Impact of Sugarcane yellow leaf virus (ScYLV) infection on physiological efficiency and growth parameters of sugarcane under tropical climatic conditions in India

Yellow leaf (YL) of sugarcane caused by Sugarcane yellow leaf virus (ScYLV, a Polerovirus of the Luteoviridae family) is a serious disease affecting the crop production and productivity in India. Although impact of the disease on cane growth is observed, no systematic study has been done so far from the tropical Asian region to establish its impact on various physiological parameters, cane yield and juice quality. We have assessed physiological parameters in symptomatic and asymptomatic plants of ten different cultivars and a genotype. In addition, similar comparisons were made between virus-infected and virus-free plants derived through meristem culture. Our studies established that among several physiological parameters, photosynthetic rate (A), stomatal conductance (g _s) and SPAD metre values were significantly reduced in cultivars severely infected with ScYLV. Virus-infected cultivars exhibited significant reduction in growth/yield parameters, viz. stalk height, stalk thickness and number of internodes. Plant growth reductions were found to be 42.9, 42.3 and 38.9 % in susceptible cultivars CoPant 84211, Co 86032 and CoC 671, respectively. In addition to reduction in stalk weight, height and girth, YL disease also reduced juice yield in the affected canes up to 34.15 %. Similarly, comparison of diseased (virus-infected) and virus-free plants derived through meristem culture also revealed a drastic reduction in cane growth/physiological parameters and juice yield due to virus infection. The present study is the first comprehensive report demonstrating that YL disease caused by ScYLV seriously affects cane and juice yield in major sugarcane varieties under tropical climatic conditions (India). Consequently, this situation warrants a massive programme to provide healthy seed material and initiate breeding for YL resistance in sugarcane.     

Differentiation of Aerobacter-Klebsiella isolated from sugarcane

Three hundred and eighty-four isolates were obtained in the completed test portion of the most probable number determinations of coliforms in sugarcane sources. Of these isolates, 88% were of the (- - + +) indole, methyl red, Voges-Proskauer, citrate (IMViC) type and were identified as Aerobacter aerogenes according to the protocol of the American Public Health Association (1). Employing 359 of these cultures, a comparative biochemical, serological, and pathogenicity study was carried out with Klebsiella pneumoniae CDC no. 2211-66 type 9. More than 86% of the organisms tested gave biochemical reactions typical of K. pneumoniae. Of the other isolates, 2% were Enterobacter aerogenes, and the remaining 12% were identified as atypical, nonmotile IMViC types. Comparable agglutination titers were also observed between A. aerogenes and the CDC strain of K. pneumoniae when several randomly selected sugarcane strains were reacted with prepared K. pneumoniae whole cell antiserum. Neither the K. pneumoniae reference organism nor selected sugarcane isolates displayed pathogenicity for mice. On the basis of all the analyses performed, it was suggested that such organisms be classified as K. pneumoniae.     

Alleviation of detrimental effects of NaCl by silicon nutrition in salt-sensitive and salt-tolerant genotypes of sugarcane (Saccharum officinarum L.)

Salt stress is a major environmental factor which adversely affects the crop yield and quality. However, adequate regulation of mineral nutrients may ameliorate the deleterious effects of salts and help to sustain crop productivity under salt stress. Salt-sensitive (SPF 213) and salt-tolerant (HSF 240) sugarcane genotypes were grown in gravel at 0 and 100 mM NaCl by supplying 0, 1.4 mM, 2.1 mM and 2.8 mM of Si as calcium silicate. Results revealed that plants treated with NaCl alone showed a significant (P?≤?0.05) reduction in dry matter production, K⁺ concentration, cane yield and juice quality in both genotypes but the magnitude of reduction was relatively more in salt-sensitive genotype than salt-tolerant. Addition of Si significantly (P?≤?0.05) reduced the uptake and translocation of Na⁺ but increased K⁺ concentrations particularly in shoots of both sugarcane genotypes. Cane yield and yield attributes were significantly (P?≤?0.05) higher where Si was added. Juice quality characteristics were significantly (P?≤?0.05) improved in salt-sensitive and salt-tolerant sugarcane genotypes with the application of Si. The results suggested that added Si interacted with Na⁺, reduced its uptake and transport to shoots and consequently improved cane yield and juice quality in salt-sensitive and salt-tolerant sugarcane genotypes under salt stress.     

Microbiological Quality of Bagged Cut Spinach and Lettuce Mixes

Analysis of 100 bagged lettuce and spinach samples showed mean total bacterial counts of 7.0 log₁₀ CFU/g and a broad range of <4 to 8.3 log₁₀ CFU/g. Most probable numbers (MPN) of ≥11,000 /g coliforms were found in 55 samples, and generic Escherichia coli bacteria were detected in 16 samples, but no E. coli count exceeded 10 MPN/g.     

Antimicrobial resistant coliform bacteria in the Gomti river water and determination of their tolerance level

The distribution of resistance to ampicillin, chloramphenicol, sulfonamides, tetracycline, and streptomycin among coliform in the Gomti river water samples was investigated. The coliform populations were isolated on Mac Conky and eosin methylene blue (EMB) agar plates supplemented with antibiotics. The incidence of resistance among the coliform population varied considerably in different drug and water sampling sites. Coliform bacteria showed lower drug resistant viable count in sampling site-III (receiving treated wastewater) as compared to more polluted site-I and site-II. Viable count of coliform population obtained on both medium was recorded higher against erythromycin from sampling site-III. Lower viable count of coliforms was recorded against tetracycline in site-II and III. Similar resistance pattern was obtained in the frequency of E. coli and Enterobacter species from all the three sampling sites. Percentage of antibiotic resistant E. coli was observed higher than Enterobacter spp among the total coliforms against all antibiotics tested without Erythromycin and penicillin in site-I and II respectively. Isolates of E. coli and Enterobacter spp. showed their tolerance level (MIC) in the range of 2-100 against the antibiotics tested. Maximum number of isolates of both genus exhibited their MICs at lower concentration range 2-5µg/ml against ciprofloxacin, tetracyclin and amoxycillin.

Abbreviations

EMB - Eosin methylene blue, IMViC tests - Indole, Methyl Red, Voges Proskauer and Citrate Utilization Tests, MIC - Minimum inhibitory concentration.     

Membrane Filter Procedure for Enumerating the Component Genera of the Coliform Group in Seawater

A facile, quantitative, membrane filter procedure (mC) for defining the distribution of coliform populations in seawater according to the component genera was developed. The procedure, which utilizes a series of in situ substrate tests to obviate the picking of colonies for identification, also provides an estimate of the total coliform density. When pure cultures of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae were suspended in seawater and held at 4 C for 24 h, between 56 and 100% of the cells which grew on nutrient agar spread plates at 35 C could be recovered by the mC procedure. Confirmation as coliforms of typical colonies from natural samples was about 95%. Assay variability was found to be insignificant. The recovery of coliforms from marine waters by the mC procedure was comparable to those obtainable by current methods. Klebsiella was differentiated by the urease reaction and E. coli by its ability to form indole. The confirmation frequencies for colonies designated as Klebsiella and E. coli by the in situ tests approached 95% for the former and 98% for the latter.     

Characterization of the coliform and enteric bacilli in the environment of calves with colibacillosis.

In the first part of the present study the coliform and enteric bacilli in the environment of calves with colibacillosis were examined. The occurrence, number, and pathogenic properties of Escherichia coli in barnyard soils were obtained from six cattle ranches. The O and K serogroups of E. coli isolates obtained from the feces of calves with colibacillosis born at these cattle ranches were determined, and their serotypes were compared with the E. coli O and K serotypes found in soils. The results showed a reservoir of potentially pathogenic E. coli in barnyard soils contaminated with bovine feces. For the second part of this study, 6 healthy calves and 51 calves with colibacillosis were studied. The numbers of total aerobic heterotrophic bacteria, total streptococci, fecal streptococci, total coliforms, and fecal coliforms in the feces of calves were determined. In addition, coliform and enteric bacilli from the feces of both healthy and diseased calves were identified, and their indole, methyl red, Voges-Proskauer, citrate (IMViC) types were described. In parallel, the IMViC types of coliform and enteric bacilli isolated from barnyard soils previously contaminated with bovine feces were compared with those isolated from uncontaminated soils. All fecal specimens were also examined for the presence of rotavirus. No significant effect on the numbers of the bacterial types was found. The results suggest that the predominant IMViC types found in the feces of calves with colibacillosis originate from the soil. From this study it is apparent that the occurrence, number, and survival of E. coli in barnyard soils is related to ranch husbandry and sanitary practices.     

Characterization of the coliform and enteric bacilli in the environment of calves with colibacillosis

In the first part of the present study the coliform and enteric bacilli in the environment of calves with colibacillosis were examined. The occurrence, number, and pathogenic properties of Escherichia coli in barnyard soils were obtained from six cattle ranches. The O and K serogroups of E. coli isolates obtained from the feces of calves with colibacillosis born at these cattle ranches were determined, and their serotypes were compared with the E. coli O and K serotypes found in soils. The results showed a reservoir of potentially pathogenic E. coli in barnyard soils contaminated with bovine feces. For the second part of this study, 6 healthy calves and 51 calves with colibacillosis were studied. The numbers of total aerobic heterotrophic bacteria, total streptococci, fecal streptococci, total coliforms, and fecal coliforms in the feces of calves were determined. In addition, coliform and enteric bacilli from the feces of both healthy and diseased calves were identified, and their indole, methyl red, Voges-Proskauer, citrate (IMViC) types were described. In parallel, the IMViC types of coliform and enteric bacilli isolated from barnyard soils previously contaminated with bovine feces were compared with those isolated from uncontaminated soils. All fecal specimens were also examined for the presence of rotavirus. No significant effect on the numbers of the bacterial types was found. The results suggest that the predominant IMViC types found in the feces of calves with colibacillosis originate from the soil. From this study it is apparent that the occurrence, number, and survival of E. coli in barnyard soils is related to ranch husbandry and sanitary practices.     

Occurrence of Escherichia coli in Wild Cottontail Rabbits

Free-ranging cottontail rabbits (Sylvilagus floridanus) from two areas in central Pennsylvania were sampled over a 4-year period. Large numbers of coliforms were isolated from the intestinal tracts of these animals; in 136 of the 141 rabbits sampled, Escherichia coli was found to be a major component of the alimentary flora. Four serogroups (O7, O77, O73, and O103) were predominant among the isolates and were considered resistant coliflora of this species of cottontail rabbit.     

Two Novel Bacterial Biosensors for Detection of Nitrate Availability in the Rhizosphere

The nitrate-regulated promoter of narG in Escherichia coli was fused to promoterless ice nucleation (inaZ) and green fluorescent protein (GFP) reporter genes to yield the nitrate-responsive gene fusions in plasmids pNice and pNgfp, respectively. While the promoter of narG is normally nitrate responsive only under anaerobic conditions, the L28H-fnr gene was provided in trans to enable nitrate-dependent expression of these reporter gene fusions even under aerobic conditions in both E. coli DH5α and Enterobacter cloacae EcCT501R. E. cloacae and E. coli cells containing the fusion plasmid pNice exhibited more than 100-fold-higher ice nucleation activity in cultures amended with 10 mM sodium nitrate than in nitrate-free media. The GFP fluorescence of E. cloacae cells harboring pNgfp was uniform at a given concentration of nitrate and increased about 1,000-fold when nitrate increased from 0 to 1 mM. Measurable induction of ice nucleation in E. cloacae EcCT501R harboring pNice occurred at nitrate concentrations of as low as 0.1 μM, while GFP fluorescence was detected in cells harboring pNgfp at about 10 μM. In the rhizosphere of wild oat (Avena fatua), the whole-cell bioreporter E.cloacae(pNgfp) or E. cloacae(pNice) expressed significantly higher GFP fluorescence or ice nucleation activity when the plants were grown in natural soils amended with nitrate than in unamended natural soils. Significantly lower nitrate abundance was detected by the E. cloacae(pNgfp) reporter in the A. fatua rhizosphere compared to in bulk soil, indicating plant competition for nitrate. Ice- and GFP-based bacterial sensors thus are useful for estimating nitrate availability in relevant microbial niches in natural environments.     

Pilot-scale citric acid production with Aspergillus niger under several conditions

Citric acid fermentations with Aspergillus niger in stirred tank reactors (500 l and 2.6 m³) were carried out using cane juice, cane molasses and a synthetic medium. Highest yields were obtained with the cane juice and synthetic medium. The rate of stirring had a pronounced influence on the morphology of A. niger.     

Predicting Salmonella Populations from Biological,Chemical, and Physical Indicators in Florida Surface Waters

Coliforms, Escherichia coli, and various physicochemical water characteristics have been suggested as indicators of microbial water quality or index organisms for pathogen populations. The relationship between the presence and/or concentration of Salmonella and biological, physical, or chemical indicators in Central Florida surface water samples over 12 consecutive months was explored. Samples were taken monthly for 12 months from 18 locations throughout Central Florida (n = 202). Air and water temperature, pH, oxidation-reduction potential (ORP), turbidity, and conductivity were measured. Weather data were obtained from nearby weather stations. Aerobic plate counts and most probable numbers (MPN) for Salmonella, E. coli, and coliforms were performed. Weak linear relationships existed between biological indicators (E. coli/coliforms) and Salmonella levels (R² < 0.1) and between physicochemical indicators and Salmonella levels (R² < 0.1). The average rainfall (previous day, week, and month) before sampling did not correlate well with bacterial levels. Logistic regression analysis showed that E. coli concentration can predict the probability of enumerating selected Salmonella levels. The lack of good correlations between biological indicators and Salmonella levels and between physicochemical indicators and Salmonella levels shows that the relationship between pathogens and indicators is complex. However, Escherichia coli provides a reasonable way to predict Salmonella levels in Central Florida surface water through logistic regression.     

Detection of sugarcane yellow leaf virus,the causal agent of yellow leaf syndrome in sugarcane by DAS-ELISA

DAS-ELISA studies were conducted on detection of sugarcane yellow leaf virus (SCYLV) causing yellow leaf syndrome (YLS) of sugarcane in leaf and juice antigens. Among the two types of antigen sources used for the virus detection, juice antigen showed high titre for the virus as compared to leaf antigen. Assay with juice samples recorded more number of varieties positive to the virus. Further DAS-ELISA studies revealed that plants raised from disease-infected planting materials recorded high titre for SCYLV as compared to those raised from symptom-free seed canes. Similarly, assaying SCYLV titre in plant and ratoon crop in the field showed that SCYLV infection was partial in plant crop and in the subsequent ratoon crop, all the samples were positive to the virus. ELISA studies also indicated that 33 of 41 cane varieties showing YLS were positive to the virus.     

Cloning and Characterization of Aerobactin Biosynthesis Genes of the Biological Control Agent Enterobacter cloacae

Five strains of Enterobacter cloacae that are biological control agents of Pythium damping-off diseases produced the hydroxamate siderophore aerobactin under iron-limiting conditions. Genes determining aerobactin biosynthesis of the biocontrol strain E. cloacae EcCT-501 were localized to a 12.3-kb region, which conferred aerobactin production to Escherichia coli DH5α. The aerobactin biosynthesis genes of E. cloacae hybridized to those of the pColV-K30 plasmid of E. coli, but restriction patterns of the aerobactin regions of pColV-K30 and E. cloacae differed. A derivative strain with a deletion in the aerobactin biosynthesis locus was as effective as strain EcCT-501 in biological control of Pythium damping-off of cucumber. Thus, aerobactin production did not contribute significantly to the biological control activity of EcCT-501 under the conditions of this study.     

Occurrence of Coliforms, Fecal Coliforms, and Streptococci on Vegetation and Insects

This study considers the sanitary significance of coliforms, fecal coliforms, and streptococci isolated from 152 species of plants and 40 samples of insects. These specimens were collected from various ecological environments and grouped into several categories. Results indicate that typical coliforms of the warm-blooded animal gut contribute a relatively small percentage of the organisms associated with vegetation (14.1%) and insects, (14.9%). A total of 1,203 coliform strains from vegetation and 1,084 coliform strains from insects were classified as to IMViC type and fecal coliform. No type was predominant in either the vegetation or insect groupings. The biochemical results for 646 streptococci from vegetation and 226 cultures from insects were reported. The predominant group, Streptococcus fecalis, as defined by Sherman criteria, constituted a majority of all strains from vegetation and insects. The “Completed Coliform Test” is recommended for the examination of plant and insect specimens to eliminate the many anaerobic and aerobic sporeforming bacteria that frequently produce false positive reactions by the “Confirmed Test” procedure. These findings support the current interpretation of the significance of the fecal coliform test for stream investigations or for surface water quality evaluations.     

Improvement of Brazilian bioethanol production – Challenges and perspectives on the identification and genetic modification of new strains of Saccharomyces cerevisiae yeasts isolated during ethanol process

In Brazil, bioethanol is produced by sucrose fermentation from sugarcane by Saccharomyces cerevisiae in a fed-batch process that uses high density of yeast cells (15–25 % of wet weight/v) and high sugar concentration (18–22 % of total sugars). Several research efforts have been employed to improve the efficiency of this process through the isolation of yeasts better adapted to the Brazilian fermentation conditions. Two important wild strains named CAT-1 and PE-2 were isolated during the fermentation process and were responsible for almost 60 % of the total ethanol production in Brazil. However, in the last decade the fermentative substrate composition was much modified, since new sugar cane crops were developed, the use of molasses instead of sugar cane juice increase and with the prohibition of burning of sugarcane prior harvest. As consequence, these previously isolated strains are being replaced by new wild yeasts in most of ethanol plants. In this new scenario the isolation of novel better adapted yeasts with improved fermentative characteristics is still a big challenge. Here, we discuss the main aspects of Brazilian ethanol production and the efforts for the selection, characterization and genetic modifications of new strains with important phenotypic traits such as thermotolerance.     

Evidence from field nitrogen balance and 15N natural abundance data for the contribution of biological N2 fixation to Brazilian sugarcane varieties

Aims

In Brazil N fertilization of sugarcane (Saccharum spp.) is low compared to most other countries. ¹⁵N-aided studies and the occurrence of many N₂-fixing bacteria associated with cane plants suggest significant contributions from biological N₂ fixation (BNF). The objective of this study was to evaluate BNF contributions to nine cane varieties under field conditions using N balance and ¹⁵N natural abundance techniques.

Methods

The field experiment was planted near Rio de Janeiro in 1989, replanted in 1999 and harvested 13 times until 2004. Soil total N was evaluated at planting and again in 2004. Samples of cane leaves and weeds for the evaluation of ¹⁵N natural abundance were taken in 2000, 2003 and 2004.

Results

N accumulation of the commercial cane varieties and a variety of Saccharum spontaneum were persistently high and N balances (60 to 107?kg?N ha^?1?yr^?1) significantly (p?<?0.05) positive. The δ¹⁵N of leaf samples were lower than any of the weed reference plants and data obtained from a greenhouse study indicated that this was not due to the cane plants tapping into soil of lower ¹⁵N abundance at greater depth.

Conclusion

The results indicate that the Brazilian varieties of sugarcane were able to obtain at least 40?kg?N ha^?1?yr^?1 from BNF.     

Pathways of Anaerobic Acetate Utilization in Escherichia coli and Aerobacter cloacae

Acetate-1-¹⁴C was added to anaerobic glucose-fermenting cultures of Escherichia coli and Aerobacter cloacae. In the E. coli culture, lactate formation occurred late in the fermentation, when the rate of production of formate and acetate had decreased. The occurrence of acetate label in the lactate indicated formation of pyruvate from acetyl-coenzyme A (CoA) and formate. In the A. cloacae cultures, substantial amounts of acetate label were found in the 2,3-butanediol formed. Evidence is presented that the label could have entered the diol only by conversion of formate and acetyl-CoA into pyruvate. The observed levels of radioactivity in the diol indicated that during diol formation the reaction yielding formate and acetyl-CoA from pyruvate CoA was operating close to equilibrium. The shift in metabolism from formation of acetate, ethyl alcohol, and formate to the formation of butanediol or lactate appears to be due basically to an approach to equilibrium of the pyruvate-splitting reaction, whatever the induction mechanism by which the shift is implemented.     

Differential Inactivation of Seed Exudate Stimulation of Pythium ultimum Sporangium Germination by Enterobacter cloacae Influences Biological Control Efficacy on Different Plant Species

This study was initiated to understand whether differential biological control efficacy of Enterobacter cloacae on various plant species is due to differences in the ability of E. cloacae to inactivate the stimulatory activity of seed exudates to Pythium ultimum sporangium germination. In biological control assays, E. cloacae was effective in controlling Pythium damping-off when placed on the seeds of carrot, cotton, cucumber, lettuce, radish, tomato, and wheat but failed to protect corn and pea from damping-off. Seeds from plants such as corn and pea had high rates of exudation, whereas cotton and cucumber seeds had much lower rates of exudation. Patterns of seed exudation and the release of P. ultimum sporangium germination stimulants varied among the plants tested. Seed exudates of plants such as carrot, corn, lettuce, pea, radish, and wheat were generally more stimulatory to P. ultimum than were the exudates of cotton, cucumber, sunflower, and tomato. However, this was not directly related to the ability of E. cloacae to inactivate the stimulatory activity of the exudate and reduce P. ultimum sporangium germination. In the spermosphere, E. cloacae readily reduced the stimulatory activity of seed exudates from all plant species except corn and pea. Our data have shown that the inability of E. cloacae to protect corn and pea seeds from Pythium damping-off is directly related to its ability to inactivate the stimulatory activity of seed exudates. On all other plants tested, E. cloacae was effective in suppressing damping-off and inactivating the stimulatory activity of seed exudates.