Patholoev GROWTH INHIBITION IN SUGARCANE UPRIGHTS INFECTED WITH RATOON STUNTING DISEASE G.T.A. Benda USDA-ARS-U.S. Sugarcane Field Laboratory, Houma, LA 70361 USA I I ABSTRACT The effects on growth of ratoon stunting disease (RSD) were measured in paired sugarcane uprights in the greenhouse. The primary shoots of uprights were cut back, and the secondary shoots from the oldest buds were grown for 14 to 18 weeks. In CP52-68, CP70-321 and CP70-330, the secondary shoots from inoculated uprights grew less tall and weighed less than those from uninoculated uprights. The results indicate that xylem function in the diseased uprights is impaired in the few available vascular elements in the bud openings of the uprights and of the primary shoot. INTRODUCTION The ratoon stunting disease, RSD, of sugarcane is caused by a xylem-inhabiting bacterium, Clavibactel. xyli var xyli (Daviss). The disease is spread readily when juice from infected stalks comes in contact with a wound on a healthy stalk, or when seed pieces of sugarcane are inoculated by dipping in the juice of diseased plants. While this is an efficient method of inoculation (Steind18), sugarcane juice itself, whether from healthy or diseased plants, may adversely affect the germination of seed pieces (Zummol0). There are no definitive external symptornr of RSD. The presence of the disease is commonly confirmed by internal stalk symptoms (Steind18) or by microscopic observation of the bacterium (Gillaspie7). The bacterium causes a characteristic wilting and eventual death in infected plants of the sorghum-sudangrass hybrid NB 280-S grown as an upright (Benda2). The effect of the disease has been measured as yield loss when infected and disease-free canes are compared in the field; differences are more pronounced when the cane has been exposed to moisture stress at some period during growth (Steind18). Disease injury has been attributed to reduced xylem water movement with the vessels plugged or their effectiveness otherwise limited. The extensive vascularisation of the sugarcane plant may be one of the reasons why the effect of the disease has been difficult to demonstrate until the plants are very large. In large plants, such demonstration of weight differences between healthy and diseased plants has been shown to depend on the number of replications (Dean6). In uprights, the xyleni in the bud opening is limited to the size and number of vascular element; driginally present so that, as the shoot of the upright grows, Keywords: Seedpiece, Saccharurn, Clavibacter 317 (?
PATHOLOGY the demand for water flow through these few elements increases (Benda2). Sugarcane uprights have been used to demonstrate growth inhibition due to RSD in the green house (Bendal), but the pairs of diseased and healthy uprights had to come from matched setts comparable in age and size, and the pots had to be so arranged on the bench as to offset the effect of position. The objective of the present study is to measure differences in yield between diseased and healthy plants by a method which permits a better understanding of the disease. To do so, variability within a pair of healthy, resp. infected, uprights was further reduced by having each pair come from adjoining nodes of the original seed cane and by having the secondg~y shoots - whose growth rates were compared - come from the oldest bud of the primary shoot of the upright. MATERIALS AND METHODS Greenhouse-grown sugarcane was harvested when 12 to 24 months old. In Experiments 1 to 3, (Table I) the stalks were grown from seed cane which had been i hot-water treated (50 "C for 2 h) at each propagation for several consecutive vegetative generations. In Experiment 4, RSD-infected cane was harvested. The sugarcane varieties selected were CP52-68, CP70-321, and CP70-330. The stalks, some more than 6 m long, were stripped of leaves, then marked into two-node seed pieces (internodes were marked 5 cm above the leaf scar of the younger node and 3 cm below the leaf scar of the older kde). The older bud of each seed piece was excised. The seed pieces were numbered consecutively, the numbering continuing from stalk to'stalk. Inoculum was prepared fresh from RSD-infected stalks of CP45-184 ground to produce juice. The seed-cane stalks were cut with shears and alternate seed pieces (the even-numbered ones) were inoculated within a minute of cutting by dipping each end of the seed piece for 10 seconds into the juice inoculum. The odd-numbered seed pieces were not dipped. All seed pieces were placed in glasses and then water to cover the lower node was added after an hour or more. After a few days, when rooting was beginning, the seed pieces were transplanted upright to 7.5 cm peat pots so that the sand-soil mixture covered the lower node. The plants were then moved into the greenhouse. When one at least, but usually two or more, leaves had developed, leaves having exposed dewlaps and laminae several centimetres long, the scale leaves were removed sequentially until the oldest axillary bud was exposed (Table I). The seed pieces were transplanted to larger pots (15 cm) filled with sand-soil mixture. The pots were arranged on the greenhouse bench so that two rows of pots with inoculated uprights alternated with two rows of pots with uninoculated uprights, and uprights from seed pieces adjacent on the seed cane stalk were opposite each other. After the oldest buds on the primary shoots had been exposed for one to four weeks (Table I), and these buds had germinated on about half the seed pieces, the primary stalk was cut back with a scalpel so as to leave the node carrying the oldest bud and the next younger node intact. Sometimes several nodes were left intact, and any axillary shoots developing from the buds of younger nodes were broken off. The young secondary shoots from thi oldest bud developed rapidly, and the plants needed to be supported by bamboo stakes. The pots were fertilised weekly I
TABLE I. Details of experiments comparing the growth of secondary shoots on uprights with or without ratoon stunting disease. - Manipulation of uprights Prepared Primary shoot Inoculated of pair Seed inoculated1 Oldest bud Younger Older Experiment Variety Cane1 uninoculated exposed Cut back Harvested sett sett - - - - - - - - - Date (1985) - - - - - - - 1 CP 70-321 X 3-07 3-25 4-16 8-29 6 7 2 CP 52-68 X 3-26 4-1 2 4-19 8-30 16 11 3 CP 70-330 X 5-30 6-28 7-1 6 10-28 8 8 4 CP 70-330 RSD 5-30 6-28 7-1 6 10-28 0 11 ' X = not infected with the ratoon stunting disease (RSD).
320 PATHOLOGY with nitrogen, and complete fertiliser was added occasionally. The plants were sprayed weekly with insecticide and the roots from the aerial plant were cut back as needed to prevent their reaching the soil. Growt-h was measured at weekly or longer intervals. Initially, all leaves with exposed dewlap and laminae longer than 1 cm in length were counted. Subsequently, at each measurement, the leaves with newly exposed dewlaps were counted and addecl to the cumulative total. The height was measured from the origin of the secondary shoot to the top-visibl'e dewlap, and axillary (tertiary) shoots were counted according to size classes. The experiments were terminated on cloudy mornings when the plants were fully t~~rgid. The entire plant was broken off the upright seed piece, and the fresh weight was determined. The disease status of the control and the inoculated plants was checked for internal symptoms or by searching for bacteria under phase-contrast microscopy (Gillaspie7). The significance of differences between means in these paired comparisons was determined according to a rank method (Wilcoxon9). RESULTS When the plants were checked for internal symptoms at harvest, the inoculated plants of Experiments 1 to 3 and all plants of Experiment 4 showed typical RSD symptoms except for one plant each of CP70-321 and CP52-68 which were without symptoms. The uninoculated plants of Experiments 1 to 3 had no symptoms except for one plant of CP52-68. These three aberrant plants were retained in the data under their original designations. TABLE I1 The effect of the ratoon stunting disease (RSD) on the ranges of growth parameters of secondary shoots on uprights at harvest. Range of values at harvest RSD Height Fresh weight Leaf total Experiment Variety inoculum (cm) (9) (No.) 1 CP 70-321 uninoculated 93-179 282-733 21-25 inoculated 68-139 176-469 17-21 2 CP 52-68 uninoculated 137-192 458-688 17-21 inoculated 94-178 237-682 15-21 3 Cp 70-330 uninoculated 81.144 248-465 16-18 inoculated 44-122 107-335 15-18 4 cp 70.330 uninoculated 41-112 117-329 14-18 inoculated 51-118 126-410 14-18 The variability of data in these growth experiments remains very high (Table 11). As the ranges of values indicate, there are areas of overlap for all varieties between the values for inoculated and uninoculated uprights. In Experiments 1 to 3, the maxima and minima of inoculated uprights are less (in height or fresh weight)
- - - - -- G.T A. BENDA 321 than those of the uninoculated uprights, although the maxima of total leaf number may be equal. Measurements between members of pairs in Experiments 1 to 3 may differ widely, but the differences in height and fresh weight within the pair mostly favour the uninoculated uprights. The differences among means for height and fresh weight are significant at the 1% level (Table 111). Differences in total leaf number, which reflects the number of nodes laid down during the period of observation, show less variability; the differences are significant for CP70-321 (1% level) and CP52-68 (5% level), but not for CP70-330. In Experiment 4 (Table 111), where the effects of inoculation or no inoculation on initially diseased plants are compared, none of the differences between means is significant. The,means for the diseased plants are close in value to the means of the inoculated uprights of Experiment 3. This result may reflect the similar conditions under which the original seed canes and the uprights were grown, Experiments 3 and 4 having been placed on the same greenhouse bench. TABLE Ill. The effect of the ratoon stunting disease (RSD) on the growth parameters of secondary shoots on uprights at harvest. Average values at harvest Pairs RSD Height Fresh weight Leaf total Experiment Variety (No.) inoculum (cm) (9) (No.) Cp 70-321 13 uninoculated 152.4 562.8 22.8 inoculated 96.7**3 309.0** 18.6** Cp 52-68 27 uninoculated 168.8 588.8 18.7 inoculated 145,8* * 467.6** 18.0* cp 70-330 16 uninoculated 111.2 343.4 17.0 inoculated 81.9** 224.4** 16.2 NS Cp 70-330 11 uninoculated 84.0 222.5 16.2 inoculated 79.8 NS 232.8 NS 16.2 NS NS means not significantly different; (*) (**), means differ significantly at the 0.05 or the 0.01 level of probability, respectively. The growth habit of these uprights embodied a dominant main shoot with smaller axillary shoots of which some died before harvest. All uprights of CP52-68, the majority of CP70-321, and less than half of CP70-330 had one or more tertiary shoots. The uninoculated uprights of CP70-321 had significantly more tertiary shoots than the inoculated ones, but such differences in CP52-68 and CP70-330 \\ere not significant. The weight of the tertiary shoots is included in the total flesh \\eight at harvest. The occurrence of significant differences in height or leaf number between inoculated and uninoculated uprights varied among the experiments. In Experiment 1, the average height and leaf number of inoculated uprights of CP70-321 were significantly below the uninoculated when first measured 24 days after the primary
322 PATHOLOGY shoot had been cut back (May 10). In Experiment 2, the differences became significant only when the plants had become large (Table IV); the differences in average height between inoculated and uninoculated uprights had become significant by 87 days after the primary shoot had been cut back; for average leaf number, the period was 132 days. In Experiment 3, with CP70-330, the height differences were not signficant on August 28, but were significant by September 5, or 51 days after the primary rhoot had been cut back; differences in leaf nuinber wele not significant. In Experiment 4, there were no significant differences in height or leaf number during the period of observation. TABLE IV. The increase with time in average height and leaf number of secondary shoots of uprights of CP 52.68 as affected by ratoon stunting disease (RSD) [Experiment 21. RSD Date measured (1985) inoci~lum (Significance)" 5-13 5-23 5-30 7-3 7-16 8-30 Average height of main shoot IJninoculated 15.3 21.3 25.3 72.3 93.0 168.8 Inoculated 14.6 21.5 25.6 ' 70.1 88.0 (significance) NS N S NS N S * 145.8 * * Average number of leaves Uninoculated 4.7 6.0 7.0 11.8 13.4 18.7 Inoculated 4.4 6.1 7.0 11.7 13.3 18.0 (significance) * NS NS NS NS NS means not significantly different; (*) (**), means differ significantly at the 0.05 or the 0.01 level of probability, respectively. Within an experiment, no effort was made to have the number of inoculated ~~prights from the older setts of the original seed canes equal the number from youngel setts. In Experiments 1 and 3, this was achieved anyway, less so in Experiment 2, and not at all in Experiment 4 where all inoculated uprights were from an older part of the seed cane than the uninoculated ones (Table I). The results suggest that between adjoining setts in these old and young seed canes, age does not affect the results decisively. There was some evidence of incipient wilting in the experiments with CP70-330 shortly before the plants were harvested. During sunny periods in the middle of the day, the two leaves just above the leaf with the top visible dewlap were observed to roll up lengthwise. This rolling was observed in the majority of plants whether inoculated or not, When the sky became clouded, the young leaves would resume their typical flat appearance. DISCUSSION The rather involved procedure used to establish the secondary shoot of the uprights makes it unlikely that any effect of the juice inoculum on germination or
G T.A BENDA 323 early growth (Z~mmo'~) would contribute to the growth differences between inoculated and uninoculated uprights. Inoculation as such appears to have no effect on growth in Experiment 4 where diseased plants served as starting material (Table 111). In Experiment 2, the gradual appearance of significant differences in height and leaf number between-inoculated and uninoculated uprights indicates that the disease is responsible for these ;iliffe;ences rather than the inoculation as such \ (Table IV). The results obtained with the uprights of the sorghum-sudangrass hybrid (~enda~) had shown that the plants of uprights infected with RSD were less able to meet the demand for water as the aerial plants grew larger; the water uptake of diseased plants decreased as the plants became progressively more wilted. A series of experiments with uprights of the sorghum-sudangrass hybrid had shown that water transport becomes blocked in the area of the bud opening, and the wilting was attributed to the few available vascular elements having become less effective in water conduction due to the disease (Benda2, 39 4). In sugarcane, with its much larger bud opening, the effects of the dis,ease are likely to be expressed both more slowly and less clearly. To amplify the effect of the disease on sugarcane, a second bud opening was added in these experiments, and the bud opening selected is one of the smallest that a stalk is likely to have, namelyl the opening of the oldest bud on the stalk. ~ k results e (Table 111) showed that under these conditions growth of the '>r' secondgry stalk, as measured by shoot height and fresh we~ght, was indeed severely affected. If the results are interpreted in the light of the sorghum-sudangrass hybrid experi&&nts, the results indicate that neither the positive root pressure at night nor the tenaon during the day can compensate for the inefficiency in water conduction of the x$em elements in the bud openings. '2 Thebexperiments were terminated while the plants were still growing, and no attempt was made to determine whether sugarcane by this method can be stressed to the point of permanent wilting. The method described in this paper cannot be used to predict field behavior because in cane grown normally, all axillary shoots tend to root and become more or less independent of the shoots from which they branched. While the results indicate that the reduced efficiency of vascular conduction in diseased plants may influence yield, it is uncertain that under field conditions the conduction efficiency would limit growth. No vascular connections as limiting to water conduction as the bud openiags in these experiments have been identified in field-grown cane. Of the varieties used, CP52-68 is considered tolerant to RSD under field conditions in Louisiana, while both CP70-321 and CP70-330 show severe losses, especially in stubble crops. Although the slow development of growth differences in CP52-68 (Table IV) may suggest a certain tolerance to the disease, even the suggestion that this is in any way correlated with field results is premature. REFERENCES 1. Benda, G.T.A. (1969). The latoon stunting disease of Saccharurn: Effect on growth. Abstr. of XI Int. Bot. Congr., Seattle, p. 12. 'i
324 PATHOLOGY I 2. Benda, G.T.A. (1971). Wilting and death in the ratoon stunting disease of sudangrass hybrid uprights. Amer. Soc. Sugar Cane Techno]., Proc. 1(NS)1.39-47. i 3. Eenda, G.T.A. (1973). The ratoon stunting disease and gro~t)~;~single-rooted plants of sitgarcane and sudangrass hybrid. Amer. Soc. Sugar Cane ~eclhdl., Proc. 2(NS):83-88. 4. Eenda, G.T.A. (1975). On the wilting of a sorghum-sudangrass hybrid infected with the ratoon stunting disease of sugarcane. Amer. Phytopathol. Soc., Proc. 2:64-65. 5. Davis, M.J., Gillaspie, A.G., Jr., Vidaver, A.K. and Harris, R.W. (1984). Clavrbacter: --. Int. J. System. Bacterial. 34:107-117. 6. Dean, J.L. (1983). Single-stool plots for estimating relative y~eld losses caused by the ratoon stunting dlsease of sugarcane. Plant Dis. 67:47-49. 7. Gillaspie, A.G., Jr., Davis, R.E. and Worley, J.F. (1973). Diagnosis of ratoon stunt~ng disease based on the presence of a spec\& microorganism. Plant Dis. Rep. 57:987-990. 8. Steindl, D.R.L. (1961). Ratoon stiinting Disease, v. 1, p. 433-459. In J.P. Martln, E.V. Abbott, and C.G. Hughes, (ed), Sugarcane Diseases of the World. Elsevler, Amsterdam. 9. W~lcoxon, F. (1949). Some Rapid Approximate Statistical Plocedures. 16 p., American Cyanimid Co., New York. 10. Zummo, N. (1974). Inhibition of germination and growth of sugarcane buds from seed pieces dipped in sugarcane julce from healthy and RSD-infected stalks. ISSCT, Proc.