. Sugar Cane 65. Chu, H.T. & Lee, S.M. (1968). Ratooon stunting disease control in Taiwan. Proc. ISSCT 13: Comstock, J.C.; Miller, J.D.; Dean, J.L. & Davis, M.J. (1991). Screening for ratoon stunting disease resistance using a tissue blot assay. Sugar Cane 6:6. (abstr.) Comstock, J.C.; Shine, Jr., J.M. & Perdomo, R. (1993). Ratoon stunting disease in Florida's non-hot-water treated sugarcane seedfields. J. Amer. Sugar Cane Technol. 13: Damann, Jr., K.E. (1988). ~lkaline-hduced metaxylem autofluorescence: A diagnostic symptom of ratoon stunting disease of sugarcane in Florida. Plant Dis. 68: Damann, Jr., K.E. (1992). Effect of sugarcane susceptibility on spread of ratoon stunting disease by the mechanical harvester. Plant Dis. 76: Damann, Jr., K.E. & Benda, G.T.A. (1983). Evaluation of commercial heat-treatment methods for control of ratoon stunting disease of sugarcane. Plant Dis. 67: Damann, Jr., K.E. & Hollier, C.A. (1991). Distribution and incidence of ratoon stunting disease in Louisiana sugarcane. Plant Dis. 75: Damann, Jr., K.E.; Ogunwolu, E.O. & Reagan, T.E. (1984). Incidence of ratoon stunting disease in Louisiana. Phytopathol. 74:627. Davis, M.J. (1985). Directcount techniques for enumerating Clavibacter xyli subsp. xyli which causes ratoon stunting disease of sugarcane. Phytopathol. 75: Davis, M.J. & Dean, J.L. (1984). Comparison of diagnostic techniques for determining incidence of ratoon stunting, disease of sugarcane in Florida. Plant Dis. 68: Davis, M.J.; Dean, J.L. & Harrison, N.A. (1988). Quantitative variability of Clavibacter xyli subsp. xyli populations in sugarcane cultivars differing in resistance to ratoon stunting disease. Phytopathol. 78: Davis, J.M.; Dean, J.L.; Miller, J.D. & Shine, Jr., J.M. (1994). A method to screen for resistance to ratoon stunting disease of sugarcane. Sugar Cane 6:9-16. Davis, M.J.; Gillaspie, Jr., A.G.; Vidaver, A.K. & Harris, R.W. (1984). Clavibacter: A new genus containing some phytopathogenic coryneform bacteria, including Clavibacter xyli subsp. xyli sp. nov., subsp. nov. and Clavibacter xyli subsp. cynodontis subsp. nov., pathogens that cause ratoon stunting disease of sugarcane and Bermudagrass stunting disease. Int. J. of Systematic Bact. 34: Dean, J.L. & Davis, M.J. (1989). Yield loss caused by ratoon stunting disease of sugarcane in Florida. J. Amer. Soc. of Sugar Cane Technol. 10: Gillaspie, Jr., A.G. & Teakle, D.S. (1989). Ratoon stunting disease. In C. Ricaud, B.T. Egan, A.G. Gillasp(e, Jr. & C.G. Hughes (eds.). Elsevier, Amsterdam, pp Grllaspie, A.G., Jr.; Davis, R.E. & Worley, J.F. (1973). Diagnosis of ratoon stunting disease based on the presence of a specific microorganism. Plant Dis. Rep. 57: Grisham, M.P. (1991). Effect of ratoon stunting disease on yield of sugarcane grown in multiple three-year plantings. Phytopathol. 81: Harrison, N.A. & Davis, M.J. (1988). Colonization of vascular tissues by Clavibacter xyli subsp. xyli in stalks of sugarcane cultivars differing in susceptibility to ratoon stunting disease. Phytopathol. 78: Harrison, N.A.; Davis, M.J. & Dean, J.L. (1986). Infectivity titrations of Clavibacter xyli subsp. xyli and sugarcane cultivars differing in susceptibility to ratoon stunting disease. Plant Dis Irey, M.S. (1986). Yield comparison of healthy and ratoon stunting disease infected cane of six commercial sugarcane cultivars in Florida. J. Amer. Soc. of Sugar Cane Technol. 6: Irvine, J.E. & Irey, M.S. (1991). A technique for field surveys for ratoon stunting disease. Proc. Inter-American Sugar Cane Seminars, pp Miller, J.D.; Davis, M.J.; Dean, J.L. & Shine, Jr., J.M. (1995). Heritability of resistance to ratoon stunting disease in sugarcane. Sugar Cane. (in press) Miller, J.D.; Tai, P.Y.P.; Glaz, B.; Dean, J.L. & Kang, M.S. (1984). Crop Sci. 24:210.

7 J.C. Comstock, et a1 Roach, B.T. (1992). Susceptibility to ratoon stunting disease in the Saccharurn complex and feasibility of breeding for resistance. Sugar Cane: Roach, B.T. & Jackson, P.A. (1992). Screening sugar cane clones for resistance to ratoon stunting disease. Sugar Cane 2:2-12. Shine, Jr., J.M. & Comstock, J.C. (1992). Digital image analysis system for detecting tissue-blot-imrnunosorbent assay results for ratoon stunting disease of sugarcane. Plant Dis Teakle, D.S.; Smith, P.M. & Steindl, D.R.L. (1973). Association of a small coryneform bacterium with the ratoon stunting disease of a sugar-cane. Aust. J. Agric. Res. 24: Wismer, C.A. (1971). A sugarcane clone apparently immune to RSD. Sugarcane Pathologists' Newsl. 6:46. CRIBLAGE POUR LA RESISTANCE AU RABOUGRISSEMENT DES REPOUSSES EN FLORIDE J.C. Comstock', J.D. Miller1, J.M. Shine, Jr.' & P.Y P. Tail USDA-ARS, Sugarcane Field Station, Canal Point, PL Florida Sugar Cane League, Canal Point, PL RESUME Un progme de criblage pour detecter la r6sistance au RSD a kt6 mis en place A la Station de Recherche sur la came ii sucre de I'USDA. Le criblage commence au stade 11, qui comprend environ 1000 varietes. Le criblage du RSD est conduit skpar6ment mais de fa~on parall2le au programme de developpement varietal. Les varietes qui sortent du stade I1 et tous les stades dont le r6sultat est bon sont criblees dam chacun des stades ultkrieurs. Environ 15 % des cultivars au stade I1 sont 6values comrne~ susceptibles et la plupart sont 6cartb. 11s montrent plus de 10 faisceaux vasculaires infest& par cea2tre de coeur de tige, ce qui est comparable aux variet6s cultivees sensibles. La sensibilit6 au RSD seule ne fournit pas un argument suffisant pour ecarter la variete dam les stades ultkrieurs, car une kvaluation gknkrale sur plusieurs caractkres conduit B au mobs un dkfaut chez presque toutes les variktes, ce qui rend difficile d'kcarter une varietc cause d'un seul'dcfaut. Les correlations entre les tests pour la sensibilitk au RSD. varient, mais elles augmentent au fur et A mesure que le nombre de tiges kchantillo~ee~ augmente. Les resultats indiquent que le niveau de resistance de la variete parentale influence le pourcentage des descendants rksistantslsensibles au RSD. C'est pourquoi il n'est pas recommand6 d'effectuer des croisements entre deux parents dont les descendants prksentent des pourcentages importants de clones sensibles au RSD.