EVOLUTION OF AERIAL INFECTION OF LEAF SCALD CAUSED BY XAAJTHOMONAS ALBZLZNEANS (ASHBY) DOWSON IN SUGARCANE

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1 EVOLUTON OF AERAL NFECTON OF LEAF SCALD CAUSED BY XAAJTHOMONAS ALBZLZNEANS (ASHBY) DOWSON N SUGARCANE S. Saumtally, H. M6dan & L.J.C. Autrey Mauritius Sugar ndustry Research nstitute (MSR), R6duit ABSTRACT The progress of aerial foliar stripes of leaf scald in sugarcane caused by Xanthomonas albilineans was followed in var. M Stripes induced by the pathogen, starting from necrotic areas at the tip of the leaf, were observed 5 mo after planting. They developed on leaves 5-14, with a higher frequency on leaves 8 & 9 (the youngest unrolled leaf being taken as leaf 1). Some stripes progressed faster than others and 5 mo after the first infection was recorded, the length of stripes in the trial varied from < 1-80 cm and spanned the entire length of a few leaves. The evolution of the stripes could not be followed further as'the old leaves bearing symptoms (usually leaf 14) dried up before the stripes entered into the leaf sheaths. t was evident, however, that the disease had entered the systemic phase of the disease as leaf scald was observed on young regrowth. n the same experiment half the plants were sprayed weekly with copper oxychloride at 1 kg ailha in an attempt to control aerial transmission. This dosage was reduced to 0.5 kg aih after 6 mo due to phytotoxic effects. Stripes were observed in the treated plots when the dosage was changed, but they were still appreciably less than in untreated plots. The percent infected stools was reduced from 67 to 11 % ui in vitro plantlets and from 41 to 17% in plants issuing from one-eye cuttings. The experiment shows that the aerial phase of leaf scald is sipilar to that of gumming disease of sugarcane and plantations established with clean planting material may become infected through the air-borne inoculum of X. albilineans. Keywords: Disease transmission, copper oxychloride, in vitro plantlets, gumming disease, Mauritius: NTRODUCTON Leaf scald caused by the bacterium Xanthomonas albilineans (Ashby) Dowson is one of the most important diseases of sugarcane and the number of countries where it has been reported is on the increase. Recent records include Louisiana (Grisharn & Legendre 1993), Mexico (rvine et a1 1993), Pakistan (Akhtar et a1 1988), Papua New Guinea (Magarey 1989) and Zimbabwe (Ricaud & Ryan 1989), bringing the number of countries where the disease has been found to 48. Leaf scald is traditionally known to be transmitted mechanically by knives and harvesters and by planting infected setts; but other mechanisms of dissemination have been observed as well. The presence of X. albilineans has been detected in rhizospheric soil of infected roots, suggesting possible transmission by root contact (Klett & Rott 1994). These authors have also found colonization of side branches of cane inflorescences although true seed transmission was not reported; thus dissemination by fuzz is suspected. The bacterium has been shown to exude from guttation droplets (Sordie & Tokeshi 1986; Klett 1992, Autrey et al 1995b), becoming air-borne to cause infection. Evidence for aerial transmission was obtained in an epidemic of the disease that occurred in Mauritius in Foliar stripes in maize hybrids that eventually developed into systemic infection were observed (Autrey et a1 1995b). Aerial infection was also found in cane var. M , M and M as well as in the unreleased clones M , M , M and M Among those varieties, infection in M was of concern as it is very high yielding and occupied 9 % of the area under sugar cane in The use of this variety had to be halted as it was severely affected by leaf scald in some areas and showed abundant foliar striping. t was therefore decided to investigate the evolution of aerial infection in that variety and determine whether its susceptibility to this mode of infection could have contributed to the high incidence of leaf scald observed. MATERALS AND METHODS Planting material One-eye cuttings of var. M were soaked for 48 h in cold running water prior to hot-water treatment at 50 C for 3 h and planted in plastic pots in the glasshouse. The treated cuttings were also used to produce in vitro plantlets from bud cultures and the plantlets transplanted into plastic pots (Dookun et al 1995). 493 i

2 S. Saumtally, et al Assessment of freedomafrom leaf scald The planting material was assessed for leaf scald at 2 mo by indirect immunofluorescent microscopy. One tiller from each plant was sectioned; and after removing the outer layers, the base was thinly sliced with a scalpel and allowed to diffuse in 2-3 ml of sterile distilled water for 30 fi. One ml of the exudate was placed in a 1.5 rnl Eppendorf tube (Sarstedt, U.K) and centrifuged at maximum speed in a microcentrifuge (Eppendorf Model 5415C) for 10 min. The supernatant was decanted and the pellet re-suspended in the small amount of liquid remaining in the tube. Twenty microliters of the suspension were heat-fixed on an imm~11ofluorescence slide and detection carried out as described by Autrey et al, (1989). Design of trial Disease-free, 4-mo-old plants were planted in a field at Beau Champ in the eastern part of the island in Feb This site was chosen because var. M 695/69 was widely grown (480 ha) there and it showed abundant foliar striping. The 275 in vitro plantlets and 350 plants issuing from one-eye cuttings were established in two separate blocks. Treatments Half of each block was sprayed weekly with copper oxychloride (Cuprox 50 %) at 1 kg ailha 1 wk after planting to assess the protective effect of the bactericide against X. albilineans. The dosage was eventually reduced to 0.5 kg ailha 6 mo later. Applications were carried out using a mist blower (Solo, Type 40123, Germany). Disease assessment was carried out weekly to determine the occurrence of infection and to monitor the evolution of aerial infection. The development of new stripes on each leaf was noted, their positions marked and their progress followed. Each infected stalk was labeled and the percent diseased stools determined on a monthly basis. Surveys were carried out in plant cane and first ratoon. The trial was harvested in plant cane by taking the necessary precautions to prevent dissemination of leaf scald. The harvest was carried out manually by disinfecting the knife in a solution of iodine at 250 ppm (odocor 50) each time a stool was cut. RESULTS All in vitro phnflets and plants issuing from oneeye cuttings that underwent the cold-soak long hot-water treatment were found to be free from leaf scald when tested by immunofluorescence microscopy. Foliar stripes were first observed in July 1993, 5 mo after planting, in both in vitro plantlets and plants issuing from one-eye cuttings that did not receive copper oxychloride applications. nfection normally started in a necrotic area at the tip of the leaf and developed to produce cream-colored to yellow stripes, about 3 mm wide. The stripes were present on leaves 5-14, the first fully unrolled leaf being considered as leaf 1. The frequency of stripes was highest on leaves 8 & 9, and one or more stripes were present on some leaves. The progression of the sqes varied even on the same leaf. Some of them evolved slowly whereas others extended as rapidly as 70 cmlmo. The most rapid progression was often noted in the first month of the formation of the stripe. The longest stripes recorded were 68 cm in in vitro plantlets and 80 cm in pl&ts derived from one-eye cuttings 5 mo after the initial infection was observed. A few stripes covered almost the entire length of the leaf. As new leaves appeared, the older ones bearing symptoms (usually leaf 14) dried out before the stripe entered into the leaf sheath and hence the evolution could no longer be followed. Some stripes did reach the leaf sheath, and evidence that systemic infection had occurred was obtained in the first ratoon. After harvest in young regrowth, a white pencil-line symptom characteristic of the systemic phase of leaf scald was detected in one tiller from three different stools issuing from oneeye cuttings and one tiller issuing from a stool that had developed from a tissue culture plantlet. A strong protective effect of copper oxychloride was initially observed in treated plots. No disease symptoms were found ui plots treated with copper oxychloride at 1.0 kg ailha. The dosage was halved, however, due to a phytotoxic effect causing a reddish mottling on the leaves. Two weeks later, aerial infection was apparent in the treated plots. The length of stripes and the percent infected stools were appreciably lower in the treated plots (Table 1). The percent infected stools was reduced from 67 to 11 % in in vitro plantlets and from 41 to 17 % in plants raised from one-eye cuttings. As no statistical design was carried out, no conclusion could be drawn on the relative susceptibility of the two types of planting material to aerial transmission of leaf scald. 494

3 Biology: Pathology Table 1. Evolution of leaf scald infection in treated and untreated in vitro plantlets and plants issuing from one-eye cutthgs. - - Maxi. Length Planting % stool infection of stripes material Jul Aug Sep Oct Nov Dec recorder (cm) n Vitro plantlets Treated' Untreated Plants from 1- Treated eye cuttings Untreated ' Weekly applications of copper oxychloride (Cuprox 50 %) at 1 kg ailha for 6 mo. after which the dosage was reduced to 0.5 kg ailha. DSCUSSON Aerial transmission of leaf scald and infection through wounds and natural openings are recent observations. Whether leaf striping may develop into systemic infection in sugarcane as is the case for gumming disease had not been shown. n gumming disease systemic infection occurs when foliar stripes progress along the length of the leaf and eventually penetrate into the leaf sheath (Ricaud & Autrey 1989). t is now evident that foliar stripes of gumming and leaf scald are very similar in appearance and behavior. t is very likely that stripes caused by X. albilineans have been confused with those of gumming disease in the past. The phenomenon of aerial dissemination of the leaf scald pathogen has an impom implication as plantations established with disease-free cuttings may become rapidly infected through leaf infection. The development of foliar leaf scald infection into a systemic phase was shown in var. M nitial symptoms appeared 5 after planting; and rapid progression of leaf stripes was observed, a few of them extending over the entire length of the leaf. Although none of the stripes extended into the leaf sheath, systemic infection was present in the regrowth. As the old leaves bearing the longest stripes started to dry out, it is possible that certain cases of stripes covering the whole length of leaves could have escaped attention. Moreover, it is laiown that the bacterium may be present up to 45 cm beyond the tip of the stripe in apparently healthy tissue (Autrey et a1 199%); thus infection could have set in the leaf sheath although the stripe was still several centimeters away from it. Among cane diseases that cause stripe symptoms, systemic infection does not automatically occur once the stripe reaches the leaf sheath. This is the case for mottled stripe, caused by Pseudomonas rubrisubalbicans, where numerous stripes reach the leaf sheath, but no systemic infection occurs. The progression of the bacterium is thus restricted, but this is not the case with X. albilineans. Of the three leaf scald serotypes (Rott et a1 1986), two have been reported in Mauritius (Autrey et a1 1995a, Dookun et a1 1994). Both serotypes (i.e., the Mascarene and African ones) have been associated with foliar stripes, and aerial transmission does not appear to be related to specific serotypes. Different resistance mechanisms to leaf scald seem to operate in aerial and mechanical transmissions. Several highly suscepable varieties cultivated in Mauritius showed little or no foliar striping, even in the extremely severe 1989 epidemic. n a strategy to control leaf scald, varieties susceptible to aerial transmission should be identified as control based on disease-free cuttings; and sanitation may not be totally adequate if the variety can be contaminated by airborne infection. n Mauritius a program of screening promising varieties by exposure to leaf scald was started three years ago; but little infection has been recorded despite planting in an area conducive to aerial infection. t is apparent that aerial transmission requires conditions that'are still elusive and perhaps only a few Mauritian varieties are susceptible to mode of transmission. Aerial stripes have been observed in a few such varieties, but they remained short. The control of aerial infection using copper oxychloride was feasible despite its phytotoxic effect. There is evidence that such reaction could be varietal and that some varieties are not affected by the dosages used in this experiment (results not shown). The effect of copper oxychloride as a bactericide against Xanthomonas spp. is well documented (Sekizawa & Wakabayashi 1990, Sigee 1993). Copper compounds cause marked changes in elemental composition; and these changes are consistent with a toxic effect at the bacterial cell surface, leading to a large-scale efflux of K'. and influx of CS' and CuZ' (Hodson & Sigee 1991). t is not intended to recodmend applications on a commercial scale, but the bactericide could be used experimentally for creating a nuclear stock of leaf scald-free

4 S. Saumtally, et a1 planting material as well as estimating the effect of the disease on yield. CONCLUSONS On monitoring the progress of aerial foliar stripes of leaf scald in cane var. M , the disease was found to develop into a systemic phase. This mode of infection could contribute to the incidence of leaf scald even when plantations have been established with disease-free cuttings. The mechanism of resistance to aerial transmission appears to be different from mechanical transmission as long foliar stripes have not been observed in certain varieties highly susceptible to leaf scald. Aerial dissemination of the pathogen could however be responsible for maintaining leaf scald inoculum in cane plantations, especially under conditions of wind-blown rain. ACKNOWLEDGMENTS We would like to thank Drs C. Ricaud and R. Julien, Director and Deputy Director respectively, for reviewing the manuscript, their helpful criticisms and permission for publication. Our thauks also go to Dr A. Dookun, Head, Biotechnology Division for providing in vitro plantlets &f var. M REFERENCES Akhtar, M.A.; Malik, K.B. & Aslam, M. (1988). Sugar cane leaf scald (Xanthomonas albilineans) in Pakistan. Trop. Agric. prinidad) 65: Autrey, L.J.C.; Dookun, A. & Saumtally, S. (1989). mproved serological methods for diagnosis of the leaf scald bacterium Xanthomonas albilineans. Proc. SSCT 20: Autrey, L. J.C.; Saumtally, S. ; Dookun, A. & MCdan, H. (1995a). Studies on variation in the leaf scald pathogen Xanthomonas albilineans (Ashby) Dowson. Proc. SSCT 21(2): ). Autrey, L.J.C.; Saumtally, S.; Sullivan, S.; Dookun, A. & Dhayan, S. (1995b). Aerial transmission of leaf scald caused by Xanthomonas albilineans (Ashby) Dowson. Proc. SSCT 21(2): Dookun, A.; Saumtally, S. & Autrey L.J.C Antigenic differences in Xanthomonas albilineans, causal agent of leaf scald disease of sugar cane. Plant Pathogenic Bacteria. Versailles, 9-12 June Ed. NRA Paris. Les colloques 66: Dookun, A.; Moutia, M.; Mulleegadoo, K. & Autrey, L.J.C Constraints in sugar cane micropropagation by tissue culture. Proc. SSCT 22 Vol. 2( ). Grisham, M.P. & Legendre, B.L. (1993). First report of leaf scald caused by Xanthomonas albilineans. Plant Dis. 177: 537. Hodson, N. & Sigee, D.C. (1991). Copper toxicity in Erwinia amylovora: Au x-ray microanalytical study. Scan. Elec. Micros. 5: rvine, J.E.; Amador, J.M.; Gallo, R.M..; Riess, C.M. & Comstock, J.C. (1993). First report of leaf scald, caused by Xatlthomonas albilineans of sugar cane in Mexico. Plant Dis Klett, P. (1992). L'Cchaudure des feuilles de came B sucre : Cvolution des populations de Xanthomonas albilinearls d m et hors de la plante h6te. DiplGme d'etudes Approfondies de Phytopathologie Universite de Paris, nstitut National Agronomique Paris, Grignon. 47 p. Klett, P. & Rott, P. (1994). Lnoculum sources for the spread of leaf scald disease of sugar cane caused by Xanthomonas albilineans. J. Phytopathol. 142: Magarey, R.C. (1989). First record of leaf scald disease it1 Papua New Guinea. SSCT Tech. Newsl. no Ricaud, C. & Autrey, L.J.C. (1989). Gumming disease. Chapt.. n C. Ricaud, B.T. Egan, A.G. Gillaspte & C.G. Hughes (eds), Diseases of sugarcane. Major diseases. Elsevier, Amsterdam, pp Ricaud, C. & Ryan, C. (1989). Leaf scald. Chapt.. n C. Ricaud, B.T. Egan, A.G. Gillaspie & C.G. Hughes (eds), Diseases of sugarcane. Major diseases. Elsevier, Amsterdam, pp Rott, P.; Arnaud, M. & Baudin, P. (1986). Serological and lysotypical variability of Xanthomonas albilineans (Ashby) Dowson, casual agent of sugar cane leaf scald disease. J. Phytopathol. 116: Sekizawa, Y. & Wakabayashi, K. (1990). Bactericides. n 2. Klement, K., Rudolph, & D.C. Sands (eds), Methods in phytobacteriology. Akademiai Kiado, Budapest. Sigee D.C. (1993). Bacterial plant pathology. Cell and molecular aspects. Cambridge Univ. Press. 325 p. Sordie, R.A. & Tokeshi, H. (1986). Presence of Xanthomonas albilineans in guttation droplets of sugar cane and sweet

5 Biology: Pathology DETECTON, TRANSMSSON ET CONTROLE DE L'ECHAUDURE DES FEULLES DE LA CANNE A SUCRE DUE A XANTHOMONAS ALBZLZNEANS (ASHBY) DOWSON S. Saumtally, H. MCdan & L.J.C. Autrey Mauritius Sugar ndustry Research nstitute (MSR), RCduit RESUME La phase de lateme de 'Cchaudure des feuilles de la came b sucre due 1 Xanthomonas albilineans a CtC suivie sur deux varictcs tr8s sensibles M et M La bactcrie a CtC d6tectce par immunofluorescence au microscope et par isolement. Dans la varict6 M 292/70,6 khantillom d'aspect sain sur 10 etaient infest& et 8 sur 10 de la varict6 M Lors de la prkparation des boutures, l'agent pathogene a CtC transmis jusqu'b la cinqui8me bouture lorsque le coulelas Ctait trempe dans utie suspension preparation bacterienne ou dans un jus infectc, et b la seconde bouture lorsque le coutelas Ctait conkunhc par la coupe de cannes malades. Au champ, l'infection a atteint la vingticme souche qui suivait sur la ligne la coupe d'un mctre de cannes infestees. On a cherche ii contr6ler la bacteriose dans sur des boutures infectkes en testant 79 traitements par la chaleur, y compris des traitements par micro-ondes. Le traitement le plus efficace est constituc d'un trempage long dans de l'eau froide puis d m de l'eau chaude (CSLHWT -3rempage des boutures pendant 48 heures dans l'eau froide suivi d'un traitement b l'eau chaude b 50" pendant 3 heures) bien qu'b 1'Cchelle industrielle un petit pourcentage de boutures sont restees infestkes. En rangeant les boutures par couches parall8les ou perpendiculaires scparces par des espaceurs en bois, les infections sont rcduites de 0.3 % b 0.03 %. Ces ttudes demontrent le risque important qui est pris lorsqu'on destine B la plantation des plants sans symptbmes, l'importance de la transmission de 1'Cchaudure par le coutelas et 'efficacitc de la mcthode CSLHWT pour Climiner la bacdrie des boutures. Mots clcs: nfection latente, immunofluorescence, traitement par la chaleur, traitement par micro-ondes, traitement 1 l'eau chaude. 497