Leifsonia xyli subsp. xyli
(sugarcane ratoon stunting disease)

Much of the early information describing the geographic distribution of this disease was based on internal stalk discoloration (not a reliable symptom) and may be inaccurate. However, it is the best information available until the presence of the pathogen is confirmed. Because of difficulties associated with diagnosis, the distribution of this disease is probably much wider than currently known.

Risk Criteria Category

Economic Importance High
Distribution Worldwide
Seedbrone Incidence Not recorded
Seed Transmitted Not recorded
Seed Treatment None

Overall Risk Low


Notes on Phytosanitary Risk

Quarantine restrictions on the disease exist in most sugarcane production areas throughout the world. However, there are no known races or other pathogenic variants of the bacterium that might increase the risk of introduction in regions where the disease already occurs, although the possibility of such variants cannot be ignored. Quarantine procedures that combine heat treatment of seed cane to eliminate the pathogen and appropriate diagnostic techniques can greatly reduce the risk of introduction. The widespread distribution of the disease can be largely attributed to international shipment of infected cuttings (Steindl, 1961; Dean, 1974).

Although sugarcane is the only known natural host of the pathogen (Saccharum spp. and Saccharum interspecific hybrids), numerous grasses have been determined to be hosts after experimental inoculation (Gillaspie and Teakle, 1989). The experimental hosts include Zea mays, Sorghum spp., Brachiaria mutica, Brachiaria miliiformis, Chloris gayana, Cynodon dactylon, Echinochloa colonum, Imperata cylindrica, Panicum maximum, Pennisetum purpureum and Rhynchelytrum repens.

The pathogen has been found only in sugarcane in nature and has no known insect vectors (Gillaspie and Davis, 1992). Infection takes place through wounds. The pathogen can be mechanically transmitted from sugarcane to sugarcane on the blades of equipment used to cultivate and harvest crops and can be spread by propagation with infected cuttings. No evidence exists for transmission in true seed. The pathogen can remain viable and infectious for several months apparently in either moribund plant debris or the soil itself, contributing to the persistence of ratoon stunting disease in areas where the disease is common (Bailey and Tough, 1992).

The pathogen systemically invades plants through the xylem. It has been detected in most vegetative parts of sugarcane where mature xylem exists. It was recovered readily from mature stalks and the leaf sheaths and lamina of the lower leaves of infected plants, but not from the midrib and lamina of upper leaves (Teakle et al., 1975). Large populations existed in mature stalks, and smaller populations were found in the growing point, leaf lamina, leaf midrib and leaf sheath (Bailey, 1977).

L. xyli subsp. xyli is not carried on true seed. See Control section for information on the importance of planting healthy seed cane and treatments for seed cane.

The pathogen is a small, xylem-inhabiting, coryneform bacterium that often can be detected in sugarcane extracts using phase-contrast or dark-field microscopy (1000x). Its cell morphology is sufficiently distinct compared to the other natural micro-organisms associated with sugarcane to permit recognition. False-positive diagnoses based on microscopic observations are rare; however, false-negative results can be a problem, especially when pathogen concentrations are low (Davis and Dean, 1984). Concentrations are greatest in the basal portion of mature stalks during the later part of the growing season, improving the chance of detection at this time (Davis et al., 1988a). Some cultivars characteristically have lower pathogen populations than others (Gillaspie et al., 1976; Bailey, 1977; Koike et al., 1982) and can, therefore, present more of a problem for diagnosis.

Isolation

The pathogen is extremely fastidious in its nutritional requirements and can only be grown in axenic culture on special media such as the SC medium (Davis et al., 1980). The following procedure can be used for primary isolation of the pathogen in culture:

- select and excise a portion of stalk containing an internode that is free of damage due to insects, cracks, etc. and thoroughly wash it with soap and running water to remove grime

- surface disinfest the internode by submersion in 70% ethanol for 1-2 min, then dipping into 95% ethanol, and finally flaming

- aseptically excise a 2-3 cm section of the internode, place it into a 50-mL, conical centrifuge tube, and centrifuge it at 1000-4000 x g for 1-5 min to extract sap

- use the sap to inoculate SC medium, and incubate the inoculated medium aerobically at 29°C for 2-3 weeks.

The bacterium is aerobic, non-motile, Gram-positive, non-spore-forming, non-acid fast, catalase-positive, and oxidase-negative.

Pathogenicity Tests

Pathogenicity tests for routine identification are rarely conducted because of the long incubation period (usually at least 3-6 months or longer) required for symptom development or detection of the pathogen in the host plant. However, in some situations it may be desirable to inoculate sugarcane with the pathogen. Crude juice from infected plants can be used as inoculum and is often a more convenient source of inoculum than pure cultures of the pathogen. However, the quality of crude juice inoculum is difficult to control and the use of such inoculum involves the risk of spreading other pathogens. Sugarcane can be inoculated by dipping freshly cut ends of single node cuttings into a suspension of the pathogen. Cuttings are then planted and developing plants are examined for internal symptoms or the presence of the pathogen after an appropriate incubation period.

Serological Techniques

Serological methods have been developed to detect L. xyli subsp. xyli (Gillaspie, 1978; Harrison and Davis, 1990; Wu et al., 1990). Considerable sensitivity has been obtained by using fluorescent-antibody staining to detect bacterial cells with epifluorescence microscopy. Staining of the bacterium in dried extracts on glass slides was at least tenfold more sensitive than phase-contrast microscopy for detection (Harris and Gillaspie, 1978). At least another tenfold increase in sensitivity was obtained when the bacterium was first stained while still suspended in sap extracts and then concentrated on membrane filters by filtration before observation (Davis and Dean, 1984). The latter method can also be used to estimate the pathogen population size and was named the fluorescent-antibody direct-count on filters (FADCF) technique (Davis, 1985).

Although not as sensitive, more rapid serological assays have been developed. An ELISA was investigated for detection of L. xyli subsp. xyli; however, problems with non-specific reactions were encountered (Gillaspie and Harris, 1979). A modified ELISA procedure, referred to as the evaporative binding enzyme immunoassay (EB-EIA), was developed (Croft et al., 1994). A tissue-blot enzyme immunoassay (EIA) was developed to enumerate vascular bundles in cross-sections of sugarcane stalks which have been colonized by the pathogen; this technique can also be used for diagnosis (Harrison and Davis, 1988). A dot-blot EIA was also developed to detect the pathogen in sap extracts. Both the dot-blot and tissue-blot EIA involve the deposition of pathogen cells either from extracts or directly from plant tissue, respectively, onto nitrocellulose membrane filters before serological detection with an indirect EIA.

DNA Techniques

Cloned DNA probes have been developed for the pathogen (Chung et al., 1994). The sensitivities of detection reported for the DNA probes was approximately equal to that reported for the FADCF technique but less than those reported for the various EIAs. A tissue-blot DNA hybridization technique (Pan et al., 1998a) and polymerase chain reaction (PCR) procedures have been reported for the sensitive and specific detection of the pathogen in sugarcane (Pan et al., 1998b; Fegan et al., 1998).

Disease symptoms are rarely adequate for diagnosis in the field and laboratory tests are almost always required for accurate diagnosis (see Diagnostic Methods).

Any malady that results in stunted growth of sugarcane and does not have characteristic symptoms might be confused with ratoon stunting disease. For example, aluminium toxicity, iron deficiency and nematode infestations may have been confused with this disease. If stunting is uniform throughout the crop, it is probably not caused by this disease. Uneven growth reduction where some plants are affected more than others is more characteristic; however, nematode infestations can also be uneven. Diagnosis of ratoon stunting disease should thus be confirmed by detection of the pathogen in the laboratory (see section on Diagnostic Methods).

Due to the variable regulations around (de)registration of pesticides, your national list of registered pesticides or relevant authority should be consulted to determine which products are legally allowed for use in your country when considering chemical control. Pesticides should always be used in a lawful manner, consistent with the product's label.

Cultural Control and Sanitary Methods

Planting healthy cane can be used to control ratoon stunting disease. Sanitation is important in keeping healthy cane from becoming infected, because L. xyli subsp. xyli is easily transmitted mechanically.

Seed cane can be monitored for freedom from the disease using appropriate diagnostic techniques. In Australia and South Africa, phase-contrast microscopy has been used to screen thousands of samples annually for L. xyli subsp. xyli (Egan, 1980; Bailey and Fox, 1984). Continued vigilance in the selection of seed cane over several years has resulted in a reduction in the incidence of ratoon stunting disease in both plantings for seed cane production and commercial crops.

Seed cane can be heat treated to help prevent the spread of L. xyli subsp. xyli from one geographic area to another and to control ratoon stunting disease within areas where it occurs (Steindl, 1961). Hot-water, hot-air, moist-air, and aerated steam treatments have been used (Benda and Ricaud, 1978). Hot-water treatment at ca 50°C for 2-3 hours has been the most commonly used method (Steindl, 1961; Gul and Hassan, 1995). The application of streptomycin and hot-water treatment at 52°C for 30 minutes suppressed disease by 22.6% and increased yields of cane and white sugar by 35.54 and 4.55 t/ha, respectively (Gul and Hassan, 1995). Different cultivars vary in their tolerance to injury by heat. Seed cane from mature plants is usually less affected and generally germinates better after hot-water treatment than that from immature or over-mature plants. Pre-treatment of young, heat-sensitive seed cane to increase germination has been reported to reduce the deleterious effects of hot-water treatment (Benda, 1972, 1978). Seed cane was cut 1-5 days before treatment, pre-treated at 50°C for 10 minutes in hot water, and then treated the following day at 50°C for 2-3 hours. Hot-air treatment, with inlet air at 58°C for 8 hours or at 50°C for 24 hours, may be less harmful to immature seed cane than hot-water treatment and is used in some areas (Steindl, 1961). Dipping seed cane in water at ambient temperatures following hot-air treatment may protect germination. Moist-air treatment at 54°C for 7 hours in a sealed chamber was developed to overcome the moisture loss associated with hot-air treatment (Shukla et al., 1974). Aerated-steam treatment at 53°C for 4 hours uses steam-moistened hot air to reach the necessary internal stalk temperatures more quickly, approaching the treatment time used with hot water (Mayeux et al., 1979).

The two major problems limiting the effectiveness of heat treatment, other than expense, are reductions in germination and lack of complete control. Procedures that have been developed to protect germination in addition to those already discussed include treating seed cane with fungicides or other chemicals during and after heat treatment, careful selection of seed cane for treatment, and leaving leaf sheaths over buds during treatment. Proper functioning of the heating unit and temperature control system and proper loading of the heat chamber can favour both germination and control of ratoon stunting disease.

Under practical conditions, however, heat treatment is often not completely curative for ratoon stunting disease (Damann and Benda, 1983), and germination rates of treated seed cane may be reduced. Moreover, the expense involved in heat treatment is prohibitive. Consequently, heat treatment is often used to establish pathogen-free nurseries which are then used to supply planting material for commercial fields. Even so, the quantity of seed cane produced in such nurseries is usually inadequate, and additional sources of relatively pathogen-free seed cane are required. Seed cane from sources with a recent history of heat treatment can be used to fill the void, especially when adequate sanitary precautions have been taken to prevent spread of the pathogen and ensure that the level of the disease is minimal.

Because L. xyli subsp. xyli is readily transmitted by mechanical means, sanitation is important in preventing its spread to healthy plants (Comstock et al., 1996). Precautions can be taken to avoid transmission of the bacterium from one field to another on contaminated agricultural equipment. Sugarcane fields that are believed to be free from, or with a lower incidence of, ratoon stunting disease can be harvested first each day. Implements which have been used in diseased sugarcane fields can be cleansed and decontaminated before entering another field. Hot water, steam, flaming, or chemicals can be used to disinfect implements (Gillaspie and Davis, 1992).

Host-Plant Resistance

Although immunity to infection by L. xyli subsp. xyli is not known to occur in sugarcane, substantial resistance to injury has been found in some cultivars and appears to have a degree of genetic determination (Roach, 1992; Miller et al., 1995). Breeding for resistance to the disease was not a part of any breeding programme until recently, because no practical methods of screening large numbers of clones for resistance to the disease were available. Selection for disease resistance on the scale required in a sugarcane breeding programme is usually made on the basis of a visually assessed parameter of severity or incidence. No such visual parameters exist for ratoon stunting disease. Yield losses have been consistently detectable statistically only in replicated yield-loss trials combined over locations and years. Thus, yield-loss trials are grossly inadequate as a screening procedure on the scale required in a breeding programme.

The tissue-blot EIA and the EB-EIA techniques (see Diagnostic Methods) have been incorporated into sugarcane breeding programmes in Florida, USA (Davis et al., 1994) and in Australia (Croft et al., 1994), respectively, to permit large-scale screening for resistance to ratoon stunting disease. The number of colonized vascular bundles in stalks is measured by the tissue-blot EIA, and population size of the pathogen in sap extracts is measured by the EB-EIA. Both parameters are measures of the extent of colonization of sugarcane by the pathogen and were found to be highly correlated (Harrison and Davis, 1988). In addition to the extent of colonization, the frequency of infection of sugarcane clones can be determined by either method. These parameters are then used as estimates of disease incidence and severity as might be encountered in commercial sugarcane production. The validity of using the estimates of pathogen colonization as a measure of disease severity is based on their correlations with yield reduction due to ratoon stunting disease. The possible negative influence on this correlation of clonal tolerance to the disease has been discussed (Davis et al., 1988b; Roach and Jackson, 1992).

It was hypothesized that disease control might best be obtained by disregarding the effects of the disease on yields and concentrating on lowering the potential for epidemics by selection of clones that are less susceptible to infection and produce less inoculum when infected (Davis et al., 1994).

Chemical Control

The application of ammonium sulfate to sugarcane crops resulted in 22.89% reduction in disease caused by L. xyli subsp. xyli and yield increases of 29.09 and 2.91 t/ha, in cane and white sugar, respectively (Gul and Hassan, 1995).