Use of simple sequence repeat markers for DNA fingerprinting and diversity analysis of sugarcane (Saccharum spp) cultivars resistant and susceptible to red rot.

Red rod is an economically important disease of sugarcane caused by the fungus Colletotrichum falcatum. We used a simple sequence repeat (SSR)-based marker system to identify and analyze genetic relationships of red rot resistant and susceptible sugarcane cultivars grown in Pakistan. Twenty-one highly polymorphic SSR markers were used for DNA fingerprinting and genetic diversity analysis of 20 sugarcane cultivars. These SSR markers were found to be highly robust; we identified 144 alleles, with 3-11 alleles per marker and a mean of 6.8. Three SSR markers were able to identify all 20 cultivars. DNAMAN(®)-generated homology tree was used to analyze genetic diversity among these cultivars; all cultivars shared 58% or more similarity. We correlated polymorphism information content and resolving power values with marker effectiveness in the process of sugarcane cultivar identification. We concluded that a small number of SSR-derived DNA markers will allow breeders to identify red rot resistant and susceptible cultivars.

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.

A diagnostic system using broad categories with clinically relevant specifiers: lessons for ICD-11.

A diagnostic system for ICD-11 is proposed which commences with broad reorganization and simplification of the current categories and the use of clinically relevant specifiers. Such changes have implications for the positioning of diagnostic groups and lead to a range of possibilities for improving terminology and the juxtaposition of individual conditions. The development of ICD-11 provides the fi rst opportunity in almost two decades to improve the validity and reliability of the international classification system. Widespread change in broad categories and criteria cannot be justified by research that has emerged since the last revision. It would also be disruptive to clinical practice and might devalue past research work. However, the case for reorganization of the categories is stronger and has recently been made by an eminent international group of researchers (Andrews et al., 2009). A simpler, interlinked diagnostic system is proposed here which is likely to have fewer categories than its predecessor. There are major advantages of such a system for clinical practice and research and it could also produce much needed simplification for primary care (Gask et al., 2008) and the developing world (Wig, 1990; Kohn et al., 2004).

Comparative study on two commercial strains of Saccharomyces cerevisiae for optimum ethanol production on industrial scale.

Two commercial strains of Saccharomyces cerevisiae, Saf-Instant (Baker's yeast) and Ethanol red (Mutant) were compared for ethanol production during hot summer season, using molasses diluted up to 6-7 degrees Brix containing 4%-5% sugars. The yeasts were then propagated in fermentation vessels to study the effects of yeast cell count and varying concentrations of Urea, DAP, inoculum size and Lactrol (Antibiotic). Continuous circulation of mash was maintained for 24 hours and after this fermenter was allowed to stay for a period of 16 hours to give time for maximum conversion of sugars into ethanol. Saccharomyces cerevisiae strain (Saf-instant) with cell concentration of 400 millions/mL at molasses sugar level of 13%-15% (pH 4.6 +/- 0.2, Temp. 32 degrees C +/- 1), inoculum size of 25% (v/v), urea concentration, 150 ppm, DAP, 53.4 ppm and Lactrol,150 ppm supported maximum ethanol production (8.8%) with YP/S = 250 L ethanol per tone molasses (96.5% yield), and had significantly lower concentrations of byproducts. By selecting higher ethanol yielding yeast strain and optimizing the fermentation parameters both yield and economics of the fermentation process can be improved.

P53 mutations in carcinoma breast--a clinicopathological study.

OBJECTIVES: To find out the incidence of p53 mutations and their possible correlation with clinicopathological presentation in females with breast carcinoma. SETTINGS: Department of Surgery, Pakistan Institute of Medical Sciences, Islamabad. PATIENTS AND METHODS: Seventy four patients with operable carcinoma breast that underwent mastectomy were included in this prospective study. Tumour tissue specimens and peripheral blood samples were examined for p53 mutations. Age, tumour size, nodal status and histopathology was assessed in patients with and without p53 mutations. RESULTS: Ten (13.5%) patients showed p53 mutations in their tumour specimens while 64 (86.48%) had normally functioning p53 gene. Patients were divided into two groups, A (normally functioning p53), and B (mutated p53). Intraductal carcinoma was the most frequent histological variant(A = 57, B = 10), while lymph nodes were involved in 67.19% (A = 47) and 60% (B = 6) cases respectively. The age of patients and clinical parameters (tumour size, nodal status and histopathological diagnosis) were compared between the two groups and no statistically significant correlation between p53 mutations and clinicopathological parameters was found. CONCLUSIONS: It was concluded that p53 mutation is present in carcinoma breast in Pakistani population but there was no significant correlation between p53 mutation and tumour aggressiveness (size, nodal status and histopathology).

Study the presence of fibronectin binding protein (FnBp) in tuberculosis (TB) patients by elisa.

Fibronectin-binding protein (FnBp) antigens are a prominent secretory protein of short term culture supernatants of M. tuberculosis and M. bovis (BCG) and is conserved within the genus Mycobacterium. The 30/31 kDa antigen of M. tuberculosis is one of the major secretory molecules and is probably routinely recognised by the host immune system in the early stage of tuberculosis infection. Serum immune complexes, prepared from TB patients and normals, were analysed for the presence of FnBp by ELISA using an anti-30/31 kDa (FnBp) monoclonal antibody (CF8) and by western blotting using Fibronectin-HRP. A significant difference was seen between normals and TB patients (p < 0.05). This test was found to have a specificity of 80% and a positive predictive value of 73%. This is a preliminary finding and the test needs to be evaluated further for its performance on a larger number of confirmed TB patients and controls.