First Report of Ratoon Stunt of Sugarcane Caused by Leifsonia xyli subsp. xyli in Pakistan.

Sugarcane (Saccharum hybrids), the second largest cash crop of Pakistan, is planted on 1.029 million ha with an annual production of 50 million tons. During a survey of the sugarcane crop in Faisalabad, Sargodha, and the Dera Ghazi Khan Division of the Punjab Province of Pakistan from 2007 to 2010, symptoms consistent with ratoon stunting, including stunted growth and reddening of the vascular bundles at the nodal regions (1), was observed on sugarcane cvs. CP77-400, SPF-241, CP72-2086, and NCo-310. CP72-2086 and NCo-310 showed severely stunted growth in both crop cycles. A chemical test was performed for detecting ratoon stunt from the field. Longitudinal sections of mature nodes were treated with a combination of hydrogen peroxide and hydrochloric acid. Healthy canes developed a blue-green color in the parenchymatous tissue around the fibrovascular bundles, diseased cane did not. This field test illustrated that as much as 25% of the plants were infected by ratoon stunt in the survey area. Aerobic bacteria were isolated from a stunted sample (NCo-310) on modified sugarcane medium (17 g of cornmeal agar, 8 g of peptone from soy meal, 1 g of K₂HPO₄, 1 g of KH₂PO₄, 0.2 g of MgSO₄·7H₂O, 0.5 g of glucose, 1 g of cysteinefree base, 2 g of bovine serum albumin, and 15 mg of bovine hemin chloride) and incubated for 3 to 4 weeks at 28°C. Light, off-white, round, and raised growth bacterial colonies (1.5 to 4.5 × 0.2 to 0.35 μm). Isolates were positive for the gram and catalase reactions and negative for oxidase, aesculin hydrolysis, urease production, and motility. The pathogen was identified as Leifsonia xyli subsp. xyli (formerly Clavibacter xyli subsp. xyli) based on its morphological characteristics (2). A direct antigen coating-ELISA was developed with antiserum raised against L. xyli subsp. xyli at the National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan. Infected or suspected to be infected plants of different cultivars were used for an ELISA test. Results showed that sugarcane cvs. NCo-310 (Log 1.342 CFU/ml) and CP72-2086 (Log 0.118 CFU/ml) had higher L. xyli subsp. xyli titres than the other cultivars tested (SPF-213 [Log 0.071CFU/ml], CPF-237 [Log 0.077CFU/ml], HSF-240 [Log 0.069 CFU/ml], NSG-555 [Log 0.060 CFU/ml], SPSG-26 [Log 0.076 CFU/ml], SPSG-79 [Log 0.074 CFU/ml], SPF-238 [Log 0.057 CFU/ml], and CP77-400 [Log 0.063 CFU/ml]). Cv. SPF-241 (Log 0.107 CFU/ml) was weakly positive for ratoon stunt (4). Axillary buds of sugarcane were injected via a sterile hypodermic syringe with an 18-gauge needle to deliver a bacterial suspension of 10⁹ cells/ml (3). Inoculated sugarcane plants were examined at intervals over 9 months for the development of symptoms and the presence of bacteria. Cultivars were evaluated on the basis of average number of colonized vascular bundles. SPF-213, CPF-237, HSF-240, NSG-555, SPSG-26, SPSG-79, SPF-238, and CP77-400 were resistant; SPF-241 showed moderate resistance and CP72-2086 and NCo-310 were highly susceptible to ratoon stunt. The pathogen was reisolated from the inoculated plants and identified as L. xyli subsp. xyli by bacteriological tests and its serological reaction. To our knowledge, this is the first report of ratoon stunt of sugarcane in Punjab Province of Pakistan. References: (1) M. J. Davis et al. Science 210:1365, 1980. (2) L. I. Evtushenko et al. Int. J. Syst. Evol. Microbiol. 50:371, 2000. (3) M. P. Nayiager et al. Phytopathol. Z. 99:273, 1980. (4) G.-P. Rao and G.-P. Singh. Sugar Tech. 2:35, 2000.