Assessment of the genetic diversity of Xylella fastidiosa isolated from citrus in Brazil by PCR-RFLP of the 16S rDNA and 16S-23S intergenic spacer and rep-PCR fingerprinting

The genetic diversity among twenty three strains of Xylella fastidiosa, isolated from sweet orange citrus, was assessed by RFLP analysis of the 16S rDNA and 16S-23S intergenic spacer and by rep-PCR fingerprinting together with strains isolated from coffee, grapevine, plum and pear. The PCR products obtained by amplification of the 16S rDNA and 16S-23S spacer region were digested with restriction enzymes and a low level of polymorphism was detected. In rep-PCR fingerprinting, a relationship between the strains and their hosts was observed by using the BOX, ERIC and REP primers. Two major groups were obtained within the citrus cluster and relationships to the geographic origin of the strains revealed. Citrus strains isolated from the States of São Paulo and Sergipe formed one group and strains from the Southern States formed another group. Distinct origins of X. fastidiosa in the Southern and Southeastern States is postulated. The pear isolate was distantly related to all of the other X. fastidiosa strains.     

Assessment of genetic diversity of Iranian Ascochyta rabiei isolates using rep‐PCR markers

Genetic diversity and population structure among 29 isolates of Ascochyta rabiei (AR) obtained from diseased chickpea plants in six different geographical origins in Iran was characterized by MAT and rep‐PCR (BOX/ERIC/REP) markers. Both mating types were found in all six populations, and the frequencies of mating types were variable between populations. The majority of the isolates belonged to Mat1‐1 (58.12%) with the remainder (41.88%) being Mat1‐2. A dendrogram was calculated with Jaccard's similarity coefficients with unweighted pair group method clustering (UPGMA) for the combination of rep‐PCR results, AR strains were differentiated into four clusters (A–D) at 60% similarity level. ERIC, REP and BOX showed a total of 19, 37 and 24 alleles per locus, respectively. Gene diversity (He) and Shannon's information index (I) were the highest in the REP (He = 0.82; I = 2.11), while the lowest values were estimated for the ERIC (He = 0.42; I = 1.3). Our result showed that among the three techniques studied, REP‐PCR produced the most complex amplified banding patterns, which reflected a high degree of diversity among the Iranian AR strains. ERIC‐PCR was the least discriminating method, and BOX‐PCR was intermediate. To the best our knowledge, this is first study of assessment of genetic diversity of AR isolates by rep‐PCR markers.     

Comparison of three molecular methods for typing Aeromonas popoffii isolates

Three typing methods, restriction fragment length polymorphism (RFLP) of the 16S-23S intergenic spacer region (ISR), PCR amplification of the enterobacterial repetitive intergenic consensus (ERIC) and of the repetitive extragenic palindromic units (REP), were evaluated for typing 26 isolates of Aeromonas popoffii from different geographical origins. When the methods were independently studied, ERIC showed the highest discriminatory power. When the methods were combined, the best combination of two methods was ERIC with REP since strains showed a tendency to cluster according to their geographical origin. However, this tendency was reinforced with the addition of ISR-RFLP. This revised version was published online in June 2006 with corrections to the Cover Date.     

Assessment of genetic relatedness among commercial and wild strains of Agaricus bisporus using repetitive extragenic palindromic sequences and polymerase chain reaction

The genetic relatedness among 18 strains of Agaricus bisporus was assessed based on the fragment pattern analysis obtained by the amplification of genomic DNA by BOX, ERIC (ERICIR-I/ERIC2) and REP (REP1RI/REP2I) gene sequences. Based on the banding patterns of PCR-amplified products, eight putative groups among the 18 commercial and wild strains were recognized. REP-PCR generated multiple distinct products showing considerable variability among the strains with ERIC and REP elements successfully enabled detection of wild and commercial A. bisporus. Strains originating from the same geographical location were not always genetically related. To our knowledge, this was the first relevance study of biodiversity in commercial and native populations of A. bisporus by using the REP-PCR technique. The results confirmed the usefulness REP-PCR typing in intraspecific genetic variation assessments of the button mushroom. High level of Iranian wild strains distance with the commercial cultivars approves their importance as a promising new source of diversity in A. bisporus breeding program.     

用rep—PCR技术研究中国花生根瘤菌的多样性

采用细菌基因组重复序列ＰＣＲ技术（简称ｒｅｐＰＣＲ）中常用的ＲＥＰＰＣＲ和ＥＲＩＣＰＣＲ,对从中国１１个省、市的２３个点、２４个花生品种采集的根瘤中分离的５９株花生根瘤菌Ｂｒａｄｙｒｈｉｚｏｂｉｕｍｓｐ．（Ａｒａｃｈｉｓ）进行多样性研究,同时对来自国外的６株花生根瘤菌及１４株参比慢生根瘤菌也进行了比较。得到的低相似性结果表明中国花生根瘤菌基因组存在显著的多样性。ＲＥＰＰＣＲ揭示,在相似性５０％上分为１１个群,而ＥＲＩＣＰＣＲ却得到２４个分群。这两种结果对菌株的分群有差异,暗示这两种短重复序列在慢生根瘤菌基因组中的分布的不同。没有发现菌株间基因组的多样性分布与花生品种、地理来源之间的必然联系。将两者电泳图谱结合并分析,得到介于上述两者间的结果。此结果进一步反映了菌株基因组间存在的多样性。同时还表明ｒｅｐＰＣＲ不仅是研究生物多样性的快速简便方法,还可应用于菌株的鉴别和生态学研究。     

Rapid identification of Salmonella typhimurium, S. enteritidis and S. virchow isolates by polymerase chain reaction based fingerprinting methods.

In this study we used and evaluated three rapid molecular typing methods for the identification of three frequent, clinically significant Salmonella serovars on the basis of the ease, simplicity and reproducibility of the chosen methods. We determined the genetic diversity among several isolates of Salmonella enteritidis, S. typhimiurium and S. virchow, and compared them with other enterobacteria by using the repetitive extragenic palindromic (REP) sequences, the enterobacterial repetitive intergenic consensus (ERIC) sequences, and the 16S-23S rDNA intergenic spacer region (ITS 1). The objective was to evaluate their potential application to discriminate among members of the species Salmonella enterica subspecies enterica using the genetic diversity of the group found by genomic fingerprinting. The three different serovars of Salmonella studied gave reproducible and distinguishable profiles using whichever of the above mentioned polymerase chain reaction (PCR) methods assayed. The conserved patterns in each serovar allowed for easy differentiation from other serovars of Salmonella.     

Digestive efficiency of the Hoatzin, Opisthocomus hoazin: a folivorous bird with foregut fermentation

The Hoatzin Opisthocomus hoazin is the only known bird with a well-developed foregut plant fermentation system; most fermentation takes place in the crop and caudal oesophagus. To test Hoatzin digestive efficiency, balance (total collection) trials with captive Hoatzins were made using two experimental diets of different composition and fibre content. Dry matter (DM) intakes were similar for the diets (mean = 62.8 g DM/kg body mass/ day). Average DM, organic matter and nitrogen digestibilities were not significantly different between diets, with average values of 72.9%, 75.0% and 78.3%, respectively. In vitro organic matter digestibilities by cow ruminal inoculum were very similar to organic matter digestibilities in live Hoatzins for both diets. Fibre digestibility was among the highest recorded for herbivorous birds. Cellulose and acid detergent fibre digestibilities were 58.8% and 52.7%, respectively. Neutral detergent fibre (NDF) digestibility differed among diets—the higher the NDF content of the diet, the higher the NDF digestibility. The NDF digestibilities were 37.9% and 70.9% for the two diets with NDF concentrations of 32.4% and 37.3%, respectively. Differences in NDF digestibility can be attributed to the different concentrations of hemicellulose in the experimental diets. The high overall digestibility by captive Hoatzins is higher than values previously reported for other avian herbivores but similar to those of foregut-fermenting mammals on similar diets. The unique digestive strategy of the Hoatzin maximizes digestion of cell wall and cell contents. The high digestive efficiency in the Hoatzin is not predicted by allometric models of fibre digestion as a function of body mass. Other nutritional benefits, such as detoxification of plant secondary compounds and microbial synthesis of essential amino acids and vitamins, may explain the evolution of foregut fermentation in this avian folivore.     

Genetic diversity of fast-growing rhizobia that nodulate soybean ( Glycine max L. Merr)

The fast-growing Rhizobium sp. strain NGR234, isolated from Papua New Guinea, and 13 strains of Sinorhizobium fredii, isolated from China and Vietnam, were fingerprinted by means of RAPD, REP, ERIC and ARDRA. ERIC, REP and RAPD markers revealed a considerable genetic diversity among fast-growing rhizobia. Chinese isolates showed higher levels of diversity than those strains isolated from Vietnam. ARDRA analysis revealed three different genotypes among fast-growing rhizobia that nodulate soybean, even though all belonged to a subcluster that included Sinorhizobium saheli and Sinorhizobium meliloti. Among S. fredii rhizobia, two strains, SMH13 and HH303, might be representatives of other species of nitrogen-fixing organisms. Although restriction analysis of the nifD–nifK intergenic DNA fragment confirmed the unique nature of Rhizobium sp. strain NGR234, several similarities between Rhizobium sp. strain NGR234 and S. fredii USDA257, the ARDRA analysis and the full sequence of the 16S rDNA confirmed that NGR234 is a S. fredii strain. In addition, ARDRA analysis and the full sequence of the 16S rDNA suggested that two strains of rhizobia might be representatives of other species of rhizobia.     

Expression of a modified Neocallimastix patriciarum xylanase in Butyrivibrio fibrisolvens digests more fibre but cannot effectively compete with highly fibrolytic bacteria in the rumen

AIMS: This study investigated the competitive abilities of two Neocallimastix patriciarum-derived xylanases constructs in Butyrivibrio fibrisolvens H17c (xynA and pUMSX) and their ability to compete in vivo. METHODS AND RESULTS: The digestibility of neutral detergent fibre (NDF) increased during co-culture of xynA or pUMSX and weakly cellulolytic, but not with highly cellulolytic, ruminococci. Competition studies among xynA, pUMSX and cellulolytic consortia demonstrated that xynA was the fittest. XynA did not persist at high levels in the rumen and was undetectable after 22 days. CONCLUSION: The construction of recombinant xylanolytic B. fibrisolvens does improve the digestibility of fibre above that of the native, but digestibility is still less than that of the most potent fibre digesters such as ruminococci. SIGNIFICANCE AND IMPACT OF THE STUDY: Fibre digestion may be improved by genetic manipulation of ruminal bacteria but ecological parameters, such as persistence in vivo and the niche of the organism, must be taken into account.     

Genetic diversity among isolates of Paenibacillus larvae from Austria

Genetic diversity of 214 Paenibacillus larvae strains from Austria was studied. Genotyping of isolates was performed by polymerase chain reaction (PCR) with primers corresponding to enterobacterial repetitive intergenic consensus (ERIC), BOX repetitive and extragenic palindromic (REP) elements (collectively known as rep-PCR) using ERIC primers, BOX A1R and MBO REP1 primers. Using ERIC-PCR technique two genotypes could be differentiated (ERIC I and II), whereas using combined typing by BOX- and REP-PCR, five different genotypes were detected (ab, aB, Ab, AB and αb). Genotypes aB and αb are new and have not been reported in other studies using the same techniques.     

ERIC and REP-PCR Banding Patterns and Sequence Analysis of the Internal Transcribed Spacer of rDNA of Stemphylium solani Isolates from Cotton

The genetic diversity of the Stemphylium solani isolates from cotton was assessed by Enterobacterial Repetitive Intergenic Consensus (ERIC) and Repetitive Extragenic Palindromes (REP)-PCR fingerprinting. Twenty eight monosporic isolates of S. solani from cotton were used along with five isolates from tomato and one isolate of Alternaria macrospora from cotton for comparison. The dendrogram obtained revealed clear differences between the cotton and tomato isolates as well as between the tomato isolates from different geographic regions. The genetic relationships among S. solani isolates were also analyzed by sequencing the internal transcribed spacer (ITS) region of four isolates representing the three ERIC and REP groups. The tomato isolate from the State of S?o Paulo showed a distinct ITS sequence from that of the cotton isolates and tomato isolate from the State of Goiás, giving evidence that it belongs to a different genotype of S. solani. This is the first report of the entire sequence of the ITS1-5.8S-ITS2 regions of S. solani.     

采用PCR-RFLP技术在不同水平上鉴定大豆根瘤菌

采用16S rRNA基因PCR扩增与限制性酶切片段多态性分析(RFLP)技术对选自弗氏中华根瘤菌(S.fredii)、大豆慢生根瘤菌(B.japonicum)和埃氏慢生根瘤菌(B.elkanii)的19株代表菌进行了比较分析,根据用3种限制性内切酶的RFLP分析结果,可将供试菌株分为S.fredii,B.japonicum, B.elkanii Ⅱ和B.elkanii Ⅱa等4种基因型。各类菌株之间没有交叉,因此本研究采用的PCR-RFLP技术不失为一种快速鉴别大豆根瘤菌的新方法。采用本技术已将分离自中国的22株快生菌和19株慢生菌分别鉴定为S.fredii和B.japonicum。对供试参比菌株和野生型菌株进行的16S～23S基因间隔DNA(IGS)的PCR-RFLP分析结果表明：S.fredii和B.japonicum菌株的IGS长度不同,所有供试S.fredii菌株的IGS为2.1 kb,而供试B.japonicum菌株则为2.0 kb。依据RFLP的差异,可将来自中国两个不同地区的S.fredii株区分为2个基因型,而来自中国东北黑龙江地区的19株B.japonicum菌株则可分为11个基因型。对上述野生型菌株还进行了REP-PCR和ERIC-PCR分析并确定其具有菌株水平的特异性。     

Electron Microscopy and Molecular Characterization of Phytoplasmas Associated with Little Leaf Disease of Brinjal (Solanum melongena L.) and Periwinkle (Catharanthus roseus) in Bangladesh

In Bangladesh little leaf disease was observed in brinjal ( Solanum melongena L.) and in periwinkle ( Catharanthus roseus ). Phloem-inhabiting phytoplasmas were consistently detected in both species of diseased plants using transmission electron microscopy (TEM) and polymerase chain reaction (PCR) techniques. The shape, size and within-tissue distribution of phytoplasmas appears to be similar in both hosts. Furthermore, the molecular characterization and identifications of observed phytoplasmas were carried out based on restriction fragment length polymorphism (RFLP) patterns of PCR-amplified products (1200 bp) using phytoplasma-specific universal primers and sequencing analysis of both 16S ribosomal DNA (rDNA) and intergenic spacer region (ISR) of 16S-23S rDNA phytoplasma genes. The patterns of RFLP analysis with seven restriction enzymes exhibited a similar pattern for both phytoplasma strains. The sequence homology between these two strains showed 100% similarity based on 16S rDNA and 16S-23S ISR. Therefore, in Bangladesh the causal agents of brinjal little leaf (BLL-Bd) and periwinkle little leaf (PLL-Bd) are probably the same or closely related phytoplasma strains. These strains, are very close or identical to the strain of brinjal little leaf phytoplasma in India (BLL-In), belonging to the clover proliferation group (Lee et al., Int. J. Syst. Bacteriol. 48, 1153–1169, 1998; Seemuller et al., J. Plant Pathol. 80, 3–26, 1998).     

rep-PCR fingerptinting as a tool for the analysis of genomic diversity in Escherichia coli strains isolated from an aqueous/freshwater environment

The rep-PCR fingerprinting method, with the support of ERIC and REP primers, was used to analyse the genomic diversity of 93 E. coli strains isolated from lake water samples drawn at two different depths. The applied UPGMA for DNA analysis did not reveale any genomic similarities between the 48 E. coli strains derived from the subsurface-zone water and the 43 of the bottom-zone water. The considerable genomic diversity of the E. coli of the surface zone was expressed as a dendrogram in the form of 8 similarity groups comprising strains isolated from samples drawn over one month. The bottom-zone strains, which display a lesser degree of genomic diversity (5 similarity groups), showed distinct common features in their DNA fingerprints. In the similarity dendrogram for the bottom-zone, strains derived in different months of sampling were segregated into the same similarity groups. Applying REP primers in rep-PCR generates more complex fingerprints increasing the discriminatory power of the analysis, whereas the ERIC primer generates less complex fingerprint patterns, and is thus clearer to interpret.     

Grass silage pulp as a dietary component for high-yielding dairy cows

Green biorefineries provide novel opportunities to use the green biomass efficiently and utilize the ecosystem services provided by grasslands more widely. The effects of the inclusion of fractionated grass silage solid fraction (pulp) on feed intake, rumen fermentation, diet digestion and milk production in dairy cows were investigated. Pulp was separated from grass silage using a screw press simulating a green biorefinery. Partial removal of liquid from forage increased DM concentration from 220 to 432 g/kg and NDF from 589 to 709 g/kg DM while CP decreased from 144 to 107 g/kg DM. A feeding trial using an incomplete changeover design with 24 Nordic Red cows and two 3-week periods was conducted. The pulp replaced grass silage in the diet at 0 (P0), 25 (P25) and 50 (P50) percentage of total forage, which was fed ad libitum with 13 kg of concentrate for all treatments. The forage DM intake was highest on P25 (14.1 kg/day) while P0 and P50 did not differ from each other (13.2 and 13.0 kg/day, respectively). There were no differences between the treatments in rumen pH or ammonia N, but the proportion of acetate increased with increasing pulp inclusion. The digestibility was measured using acid insoluble ash and indigestible NDF (iNDF) as internal markers. Neither of the markers detected differences in NDF digestibility, but according to iNDF, apparent total tract organic matter digestibility decreased with increasing pulp inclusion. The cows maintained milk production at P25, but it showed some decline at P50 (energy-corrected milk at P0 and P25 was 39.8 kg/day while for P50, it was 38.5 kg/day, P = 0.056) and the milk protein yield significantly declined with higher pulp inclusion. Simultaneously, the nitrogen use efficiency in milk production increased. It seems that the fibrous grass-based fraction from a biorefinery process has potential to be used as a feed for ruminants.     

The effects of and interactions between the maturity of grass silage and concentrate starch source when offered as total mixed rations on the performance of dairy cows

A 2 × 2 factorial feeding experiment was conducted to examine the effects of varying the maturity level of the grass used to prepare silage and the nature of concentrate starch source and their interactions on dry matter intake (DMI), diet digestibility, energy corrected milk (ECM) production and milk composition in dairy cows. Twenty-eight multiparous Swedish Red dairy cows, 133 ± 45 days in milk (DIM), with an average milk yield of 30 ± 4 kg/day and a live weight of 624 ± 69 kg were blocked by DIM and randomly assigned to seven replicated balanced 4 × 4 Latin squares with four 21-day experimental periods. The experimental diets consisted of four total mixed rations (TMR) consisting of early-cut grass silage (EGS) supplemented with either barley- or maize-based concentrate and late-cut grass silage (LGS) supplemented with either barley- or maize-based concentrate. All TMR contained identical proportions of forage (51%) and concentrate (49%). Total tract digestibility was estimated by determining indigestible NDF (iNDF) concentrations in feeds and faeces and using iNDF as an internal marker. The feeds’ ruminal degradation parameters were determined using both in situ (nylon bag) and in vitro (gas production (GP)) techniques. Cows offered diets containing EGS had greater (P < 0.001) daily dry matter (DM) intakes, ECM yields and total tract digestibilities for DM and organic matter (OM), but these were not affected by the nature of the concentrate starch source. No interaction between the maturity of the silage and the nature of the concentrate starch source was observed for DMI, diet digestibility or ECM yield. Both grass silages and concentrates had similar rates of ruminal degradation of NDF when measured in situ. The in situ DM (P < 0.001) and starch (P = 0.001) degradation rates of barley-based concentrate were greater than those for maize-based concentrate. In vitro OM GP rates and extents were similar for both concentrate feeds. The results showed that diets containing EGS offered better animal performance and diet digestibility than diets containing LGS. The concentrate starch source did not affect animal performance, but total NDF digestibility was better with diet containing barley- than maize-based concentrate.     

新疆土著大豆根瘤菌种群遗传结构的初步分析

应用重复序列REP（repetitive extragenic palindromic,重复基因外回）和ERIC（ente-robaterial repetitive intergenic consensus,肠细菌重复基因间共有序列）结合聚合酶链式反应（ERP－PCR和ERIC－PCR）对从新疆有集的27株土大豆根瘤菌染色体进行指纹分析,发现在相似水平0.5时可基本分为两大聚类群,一个类群主要包括慢生型根瘤菌,另一类群为快生型根瘤菌,来自同一地区的根瘤菌具有较高的遗传相似性,以上结果表明该技术中对大豆根瘤菌进行种群结构和遗传多样性分析的有效手段。     

Composition and enzymatic digestibility of Oregon grass straws

Samples of nine species or types of commercial grass seed straws grown in Oregon were analyzed and treated with anhydrous ammonia in the laboratory. Enzymatic digestibility of treated and untreated materials was determined using commercial cellulase and protease. The mean composition of all nine species/types (based on six each within-group replicates) was: crude protein, 5.4%; NDF, 72.2%; ADF, 43.6%; hemicellulose, 26.4%; cellulose, 34.8%; and lignin, 6.9%. Significant species/type compositional differences were found. From their composition, several of the grass straws appeared to have better nutritional value than most cereal straws. Enzymatic digestibility of all straws was improved by ammoniation (average increase, 62%). Enzymatic digestibility ranged from about 27–42% for untreated materials to 43–66% for NH₃-treated products.     

Seasonal variations in the chemical composition and dry matter degradability of exclosure forages in the semi-arid region of northern Ethiopia

The present study was conduced at two sites (Tembien and Wukro) in the semi-arid region of Tigray in northern Ethiopia to investigate the seasonal dynamics in the chemical composition and dry matter digestibility of grass and browse species of exclosures. The browse species studied in Tembien and Wukro had a mean crude protein (CP) value of 166 and 117 g/kg dry matter (DM), respectively. The mean in vitro dry matter digestibility (IVDMD) coefficient and predicted metabolizable energy (ME) density of the browse species were 0.72 and 9.83 MJ/kg DM, respectively at Tembien, 0.62 and 8.38 MJ/kg DM, respectively, at Wukro. Neutral detergent fibre (NDF) and acid detergent fibre (ADF) values of the browse species varied from 192 to 437 and 127 to 391 g/kg DM, respectively. Acid detergent lignin (ADL) values ranged from 36 to 190 g/kg DM. The mean CP of the grass species in Tembien and Wukro during the long rainy season was 76 and 73 g/kg DM, respectively and values declined below a critical maintenance level during the dry and short rain seasons. Mean IVDMD and ME values for the two sites were 0.41 and 0.47, and 5.38 and 6.11 MJ/kg DM, respectively. The NDF, ADF, and lignin values of the grass species were generally above 700, 400, and 70 g/kg DM, respectively. The CP, IVDMD and ME values of the mixed grass samples differed (P<0.05) among harvesting months and values ranged from 20 to 103 g/kg DM, 0.47 to 0.72 MJ/kg DM, and 6.16 to 9.91 MJ/kg DM, respectively. The browse species could be used as useful dry season protein supplements to the N deficient native grass species. Especial emphasis should be given to propagate Maerua angolensis and Cadaba farinosa at community nursery sites. Harvesting in September, rather than the current extended harvest period that took place in October and November, can considerably improve the feeding value of native grass hay for smallholder ruminant production systems.     

Genetic diversity in pseudomonads associated with cereal cultures infected with basal bacteriosis

The genetic properties of 45 pseudomonad strains isolated from cereal cultures exhibiting symptoms of basal bacteriosis have been investigated. Considerable genetic diversity has been demonstrated using DNA fingerprints obtained by amplification with REP, ERIC, and BOX primers. Restriction analysis of the 16S-23S internal transcribed spacer (ITS1) allowed the strains to be subdivided into two major groups. In a phylogenetic tree, the ITS1s of these groups fell into two clusters, which also included the ITS1 of Pseudomonas syringae ("Syringae" cluster) and the ITS1 of P. fluorescens, P. tolaasii, P. reactans, P. gingeri, and P. agarici ("Fluorescens" cluster) from the GenBank database. Comparison of the ITS1 divergence levels within the "Fluorescens" cluster suggests expediency of treating P. tolaasii, P. reactans, various P. fluorescens groups, and, possibly, P. gingeri and P. agarici as subspecies of one genospecies. The intragenomic heterogeneity of ITS1s was observed in some of the pseudomonad strains studied. The results of amplification with specific primers and subsequent sequencing of the amplificate suggest the possibility of the presence of a functionally active syrB gene involved in syringomycin biosynthesis in the strains studied.