INTERUNIVERSITY NETWORK FOR THE DEVELOPMENT OF THE SUGAR ENERGY SECTOR
FEDERAL UNIVERSITY OF SÃO CARLOS
SUGARCANE BREEDING PROGRAM
RATOON STUNTING DISEASE
Ratoon stunting (RSD) analysis
Introduction
Ratoon stunting (RSD) is caused by the bacterium Leifsonia xyli subsp . xyli . The disease is widespread in all Brazilian states and affects all varieties. It is considered an invisible and insidious disease, as it presents no symptoms that identify it in the field, and its correct identification is possible only in a laboratory. Despite this difficulty, its damage is noted through reduced productivity and early sugarcane field reform. Its only method of entry into a new area is through the planting of contaminated, previously unidentified stems, from which it spreads to other plants by the harvester. Producers only notice the disease in adult plants already established in the field, when there is no longer any possibility of controlling the disease.
Importance of the disease:
There is no data on the damage the disease causes to currently cultivated Brazilian varieties. However, research results from other sugarcane-producing countries clearly show that ratoon stunting is the main disease affecting the crop. The table below shows the decline in productivity in these countries:
Spread of disease by the harvester:
The figure below shows that the spread of the disease from a contaminated stem varied from 3.9 to 7.3 meters in the first cut and from 8.8 to 11.5 m in the second cut.
Purpose of assessments
Sugarcane ratoon stunting can only be controlled by planting seedlings known to be free of the bacteria. Since reliable identification of the disease is only possible in a laboratory, the success of a sugarcane plantation is closely linked to the diagnostic testing of propagation material before planting. Thus, the productivity of a sugarcane plantation depends on the prior examination of the stems used in planting.
Recommendations for collecting samples for diagnosis
Each sample should consist of up to 100 stalks per area (plot, plot, section, etc.) for each variety. Samples should be collected from sugarcane fields over nine months old. Although there is no known difference between randomly harvested stalks and selected fine stalks, it is recommended that samples be directed to tillers suspected of being infected by the causative agent. The bacterium is most present in the basal, more mature part of the stalk—that is, in the first nodes from the base of adult plants. It is recommended not to use cutting instruments to collect the material. This should be done by breaking the canes at the base. Stems with borer holes should also be avoided.
The table below shows the information that must accompany the samples. It is very important to complete all fields (Variety, Cut, Section/Plot, Whether Heat Treatment Was Performed, and Number of Samples). This information is extremely useful for monitoring areas over the years as well as disseminating it to neighboring areas.
Table 1. Procedure for specifications of material sent for diagnosis.
Methodology for extracting xylem sap
To extract vascular fluid from each of the sampled stems, the following steps must be followed correctly:
1. Clean the stalks with a damp cloth, avoiding contamination by soil particles and other impurities;
2. Between the second and third internode from the base of the stem upwards, make a bevel cut at the bottom and a cross cut at the top. The end cuts should be made on individual stems for later extraction of xylem sap.
3. A low-pressure compressor is used with a rubber teat (milking machine type) fitted to the hose end to facilitate the attachment of the stem and facilitate sap extraction. Stems collected in the morning produce a larger volume.
Figure 2. Compressor adapted with rubber teat cup (milking machine type).
4. Collect at least 0.5 ml of vascular fluid from each sampled stem in plastic microtubes with a capacity of 1.5 ml.
4. Collect at least 0.5 ml of vascular fluid from each sampled stem in plastic microtubes with a capacity of 1.5 ml.
Figure 3. Packaging of vascular fluid in 1.5 ml microtubes.
5. Place alkyldimethylbenzylammonium chloride (a commercial quaternary ammonium product) in each microtube to preserve the sample. If the volume of sugarcane sap is 0.5 ml, the amount of pure commercial product (containing 3% of the active ingredient) to be added to each microtube is two drops. Send as soon as possible for laboratory analysis.
Methods for assessing the presence of bacteria in samples
The serological method is used worldwide for routine analysis of ratoon stunting. Within this technique, the dot blot is used in LAGEM analyses:
a) Dot blot serology
Using this method, it is possible to determine the different levels of infection:
Do not detect or clean: below 106 bacterial cells per mL of vascular sap;
N3 – low: occurrence of 107 bacterial cells per mL of vascular sap;
N2 - medium: occurrence of 10 8 bacterial cells per mL of vascular sap;
N1 - high: occurrence of more than 109 bacterial cells per mL of vascular sap.
Figure 4. Membrane with various levels of infection.
5. Add Alkyl Dimethyl Benzyl Ammonium Chloride (commercial product: quaternary ammonium) into each microtube for sample preservation. If the sugarcane sap volume is 0.5 ml, the amount of the pure commercial product (containing 3% active ingredient) to be added to each microtube is two drops. Send the samples as soon as possible for laboratory analysis.
Methods for assessing the presence of the bacterium in the samples
The serological method is the one employed worldwide for routine analysis of ratoon stunting disease. Within this technique, the “dot blot” is the method used for analyses at LAGEM:
a) Serology – “dot blot”
Through this method it is possible to determine different levels of infection:
Not detected or clean: below 10⁶ bacterial cells per mL of vascular sap;
N3 – low: occurrence of 10⁷ bacterial cells per mL of vascular sap;
N2 – medium: occurrence of 10⁸ bacterial cells per mL of vascular sap;
N1 – high: occurrence above 10⁹ bacterial cells per mL of vascular sap.
Final considerations
This disease is the most important one affecting sugarcane, and humans play a crucial role in its occurrence. The bacterium does not survive in the soil, allowing replanting of a contaminated area to be carried out immediately. The bacterium does not shelter in weeds present in the sugarcane field. No insect has the ability to transmit the disease. The only way the pathogen can be introduced into a new area is through human action by planting contaminated setts. Subsequently, the disease spreads within a field through agricultural implements. Diagnostic examination serves to guide future multiplication of clones or varieties, establishing the real need for heat treatment to control ratoon stunting disease. Even in areas where the infection level of sugarcane is low, in future multiplications it is necessary to apply heat treatment and disinfect harvesters and machetes used in cutting and chopping the seed setts, so as to prevent an increase in disease incidence within the same field or in neighboring plots.
Samples must always be sent to LAGEM/UFSCar – Araras. When the number of samples is large, scheduling is recommended!
Note:
LAGEM can provide a limited number of microtubes and preservation solution. It is advisable to call in advance to check availability. The microtubes will be returned cleaned and sterilized after the analysis, together with the delivery of the reports, or may be retrieved upon delivery of the next batch of samples.
Team:
Responsible: Prof. Alfredo Seiiti Urashima (PhD in Plant Protection)
Technical team: Nathalia Fadel
Address for sample submission:
Federal University of São Carlos
Center of Agricultural Sciences
Department of Biotechnology and Plant and Animal Production
Rodovia Anhanguera, SP-330, KM 174, CP 153
ZIP Code 13600-970 Araras, SP – Brazil
Phone: +55 (19) 3543-2656
E-mail: alfredo.urashima@ufscar.br