Genetic Diversity of African and Worldwide Strains of Ralstonia solanacearum as Determined by PCR-Restriction Fragment Length Polymorphism Analysis of the hrp Gene Region

The genetic diversity among a worldwide collection of 120 strains of Ralstonia solanacearum was assessed by restriction fragment length polymorphism (RFLP) analysis of amplified fragments from the hrp gene region. Five amplified fragments appeared to be specific to R. solanacearum. Fifteen different profiles were identified among the 120 bacterial strains, and a hierarchical cluster analysis distributed them into eight clusters. Each cluster included strains belonging to a single biovar, except for strains of biovars 3 and 4, which could not be separated. However, the biovar 1 strains showed rather extensive diversity since they were distributed into five clusters whereas the biovar 2 and the biovar 3 and 4 strains were gathered into one and two clusters, respectively. PCR-RFLP analysis of the hrp gene region confirmed the results of previous studies which split the species into an “Americanum” division including biovar 1 and 2 strains and an “Asiaticum” division including biovar 3 and 4 strains. However, the present study showed that most of the biovar 1 strains, originating from African countries (Reunion Island, Madagascar, Zimbabwe, and Angola) and being included in a separate cluster, belong to the “Asiaticum” rather than to the “Americanum” division. These African strains could thus have evolved separately from other biovar 1 strains originating from the Americas.     

Repeat Domain Diversity of avrBs3-Like Genes in Ralstonia solanacearum Strains and Association with Host Preferences in the Field

Genes homologous to avrBs3 of Xanthomonas were detected in 309 strains of Ralstonia solanacearum biovars 3, 4, and 5 but not biovar 1 or 2. A statistically significant association between the originating plant species and internal repeats of the gene was found. Sequences of repeats and variation between nearly clonal strains revealed evidence of frequent recombination.     

Genetic structure and phylogenetic relationships of Ralstonia solanacearum strains from diverse origins in Guangdong Province,China

Bacterial wilt, caused by Ralstonia solanacearum species complex is a key yield‐limiting factor on crops in Guangdong province, China. The genetic diversity of 110 R. solanacearum strains collected from 16 host plants in different areas of Guangdong province was analysed using biovar and phylotype classification schemes. Of 110 strains, fifty‐five strains belong to biovar 3, fifty‐two strains belong to biovar 4, two strains belong to biovar 2 and one strain belonged to biovar 1. Phylotype‐specific multiplex PCR showed that 108 strains belonged to phylotype I (biovars 1, 3, 4) and two strains belonged to phylotype II (biovar 2). The result of phylogenetic relationships analysis based on egl gene sequences demonstrated that 108 strains of phylotype I were grouped into nine previously described sequevars and a new sequevar 57, and two strains of phylotype II were grouped into sequevar 1. Sequevars 15, 34 and 44 widely distributed in Guangdong were predominant sequevars. Sequevar 45 was first reported on potato and pumpkin in China. These results revealed the genetic structure and phylogenetic relationships of R. solanacearum population in Guangdong and will be helpful in bacterial wilt‐resistance breeding.     

Effects of Carbohydrates in Growth Medium on Agglutination of Several Species of Salmonella with Polyvalent H Antiserum

The effect of various carbohydrates in the growth medium on agglutination of salmonellae with polyvalent H antiserum was studied. There appeared to be a relationship between fermentation of the carbohydrate by the organism and resultant agglutination with the antiserum. It is recommended that the tube test for flagellar antigens be allowed to remain in a water bath for 2 hr before the final observation is made. Sorbitol, dulcitol, mannose, maltose, rhamnose, or trehalose, when included in the growth medium for Salmonella, yielded high percentages of positive agglutinations with all conditions of the experiment.     

The effects of carbohydrates upon the survival and reproduction of adult female Pimpla turionellae L. (Hym., Ichneumonidae)

In this study the effects of 23 carbohydrates belonging to various groups upon the survival egg production and egg of female adult Pimpla turionellae L. were investigated. The best results among the carbohydrates tested was obtained with sucrose which was also employed as control. Glucose, maltose, trehalose and melezitose on the other hand showed no significant effect. The egg production was observed to be unaffected by glucose and maltose although it showed significant increase with trehalose and significant decrease with melezitose. Fructose and sorbitol caused a significant decrease in the survival of the insect. Fructose and sorbitol did not have any significant effect upon egg production whereas galactose caused a significant decrease. With the exception of galactose, no carbohydrate caused any significant effect upon egg hatching. Although they did not produce any eggs the female insects survived for 13.17, 15.13, 11.58 and 15.83 days in mannose, melibiose, raffinose and mannitol, respectively. The shortest life span was observed in arabinose followed by α-methyl- D -glucoside, dulcitol, rhamnose, cellobiose, xylose, starch, lactose, sarbose, ribose and glycogen.     

Detection of Ralstonia solanacearum Strains with a Quantitative, Multiplex, Real-Time, Fluorogenic PCR (TaqMan) Assay

A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5′ nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to ≥10² cells ml⁻¹ was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.     

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60 °C and 4.0, respectively. The enzyme was stable for 1 h at 55 °C, showing a t₅₀ of 53 min at 60 °C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K_m of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).     

Geographic differences on accumulation of sugars and polyols in locust eggs in response to cold acclimation

The accumulation of low molecular weight sugars and polyols is one of major mechanisms hypothesized to increase cold tolerance in overwintering insects. But little is known about whether these sugars and polyols are involved in geographic variation of cold tolerance. In this study, we investigated accumulation patterns of eight low molecular weight sugars and polyols of eggs in tropical and temperate populations of the migratory locust, which exhibits between-population variation in cold tolerance, in response to cold acclimation (5, 0 and −5 °C). Excluding erythritol, the other seven carbohydrates were identified as possible cryoprotectants in locust eggs. Basal maximal and minimal concentrations were 45 μg/g wet weight for trehalose and 0.59 μg/g wet weight for glycerol. Most sugars and polyols were elevated after a −5 °C exposure. In a tropical population, fructose, glucose, sorbitol and myo-inositol were significantly accumulated by low temperature treatments, but glycerol was not. In the temperate population, glycerol, glucose, mannitol, sorbitol, myo-inositol were significantly accumulated but trehalose did not increase. Our results suggest different accumulation patterns of these carbohydrates of locust eggs between tropical and temperate populations and highlighted possible roles for them in geographic variation of cold tolerance in the migratory locust.     

Polygalacturonase Production by Agrobacterium tumefaciens Biovar 3

Agrobacterium tumefaciens biovar 3 causes both crown gall and root decay of grape. Twenty-two Agrobacterium strains representing biovars 1, 2, and 3 were analyzed for tumorigenicity, presence of a Ti plasmid, ability to cause grape seedling root decay, and pectolytic activity. All of the biovar 3 strains, regardless of their tumorigenicity or presence of a Ti plasmid, caused root decay and were pectolytic, whereas none of the biovar 1 and 2 strains had these capacities. Isoelectrically focused gels that were activity stained with differentially buffered polygalacturonate-agarose overlays revealed that all of the biovar 3 strains produced a single polygalacturonase with a pH optimum of 4.5 and pIs ranging from 4.8 to 5.2. The enzyme was largely extracellular and was produced constitutively in basal medium supplemented with a variety of carbon sources including polygalacturonic acid. Lesions on grape seedling roots inoculated with A. tumefaciens biovar 3 strain CG49 yielded polygalacturonase activity with a pI similar to that of the enzyme produced by the bacterium in culture. These observations support the hypothesis that the polygalacturonase produced by A. tumefaciens biovar 3 has a role in grape root decay.     

Random Genome Sequencing of Ralstonia solanacearum Strain IVIA 1602 and Comparative Analysis with Strain GMI1000

Ralstonia solanacearum is a β‐proteobacterium which affects several hundred plant species and provokes important agronomic losses. Five biovars of this bacterium have been described and they show behavioural differences. In this study a random sequencing of the genome of R. solanacearum strain IVIA 1602 (race 3, biovar 2), isolated from potato, was performed. The resulting 730 Genomic Survey Sequences (GSSs), representing 6.38% of the complete genome, were compared against the completely sequenced genome of strain GMI1000 (race 1, biovar 3), isolated from tomato, which is the only strain of this species sequenced until now. This comparative analysis showed, as expected, a high degree of similarity, but it also revealed strain‐specific regions of the genome as well as a number of insertion/deletion events and chromosomal rearrangements. All together, this comparative analysis gives an overview of the genomic divergence between these two biovars of the R. solanacearum species complex.     

Complete Genome Sequence of Brucella abortus Strain BCB034, a Strain of Biovar 2 Isolated from Human

Brucella abortus is divided into eight biovars, of which biovars 1 to 3 are the most frequently represented biovars in strains isolated from humans. Here, we report the genome sequence of B. abortus strain BCB034, a strain isolated from a human patient and that belongs to biovar 2.     

The effect of supplementation of cryopreservation diluents with sugars on the post-thawing fertility of ram semen

This study was conducted to elucidate the effect of increasing the osmolality of a basic Tris, extender supplemented with sucrose, trehalose or raffinose on post-thawing ram semen quality (sperm motility, viability, acrosome integrity, total sperm abnormalities and membrane integrity). After primary evaluation of the collected ejaculates, only semen samples with more than 70% motile sperm, and a sperm concentration of higher than 3 × 10⁹ sperm/ml were used for cryopreservation. The semen samples were pooled and diluted (1:4) with a Tris-citric acid-fructose-yolk extender, supplemented with different concentrations (50, 70 or 100 mM) of sucrose, trehalose or raffinose. As control, semen was diluted and frozen in the base diluent, without additional sugars. Pooled semen samples were aspirated into 0.25 ml straws, cooled to 5 °C within 90 min and frozen by exposure to liquid nitrogen vapor (4-5 cm above the liquid nitrogen surface) for 10 min - before plunging into liquid nitrogen, for storage. After 24 h, straws were thawed in a water bath (37 °C) for 30 s. The frozen-thawed sperm characteristics were improved significantly (P < 0.05) by increasing the level of the sugars. Optimal results being obtained with 70 and 100 mM trehalose or raffinose. All extenders containing supplemental sugars were superior in terms of sperm quality to the control (P < 0.01) group. The highest sperm motility (60.6 ± 1.9%), viability (60.6 ± 2.5%) and membrane integrity (58.2 ± 2.1%) were recorded using 100 mM trehalose and the lowest with 50 mM sucrose (48.6 ± 1.9%, 51.4 ± 2.5% and 47.9 ± 2.1%, respectively). All sugar concentrations decreased the percentage of acrosomal and total sperm abnormalities (P < 0.05). The extenders containing 100 mM trehalose or raffinose significantly (P < 0.05) decreased the occurrence of sperm abnormalities, compared to the other treatments. The fertility rates obtained after cervical insemination of the frozen-thawed sperm were 46.8%, 44.1% and 16.7% for 100 mM trehalose, 100 mM raffinose and the control with supplementation of the diluents, respectively. The study showed that ram sperm can tolerate hyperosmotic diluents, and that a range of sugar concentrations (50-100 mM) may successfully be incorporated in the ram semen cryopreservation diluents, although further research is warranted.     

Bacillus mojavensis strain 32A, a bioflocculant-producing bacterium isolated from an Egyptian salt production pond

Bacillus mojavensis strain 32A that exhibited 96.11% flocculation efficiency for clay suspensions was selected from other 15 comparative strains. Under growth condition, strain 32A was able to produce 5.2 g/L of purified biopolymer. Its constituent was mainly polysaccharide and protein with proportional of 98.4-1.6% respectively. FTIR spectrum was confirming its chemical analysis. This biopolymer attain very fast sedimentation rate. The cost-effective biopolymer and CaCl₂ dosages were 3 mg/L and 5 ml/L respectively that posed 89.7% flocculation efficiency. These dosages were suitable only for clay concentrations ?5 g/L. The maximum flocculation efficiency of the biopolymer recorded at pH 1.0 of clay suspension. The too high (>75 °C) or too low (<25 °C) clay suspension temperature was unfavorable for the biopolymer flocculation performance. The biopolymer solution utilized high thermal stability over the temperature range of 5-60 °C. Furthermore, its pH stability recorded at pH range of 5-9.     

Effects of trehalose-loaded liposomes on red blood cell response to freezing and post-thaw membrane quality

We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of trehalose into mammalian cells. This study focuses on the effects trehalose-containing liposomes improve the recovery and membrane quality of human RBCs following cryopreservation. Unilamellar liposomes consisting of a lipid bilayer composed of DPPC, PS and cholesterol (60:30:10 mol%) were synthesized using an extrusion method. Liposome-treated RBCs (l-RBCs) were resuspended in either physiological saline, 0.3 M trehalose or liposome solution, then cooled with slow (0.95 ± 0.02 °C/min), medium (73 ± 3 °C/min) and fast (265 ± 12 °C/min) cooling rates and storage in liquid nitrogen, followed by a 37 °C thawing step. RBC post-thaw quality was assessed using percent recovery, RBC morphology, PS and CD47 expression. Liposome treatment did not adversely affect the RBC membrane. Post-thaw recovery of l-RBCs was significantly higher (66% ± 5% vs 29% ± 4%) compared to control RBCs (c-RBC, p = 0.003). Medium and high cooling rates resulted in significantly higher cell recovery compared to a slow cooling rate (p = 0.039 and p = 0.041, respectively). The recovery of l-RBCs frozen in liposome solution and trehalose solution was significantly higher than that of l-RBCs frozen in NaCl solution for all three cooling rates (p = 0.021). Flow cytometry and morphology assessment showed that liposome treatment resulted in improved post-thaw membrane quality. There was no statistically significant difference in the post-thaw recovery between RBCs treated with liposomes containing trehalose in their aqueous core and RBCs treated with liposomes containing saline in their aqueous core (p = 0.114). Liposome treatment significantly improves the recovery and membrane integrity of RBCs following low temperature exposure.     

Tropical Strains of Ralstonia solanacearum Outcompete Race 3 Biovar 2 Strains at Lowland Tropical Temperatures

Bacterial wilt, caused by members of the heterogenous Ralstonia solanacearum species complex, is an economically important vascular disease affecting many crops. Human activity has widely disseminated R. solanacearum strains, increasing their global agricultural impact. However, tropical highland race 3 biovar 2 (R3bv2) strains do not cause disease in tropical lowlands, even though they are virulent at warm temperatures. We tested the hypothesis that differences in temperature adaptation and competitive fitness explain the uneven geographic distribution of R. solanacearum strains. Using three phylogenetically and ecologically distinct strains, we measured competitive fitness at two temperatures following paired-strain inoculations of their shared host, tomato. Lowland tropical strain GMI1000 was only weakly virulent on tomato under temperate conditions (24°C for day and 19°C for night [24/19°C]), but highland tropical R3bv2 strain UW551 and U.S. warm temperate strain K60 were highly virulent at both 24/19°C and 28°C. Strain K60 was significantly more competitive than both GMI1000 and UW551 in tomato rhizospheres and stems at 28°C, and GMI1000 also outcompeted UW551 at 28°C. The results were reversed at cooler temperatures, at which highland strain UW551 generally outcompeted GMI1000 and K60 in planta. The superior competitive index of UW551 at 24/19°C suggests that adaptation to cool temperatures could explain why only R3bv2 strains threaten highland agriculture. Strains K60 and GMI1000 each produced different bacteriocins that inhibited growth of UW551 in culture. Such interstrain inhibition could explain why R3bv2 strains do not cause disease in tropical lowlands.     

Physiological Role of β-Phosphoglucomutase in Lactococcus lactis

A β-phosphoglucomutase (β-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of β-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h⁻¹, while the deletion of β-PGM resulted in a maximum specific growth rate of 0.05 h⁻¹ on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as β-glucose 1-phosphate in the medium. Furthermore, the β-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of α-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the β-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded β-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.     

Characteristics of Ralstonia solanacearum Biovar N2 Strains in Asia

Ralstonia solanacearum biovar N2 strains isolated in Asia were compared by biochemical tests with biovar N2 strains from South America and biovar 2 (race 3) strains from Africa, America, Asia and Europe. Distinct differences were found between Asian and South American strains of biovar N2, and between Asian biovar N2 and biovar 2 strains with respect to their ability to utilize several carbon sources. Using cluster analysis based on repetitive sequence‐based polymerase chain reaction (rep‐PCR) genomic fingerprints, the Asian biovar N2 strains were divided into two groups, group 1 containing Japanese strains and group 2 containing Indonesian and Philippine strains. The fingerprints showed the genetic diversity of biovar N2 strains in Asia.     

The effect of dietary protein and carbohydrate concentration on the biochemical composition and gametogenic condition of the sea urchin Lytechinus variegatus

Previously starved urchins, Lytechinus variegatus, (36.0 ± 0.8 (SE) mm test diameter) were held in replicated (3) 10-L aquaria with artificial seawater at 22 ± 2  °C and 32‰ salinity and fed three diet treatments. Urchins were fed diets containing 9 : 35, 20 : 23 or 31 : 12% dry protein: % dry carbohydrate (P : C) ad libitum for a 65-day period. Gonads from urchins fed the 9 : 35 P : C diet had similar organic, lower ash, and lower water content than urchins fed the 31 : 12 P : C protein diet. Water content varied with both diet and nutritional history; consequently, water content may have limited value as a predictor of gonad nutritional status. Protein and carbohydrate concentrations in the gonad were directly related to the dietary composition of these nutrients; gonad lipids did not vary with diet. Excess carbohydrates are frequently stored as fats in fish and mammals but this does not appear to be the case in L. variegatus. Test carbohydrate storage and gut protein storage also reflected dietary composition. Image analysis of ovaries indicated decreased nutritive phagocyte volume, increased germinal epithelium volume and larger oocyte diameters in urchins fed high protein, low carbohydrate diets. Analysis of testes also indicated decreased nutritive phagocyte volume and increased gamete volume with urchins fed high protein, low carbohydrate diets, but differences among treatments were less obvious than in ovaries. This study suggests that high protein, low carbohydrate diets promote gamete growth and development. In addition, the biochemical and gametic composition of gonads can be altered by manipulating dietary composition. This could affect the quality and value of sea urchin roe for human consumption.     

Purification and characterization of a thiol amylase over produced by a non-cereal non-leguminous plant, Tinospora cordifolia

A 43 kDa α-amylase was purified from Tinospora cordifolia by glycogen precipitation, ammonium sulfate precipitation, gel filtration chromatography, and HPGPLC. The enzyme was optimally active in pH 6.0 at 60 °C and had specific activity of 546.2 U/mg of protein. Activity was stable in the pH range of 4-7 and at temperatures up to 60 °C. PCMB, iodoacetic acid, iodoacetamide, DTNB, and heavy metal ions Hg²⁺ > Ag⁺ > Cd²⁺ inhibited enzyme activity while Ca²⁺ improved both activity and thermostability. The enzyme was a thiol amylase (3 SH group/mole) and DTNB inhibition of activity was released by cysteine. N-terminal sequence of the enzyme had poor similarity (12-24%) with those of plant and microbial amylases. The enzyme was equally active on soluble starch and amylopectin and released maltose as the major end product.     

A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent) Strains of Ralstonia solanacearum

Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.