Persistence and recovery of introduced Rhizobium ten years after inoculation on Leucaena leucocephala grown on an Alfisol in southwestern Nigeria

Establishment of Leucaena leucocephala was poor at Ibadan (Transition forest-savanna zone) and Fashola (savanna zone, 70 km north of Ibadan) in southwestern Nigeria as a result of low soil fertility and the presence of only a few native rhizobia capable of nodulating it. Inoculation with L. leucocephala at these two locations in 1982 resulted in striking responses with Rhizobium strains IRc 1045 and IRc 1050 isolated from L. leucocephala grown in Nigeria. The persistence of inoculated effective Rhizobium strains after inoculation is desirable since it removes the need for reinoculation. Because of the perennial nature of L. leucocephala and its use in long-term alley farming experiments, we examined the persistence of inoculated rhizobial strains after inoculation, and their ability to sustain N₂-fixation and biomass production at Ibadan. In 1992, ten years after Rhizobium introduction, uninoculated, L. leucocephala fixed about 150 kg N ha^-1 yr^-1 or about 41% of total plant N compared to 180 kg N ha^-1 yr^-1 or 43% measured in 1982. Serological typing of the nodules using the Enzyme-Linked-Immunosorbent Assay (ELISA) and intrinsic resistance to the streptomycin test revealed that most of the nodules (96%) formed on L. leucocephala in 1992 were by Rhizobium strains IRc 1045 and IRc 1050, which were inoculated in 1982. Nodules were absent on uninoculated L. leucocephala grown on the adjacent field with no history of L. leucocephala cultivation. We conclude that the N₂ fixed by Rhizobium strains IRc 1045 and IRc 1050 persisted for many years in the absence of L. leucocephala and sustained effectively fixed N₂ which growth and yield of L. leucocephala after several years, thus encouraging a possible low-input alley farming system by smallholder farmers in Nigeria.     

Comparison of N(2) Fixation and Yields in Cajanus cajan between Hydrogenase-Positive and Hydrogenase-Negative Rhizobia by In Situ Acetylene Reduction Assays and Direct N Partitioning

Pigeon peas [Cajanus cajan (L.) Millsp.] were grown in soil columns containing ¹⁵N-enriched organic matter. Seasonal N₂ fixation activity was determined by periodically assaying plants for reduction of C₂H₂. N₂ fixation rose sharply from the first assay period at 51 days after planting to a peak of activity between floral initiation and fruit set. N₂ fixation (acetylene reduction) activity dropped concomitantly with pod maturation but recovered after pod harvests. Analysis of ¹⁵N content of plant shoots revealed that approximately 91 to 94% of plant N was derived from N₂ fixation. The effect of inoculation with hydrogenase-positive and hydrogenase-negative rhizobia was examined. Pigeon peas inoculated with strain P132 (hydrogenase-positive) yielded significantly more total shoot N than other inoculated or uninoculated treatments. However, two other hydrogenase-positive strains did not yield significantly more total shoot N than a hydrogenase-negative strain. The extent of nodulation by inoculum strains compared to indigenous rhizobia was determined by typing nodules according to intrinsic antibiotic resistance of the inoculum strains. The inoculum strains were detected in almost all typed nodules of inoculated plants.

Gas samples were taken from soil columns several times during the growth cycle of the plants. H₂ was never detected, even in columns containing pigeon peas inoculated with hydrogenase-negative rhizobia. This was attributed to H₂ consumption by soil bacteria. Estimation of N₂ fixation by acetylene reduction activity was closest to the direct ¹⁵N method when ethylene concentrations in the gas headspace (between the column lid and soil surface) were extrapolated to include the soil pore space as opposed solely to measurement in the headspace. There was an 8-fold difference between the two acetylene reduction assay methods of estimation. Based on a planting density of 15,000 plants per hectare, the direct ¹⁵N fixation rates ranged from 67 (noninoculated) to 134 kilograms per hectare, while grain yields ranged from 540 to 825 kilograms per hectare. Grain yields were not increased with N fertilizer.

     

Short communication: Screening of native and foreign Bradyrhizobium japonicum strains for high N2 fixation in soybean

Four local rhizobia isolates selected after two screening experiments and five USDA Bradyrhizobium japonicum strains were estimated for N₂ fixation in soybean using the ¹⁵N isotope dilution technique. Strain USDA 110 was superior to the local isolates in nodulation and N₂ fixation when inoculated onto soybean cv TGX 1497-ID in a Nigerian soil and could therefore be used as an inoculant for enhanced N₂ fixation in soybean in Nigeria.     

Classification des souches du groupeBacillus thuringiensis par la détermination de l'antigène flagellaire

Summary The serological study of 26 new strains ofBacillus thuringiensis (of which the biochemical features are also given) makes it possible to classify these strains into: 1^o Strains which are in concordance with the six serotypes previously described. 2^o Strains which have a new H antigen. Here, we describe two new serotypes: serotype 7 (aizawai), serotype 8 (morrisoni). On the other hand, the serological study of five new strains ofB. thuringiensis belonging to serotype 4 shows that the H₄ antigen must be divided into ?sub-factors?: ?4 a, 4b? to be found in the strains sotto, dendrolimus, T.84-A, L (Grig) and ?4 a, 4 c? to be found in the strains Pil 94, 1748 and Rhodesia. Table 6 gives the present statute of theB. thuringiensis strains' classification by the flagellar agglutination technic.

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Avec la collaboration technique deM. Lechevallier etT. Le Borgne. Nous remercions vivement les collègues qui ont bien voulu nous adresser des cultures et nous nous tenons à la disposition de tous ceux qui seraient intéresés par la détermination de l'antigène H de leurs souches. Nous remercions également notre collègueLe Minor pour ses précieux conseils.     

Assessment of genetic variability for N2 fixation between and within provenances of Leucaena leucocephala and Acacia albida estimated by 15N labelling techniques

Nitrogen fixed in 13 provenances of Acacia albida and 11 isolines of Leucaena leucocephala inoculated with effective Rhizobium strains was measured by ¹⁵N techniques and the total N difference method. In the test soil, on the average, L. leucocephala derived about 65% of its total N from atmospheric N₂ fixation compared to about 20% by A. albida. Significant differences in the percentage of N derived from atmospheric N₂ (% Ndfa) occurred, between provenances or isolines within species. The % Ndfa ranged from 37 to 74% within L. leucocephala and from 6 to 37 within A. albida; (equivalent to 20–50 mg N plant^–1 and 4–37 mg N plant^–1 for the two species over three months, respectively) and was correlated with the nodule mass (r=0.91). The time course of N₂ fixation of three selected provenances (low, intermediate and good fixers) was followed at 12 weekly intervals over a 36 week period. The % Ndfa of all provenances and isolines increased with time; and except for one of the L. leucocephala provenances, % Ndfa was similar within species at the 36 weeks harvest. There was a significant correlation between % Ndfa and the amount of N₂ fixed (r=0.96). Significant interactions occurred between provenances and N treatments and often growth of uninoculated but N fertilized plants was less variable than for inoculated unfertilized plants.     

Identification and Characterization of Nucleotide Sequence Differences in Three Virulence-Associated Genes of Listeria monocytogenes Strains Representing Clinically Important Serotypes

Listeria monocytogenes is a Gram-positive, facultative intracellular bacterium that causes invasive, often fatal, disease in susceptible hosts. As a foodborne pathogen, the bacterium has emerged as a significant public health problem and has caused several epidemics in the United States and Europe. Three serotypes (1/2a, 1/2b, 4b) of L. monocytogenes are responsible for nearly 95% of all reported cases of human listeriosis. L. monocytogenes serotype 4b has caused all well-characterized foodborne epidemic outbreaks in North America and Europe between 1981 and 1993. However, most of the genetic studies to characterize virulence factors of L. monocytogenes have been done by using serotypes 1/2a and 1/2c. In this investigation, we examined three virulence-associated genes (hly encoding listeriolysin, plcA encoding phosphotidylinositol-specific phospholipase C, and inlA encoding internalin) of two serotype 4b and two serotype 1/2b strains. We chose these virulence-associated genes on the basis of published sequence differences among strains from Listeria subgroups containing serotypes 1/2a and 1/2c versus 4b, respectively. They correspond to sequence homologies that include very highly conserved (hlyA), highly conserved (plcA) and mostly conserved (inlA). We found by using nucleotide sequence analysis of the hly, plcA, and inlA genes, the two L. monocytogenes strains (including a strain associated with a foodborne disease outbreak in California in 1985) in this study, two serotype 1/2b strains from a study that we recently reported, and other similar published data for serotypes 1/2a, 1/2c, and 4b, had a high degree of sequence conservation at the gene and protein levels for all three genes. However, the sequences for the hly gene of L. monocytogenes strains of serotypes 1/2b and 4b were more closely related to each other and showed significant divergence from serotypes 1/2a and 1/2c. A unique nonsynonymous mutation was found in the hly gene of L. monocytogenes isolates that were associated with the 1985 California outbreak and were the epidemic phage type. When 158 L. monocytogenes isolates from the collection at the Centers for Disease Control and Prevention were screened, the mutation was found only in one other strain that had been isolated in California 3 years before the epidemic. Although the California epidemic clone was lactose negative, other L. monocytogenes serotype 4b isolates that were lactose negative did not possess the unique mutation observed in that epidemic clone. Received: 18 June 1997 / Accepted: 4 December 1997     

Structural variation in the O-specific polysaccharides of Klebsiella pneumoniae serotype O1 and O8 lipopolysaccharide: evidence for clonal diversity in rfb genes

The O-polysaccharide fraction of the lipopolysaccharide from Klebsiella pneumoniae serotype O8 was found to comprise two galactose-containing homopolymers. Structural analysis, using chemical and high-field nuclear magnetic resonance (NMR) techniques, established that the K. pneumoniae O8 polysaccharides are composed of the linear, disaccharide repeating units OAc 1 2/6 →3)-β-d -Galf-(1 →3)-α- d -Galp-(1→d -Galactan I-OAc →3)-α-d -Galp-(1 →3)-β-d -Galp-(1→d -Galactan II. K. pneumoniae O8 mutant RFK-1 was isolated by resistance to phage KO1-2; strain RFK-1 expressed only d -galactan I-OAc. The ¹H- and ¹³C-NMR resonances from this O-polysaccharide indicate that all of the O-acetyl groups within the K. pneumoniae O8 polysaccharide are carried on d -galactan I and O-acetylation occurs only on the β- d -galactofuranose residues; 60% of the available β- d -galactofuranose residues are non-acetylated. The O-acetylation of the remaining residues is equally distributed between the O-2 and O-6 positions. The carbohydrate backbone structures in the O8 polysaccharide are identical to d -galactan I and II expressed by K. pneumoniae O1, accounting for the antigenic cross-reaction between strains belonging to serotypes O1 and O8. However, the O1 polysaccharides are not acetylated and the O-acetyl groups present in the K. pneumoniae serotype O8 polysaccharides provide a structural basis for their recognition as distinct serotypes. The rfb (O-polysaccharide biosynthesis) gene cluster of K. pneumoniae serotype O1 determines the synthesis of d -galactan I. rfb_Kpo1-specific gene probes were used to examine conservation in the rfb gene clusters of other K. pneumoniae serotypes which produce d -galactan I. Six O1 strains were examined and all showed hybridization with rfb_KpO1 probes under conditions of high stringency. Three serotype O2 strains produce d -galactan I and these strains also contained DNA sequences recognized by rfb_KpO1 probes under high stringency. The physical maps of these homologous rfb chromosomal regions showed some polymorphism. Surprisingly, the rfb_KpO8 region from K. pneumoniae serotype O8 was only recognized by rfb_KpO1 probes under low-stringency hybridization conditions, providing evidence for two substantially different clonal groups of rfb genes from K. pneumoniae strains with structurally related O-antigens.     

Field evaluation of symbiotic nitrogen fixation by rhizobial strains using15N methodology

Summary Small differences in N₂ fixation by nodulated soybeans (Glycine max. (L.) Merr.), inoculated with various strains ofRhizobium japonicum, were assessed in field experiments using¹⁵N methodology, and compared with yields of plant dry matter and total N. Percentage of plant-N derived from atmospheric N₂ and from fertilizer, and values of %¹⁵N atom excess had lower coefficients of variation than did total N and dry matter yield. Nevertheless the precision of estimates of kg N/ha fixed were sufficient to differentiate only the extremes of the range of strains tested, and there were discrepancies between ranking of strains based on % N derived from fertilizer and on total N yield.     

The growth limits of a large number of Listeria monocytogenes strains at combinations of stresses show serotype--and niche-specific traits

Aims: The aim of this study was to associate the growth limits of Listeria monocytogenes during exposure to combined stresses with specific serotypes or origins of isolation, and identify potential genetic markers. Methods and Results: The growth of 138 strains was assessed at different temperatures using combinations of low pH, sodium lactate, and high salt concentrations in brain heart infusion broth. None of the strains was able to grow at pH ≤ 4·4, a_w ≤ 0·92, or pH ≤ 5·0 combined with a_w ≤ 0·94. In addition, none of the strains grew at pH ≤ 5·2 and NaLac ≥ 2%. At 30°C, the serotype 4b strains showed the highest tolerance to low pH and high NaCl concentrations at both pH neutral (pH 7·4) and mild acidic conditions (pH 5·5). At 7°C, the serotype 1/2b strains showed the highest tolerance to high NaCl concentrations at both pH 7·4 and 5·5. Serotype 1/2b meat isolates showed the highest tolerance to low pH in the presence of 2% sodium lactate at 7°C. ORF2110 and gadD1T1 were identified as potential biomarkers for phenotypic differences. Conclusions: Differences in growth limits were identified between specific L. monocytogenes strains and serotypes, which could in some cases be associated with specific genetic markers. Significance and Impact of the Study: Our data confirm the growth limits of L. monocytogenes as set out by the European Union for ready-to-eat foods and provides an additional criterion. The association of L. monocytogenes serotypes with certain stress responses might explain the abundance of certain serotypes in retail foods while others are common in clinical cases.     

Comparative study of the growth of two strains ofNitrobacter in batch and continuous culture

Growth kinetics of 2Nitrobacter strains (N.w. and L) coexisting in the same soil are studied in batch and continuous culture. Monod's parameters are estimated numerically from experimental data in the case of the batch experiment, and from steady-state equations in the case of the chemostat. In both cases, the 2 strains show different values for their growth parameters. N.w. may be characterized by its high _max-K_s values, relative to strain L. But for each strain, _max is significantly lowered between batch and continuous culture. In this latter case, at N-NO₂ ^– concentrations less than 1.5g·ml^–1, the 2 strains exhibit similar growth rates showing that for concentrations of the limiting substrate prevailing in the soil, they may compete for this substrate.     

The effect of inoculation with Azospirillum brasilense on growth and nitrogen utilization by wheat plants

Azospirillium brasilense is a rhizosphere bacteria that has been reported to improve yield when inoculated on wheat plants. However, the mechanisms through which this effect is induced is still unclear. In the present work, we have studied the effects of inoculating a highly efficient A. brasilense strain on wheat plant grown in 5 kg pots with soil in a greenhouse, under three N regimes (0, 3 or 16 mM NO₃ ^–, 50 ml/pot once or twice-a -week), and in disinfected or non-disinfected soil. At the booting stage, the inoculated roots in both soils showed a similar colonization by Azospirillum sp. that was not affected by N addition. The plants grown in the disinfected soil showed a higher biomass, N content and N concentration than those in the non-disinfected soil, and in both soils the inoculation stimulated plant growth, N accumulation, and N and NO₃ ^– concentration in the tissues.At maturity, the inoculated plants showed a higher biomass, grain yield and N content than the uninoculated ones in both soils, and a higher grain protein concentration than the uninoculated. It is concluded that in the present experiments, A. brasilenseincreased plant growth by stimulating nitrogen uptake by the roots.     

The Role of the <Emphasis Type="Italic">sigB</Emphasis> Gene in the General Stress Response of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> Varies between a Strain of Serotype 1/2a and a Strain of Serotype 4c

The ability of Listeria monocytogenes to resist many adverse environmental conditions has been attributed in part to activation of the alternative sigma factor ς^B, encoded by the sigB gene. The ability of this pathogen to survive and grow under stress conditions varies between strains within the species. The current study was undertaken to determine whether the role played by the sigB gene in the stress response varies among strains of different serotypes. Null mutations were generated in the sigB genes of L. monocytogenes L61 (serotype 1/2a) and L99 (serotype 4c), and the survival of the resulting mutants was compared with that of the wild-type strains under osmotic, oxidative, and carbon starvation stress conditions and on exposure to bacteriocins, ethanol, acid, and heat. Except in a few cases, strain L61 displayed greater dependence on the sigB products for survival of adverse conditions than did strain L99. The results of this study indicated that the relative importance of the sigB gene in the stress response is not the same in all strains of L. monocytogenes, and this difference may be specific to serotype groupings within the species. Received: 8 May 2002 / Accepted: 27 August 2002     

Early seedling and seasonal N2-fixing symbiotic activity of two soybean [Glycine max (L.) Merr.] cultivars inoculated with Bradyrhizobium strains of diverse origin

In areas with a short growing season the poor adaptability of soybean [Glycine max Meer. (L.)] to cool soil conditions is considered the primary yield limiting factor. Soybean requires temperatures in the 25 to 30°C range for optimum N₂-fixation and yield. Field studies were conducted in 1990 and 1991 at Montreal, Quebec to determine whether adaptability to cool soil conditions, with respect to earlier symbiosis establishment and function, existed among either Bradyrhizobium strains or soybean genotypes. An early maturing isoline of the soybean cultivar Evans and the cultivar Maple Arrow were inoculated with one of four strains isolated from the cold soils of Hakkaido, northern Japan, or the commercially used strains 532C or USDA110, at two planting dates. Plot biomass and nodulation were assessed at seedling (V2), and flowering(R2) growth stages and harvest maturity. Soybean genotypes did not differ for pre-flowering nodulation or N₂-fixation in the cool spring conditions of the first year. Seasonal N₂-fixation rates were also determined at the final harvest by the N-balance and ¹⁵N-isotope dilution methods. Significantly higher symbiotic activity was found for two of the four Hakkaido strains and was reflected in higher final soybean seed yield and total N₂-fixation for the growing season, as compared to the two commercial strains. Planting 14 days earlier resulted in greater early vegetative and total seasonal N₂ fixation and yield in the second year when soil temperatures were warmer, emphasizing the need for the development of soybean-Bradyrhizobium combinations superior in nodule development and function under cool soil conditions.     

Characterization of Bacillus thuringiensis ser. balearica (Serotype H48) and ser. navarrensis (Serotype H50): Two Novel Serovars Isolated in Spain

The novel strains of Bacillus thuringiensis PM9 and NA69, isolated from soil samples in Spain, were classified and characterized in terms of their crystal proteins, plasmid profile, cry genes content, and their toxicological properties against several species of Lepidoptera, Coleoptera, and Diptera. Both strains share morphological and biochemical characteristics with previously described B. thuringiensis strains, although their unique H antigens identify them as two new serotypes. Two new serovar names, B. thuringiensis serovar balearica (H serotype 48) and B. thuringiensis serovar navarrensis (H serotype 50) are proposed for the type strains PM9 and NA69, respectively. Received: 22 June 1999 / Accepted: 2 August 1999     

Hydrogen emission from nodulated soybeans [Glycine max (L.) Merr.] and consequences for the productivity of a subsequent maize (Zea mays L.) crop

Hydrogen (H₂) is a by-product of the symbiotic nitrogen fixation (N₂ fixation) between legumes and root-nodule bacteria (rhizobia). Some rhizobial strains have an uptake hydrogenase enzyme (commonly referred to as Hup⁺) that recycles H₂ within the nodules. Other rhizobia, described as Hup^?, do not have the enzyme and the H₂ produced diffuses from the nodules into the soil where it is consumed by microorganisms. The effect of this phenomenon on the soil biota and on the soil itself, and consequent stimulation of plant growth, has been demonstrated previously. Soybeans [Glycine max (L.) Merr.] cv. Leichhardt, inoculated with either a Hup⁺ strain (CB1809) or one of two Hup^? strains (USDA442 or USDA16) of Bradyrhizobium japonicum and uninoculated soybeans, plus a non-legume control [capsicum (Capsicum annuum L.)] were grown in the field at Ayr, North Queensland, Australia. The objectives were to examine (1) relationships between N₂ fixation and H₂ emission, and (2) the influence H₂-induced changes in soil might have during the legume phase and/or on the performance of a following crop. Strains CB1809 and USDA442 were highly effective in N₂ fixation (“good” fixers); USDA16 was partly effective (“poor” fixer). The soil had a large but non-uniformly distributed naturalised population of B. japonicum and most uninoculated control plants formed nodules that fixed some N₂. These naturalised strains were classified as “poor fixers” of N₂ and were Hup⁺. H₂ emissions from nodules were assessed for all treatments when the soybean crop was 62 days old. Other parameters of symbiotic N₂ fixation and plant productivity were measured when the crop was 62 and 96 days old and at crop maturity. Immediately after final harvest, the land was sown to a crop of maize (Zea mays L.) in order to determine the consequences of H₂ emission from the soybean crop on maize growth. It was estimated that soybeans inoculated with USDA442, the highly effective Hup^– strain of B. japonicum, fixed 117 kg shoot N/ha (or about 195 kg total N/ha if the fixed N associated with roots and nodules was taken into account), and contributed about 215,000 l H₂ gas per hectare to the ecosystem over the life of the crop. The volume of H₂ evolved from soybeans nodulated by the Hup⁺ strain CB1809 was only 6% of that emitted by the USDA442 treatment, but there was no indication that soybean inoculated with USDA442 benefited from the additional H₂ input. The shoot biomass, grain yield, and amounts of N fixed (105 kg shoot N/ha, 175 kg total N/ha) by the CB1809 treatment were little less than for USDA442 plants. Three days after the soybean crop was harvested, the plots were over-sown with maize along the same row lines in which the soybeans had grown. This procedure exposed the maize roots to whatever influence soybean H₂ emission might have had on the soil and/or the soil microflora immediately surrounding soybean nodules. The evidence for a positive effect of soybean H₂ emission on maize production was equivocal. While the consistent differences between those pre-treatments that emitted H₂ and those that did not indicated a trend, only one difference (out of the 12 parameters of maize productivity that were measured) was statistically significant at P?<?0.05. The findings need substantiation by further investigation.     

Effect of inoculation withKlebsiella oxytoca andEnterobacter cloacae on dinitrogen fixation by rice-bacteria associations

The influence ofKlebsiella oxytoca andEnterobacter cloacae inoculation on dinitrogen fixation by the rice-bacteria association was examined in pots in a greenhouse. For inoculation,K. oxytoca NG13 isolated from a Japan paddy soil,E. cloacae E26 isolated from a China soil and following three strains were employed.K. oxytoca NG1389 is a mutant from NG13 and has no nitrogenase activity (nif). K. oxytoca NG13/pMC71A andE. cloacae E26/pMC71A were produced by inserting anif A containingK. pneumoniae plasmid (pMC71A) to NG13 and E26, respectively. These two strains were able to fix dinitrogen fixation in the presence of ammonium, whereas nitrogenase activity of wild strains (NG13 and E26) were repressed under this condition. Inoculation effects were tested on two rice (Oryza sativa L.) varieties, a Indica type C5444 and a Japonica type T65. Rice seedlings were planted to nonsterilized potted soil, and grown under flooded conditions. Upon inoculation with NG13 and E26, growth and N increment of plants particularly in T65 were stimulated above NG1389 inoculated plants. Assay by¹⁵N dilution and acetylene reduction techniques indicated that this N increment may be due to fixed N. Inoculation with NG13/pMC71A and E26/pMC71A resulted in more dry weight and fixed N than those of NG13 and E26 inoculated plants. Dr Y. Hirota died on 23 December 1986.     

苏北地区规模化猪场产肠毒素大肠杆菌的分离鉴定及灭活疫苗效果评估

[目的]产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)是引起仔猪腹泻的重要病原菌,本研究通过调查苏北地区规模化猪场ETEC的流行情况,分析其生物学特性,研制具有免疫保护效果的优势血清型菌株的灭活疫苗,以期对苏北地区ETEC的防控提供参考。[方法]从苏北地区规模化猪场采集3-30日龄的仔猪新鲜粪样、肛拭子及小肠组织样,分离出ETEC,对分离菌株进行血清型鉴定、耐药性测定、小鼠致病力测定;最后通过动物免疫试验研究优势血清型菌株灭活疫苗对小鼠的免疫保护效果。[结果]从21个规模化猪场采集病料562份,通过PCR鉴定及测序得到141株ETEC;血清凝集试验鉴定出85株菌的O抗原血清型,其中08、0101和0128为优势血清型,占定型菌株的61.2%(52/85),其他血清型包括09、03、020、0148、0149等;分析141株ETEC对14种常见抗生素的耐药情况,得出分离株对新霉素、红霉素、四环素、庆大霉素、强力霉素、阿莫西林、甲氧苄啶/磺胺甲恶唑高度耐药,耐药率均高达80%以上;对恩诺沙星敏感性较高,敏感率达50.4%(71/141);对多粘菌素B和头孢噻肟中介耐药,占比分别为66%(93/141)和51.8%(73/141);多重耐药现象严重,其中10重耐药的菌株占比最大,为19%(27/141);小鼠攻毒试验测得08血清型强毒株YC-6的半数致死量(median lethal dose,LD50)为1.4×10^7 CFU/只,最低致死量(minimum lethal dose,MLD)为3×10^7 CFU/只;08血清型强毒株YC-6和0101血清型强毒株LYG-3制备的单价灭活疫苗对小鼠的保护率均达到100%,因此利用08血清型强毒株YC-6和0101血清型强毒株LYG-3研制二价灭活疫苗,结果显示该二价疫苗对感染不同血清型ETEC小鼠的保护率在83%以上。[结论]本研究通过对苏北地区ETEC的流行病学调查,得出其优势血清型,并研制出针对对优势血清型免疫保护效果较好的二价灭活疫苗,给临床ETEC的监测和防控提供参考。     

Autecology in Rhizospheres and Nodulating Behavior of Indigenous Rhizobium trifolii

Indigenous serotype 1-01 of Rhizobium trifolii occupied significantly fewer nodules (6%) on plants of soil-grown noninoculated subterranean clover (Trifolium subterraneum L.) cv. Woogenellup than on cv. Mt. Barker (36%) sampled at the flowering stage of growth. Occupancy by indigenous serotype 2-01, was not significantly different on the two cultivars (16 and 26%). Serotype-specific, fluorescent-antibody conjugates were synthesized and used to enumerate the indigenous serotypes in host (clovers) and nonhost (annual rye-grass, Lolium multiflorum L.) rhizospheres and in nonplanted soil. The form and concentration of Ca²⁺ in the flocculating mixture and the presence of phosphate anions in the extracting solution were both critical for enumerating R. trifolii in Whobrey soil. The two serotypes were present in similar numbers in nonplanted soil (ca. 10⁶ per g of soil) and each represented ca. 10% of the total R. trifolii population. Although host rhizospheres did not preferentially stimulate either serotype, the mean population densities of serotype 2-01 were significantly greater (P = 0.05) than those of serotype 1-01 in clover rhizospheres on 8 of 14 samplings made between the time of seeding and the appearance of nodules (day 12). In this experiment, and in contrast to our earlier findings, serotype 1-01 occupied significantly fewer (P ≤ 0.05) of the nodules (7 to 16%) on both cultivars than serotype 2-01 (51%) when sampled at 4 weeks. Differences between cultivars became apparent as the plants matured. There was a threefold increase (7 to 21%) in nodules occupied by serotype 1-01 on cv. Mt. Barker between 4 and 16 weeks. This was accompanied by increases in nodules coinhabited by both nonidentifiable occupants and either serotype 1-01 (0 to 20%) or 2-01 (11 to 51%). No increases in either of these parameters were observed on cv. Woogenellup.     

Establishment of phosphate-solubilizing strains of Azotobacter chroococcum in the rhizosphere and their effect on wheat cultivars under green house conditions

A pot experiment was conducted in the green house to investigate the establishment of phosphate solubilizing strains of Azotobacter chroococcum, including soil isolates and their mutants, in the rhizosphere and their effect on growth parameters and root biomass of three genetically divergent wheat cultivars (Triticum aestivum L.). Five fertilizer treatments were performed: Control, 90 kg N ha^—1, 90 kg N + 60 kg P₂O₅ ha^—1, 120 kg N ha^—1 and 120 kg N + 60 kg P₂O₅ ha^—1. Phosphate solubilizing and phytohormone producing parent soil isolates and mutant strains of A. chroococcum were isolated and selected by an enrichment method. In vitro phosphate solubilization and growth hormone production by mutant strains was increased compared with soil isolates. Seed inoculation of wheat varieties with P solubilizing and phytohormone producing A. chroococcum showed better response compared with controls. Mutant strains of A. chroococcum showed higher increase in grain (12.6%) and straw (11.4%) yield over control and their survival (12—14%) in the rhizosphere as compared to their parent soil isolate (P4). Mutant strain M37 performed better in all three varieties in terms of increase in grain yield (14.0%) and root biomass (11.4%) over control.     

Identification of surface protective antigen (spa) types in Erysipelothrix reference strains and diagnostic samples by spa multiplex real‐time and conventional PCR assays

Aim: To develop spa multiplex real‐time and conventional PCR assays to detect and differentiate between spaA, spaB and spaC genes within Erysipelothrix spp. Methods and Results: For evaluation of the assays, 28 Erysipelothrix spp. reference strains, 25 tissues from pigs inoculated with reference strains of serotypes 1, 2, 5, 10 or 18, and 15 diagnostic samples were used. SpaA was found to be present in Erysipelothrix rhusiopathiae serotypes 1a, 1b, 2, 5, 9, 12, 15, 16, 17, 23 and N; spaB was detected in E. rhusiopathiae serotypes 4, 6, 8, 11, 19 and 21 and spaC was detected in E. sp. strain 2 serotype 18. Spa‐related genes were not detected in E. tonsillarum strains (serotypes 3, 7, 10, 14, 20, 22, 24, 25, 26) or E. sp. strain 1 (serotype 13). With the spa multiplex real‐time PCR assay, it was also possible to further differentiate spaB into spaB1 (serotypes 4, 6, 8, 19 and 21) and spaB2 (serotype 11). Overall, spaA was detected in seven experimental tissue samples and six diagnostic tissue samples, and spaC in two experimental tissue samples. The detection limits were determined to be five colony‐forming units (CFU) per reaction for the spa multiplex real‐time PCR assay and 4000 CFU per reaction for the conventional PCR assay. Conclusions: Both spa PCR assays were specific and reproducible in the identification of spa types in Erysipelothrix spp. Significance and Impact of the Study: The described spa PCR assays may be useful tools for investigating spa prevalence among strains isolated from field tissues and to determine the role of the Spa proteins in vaccine protection and pathogenesis.