线虫寄生菌巴斯德杆菌遗传多样性分析

应用16S rDNA-PCR技术快速检测不同根系样品中的巴斯德杆菌(Pasteuria spp.),并采用PCR-RFLP和PCR-SSCP法初步分析这些样品中巴斯德杆菌群体的遗传多样性,结果表明,来自福建、广东的30份感染根结线虫病的根系中,有9份样品含有巴斯德杆菌;PCR-RFLP分析表明,克隆子的EcoH I酶切带型分为5类,其中2类占相对优势,PCR-SSCP带型分类的情况与PCR-RFLP的基本一致,但表现出更大的差异性.选取12个克隆子进行测序分析,结果显示,克隆子的16S rDNA序列与穿刺巴斯德杆菌(Pasteuria penetrans)的相应序列具有较高的同源性(97.8%～99.7%);系统进化关系分析进一步表明,不同根系样品中的巴斯德杆菌(Pasteuria spp.)16S rDNA序列与GenBank已收录的穿刺巴斯德杆菌(P.penetrans)序列形成1个主要分支和7个独立分支,具有一定的遗传差异.     

The effect of a pathogen epidemic on the genetic structure and reproductive strategy of the crustacean Daphnia magna

Host–parasite coevolution is potentially of great importance in producing and maintaining biological diversity. However, there is a lack of evidence for parasites directly driving genetic change. We examined the impact of an epidemic of the bacterium Pasteuria ramosa on a natural population of the crustacean Daphnia magna through the use of molecular markers (allozymes) and laboratory experiments to determine the susceptibility of hosts collected during and after the epidemic. Some allozyme genotypes were more heavily infected than others in field samples, and the population genetic structure differed during and after the epidemic, consistent with a response to parasite‐mediated selection. Laboratory studies showed no evidence for the evolution of higher resistance, but did reveal an intriguing life‐history pattern: host genotypes that were more susceptible also showed a greater tendency to engage in sex. In light of this, we suggest a model of host–parasite dynamics that incorporates the cycles of sex and parthenogenesis that Daphnia undergo in the field.     

The history of the Nematology Department at Rothamsted

This is a review of the activities of what rapidly became the leading plant nematology department in the world, based in what was at that time not only the most important but also the most distinguished agricultural research station in the world. We first briefly review the research done in the period under each head of department before recording in more detail some of the long‐term research programmes, including work on potato cyst nematode hatching factors, chemical control and biological control. These strong research activities flourished until the radical funding constraints that were introduced nationally following release of the Rothschild Report in 1973 forced the adoption of various management actions at research stations. The changed pattern of research funding systems, which evolved gradually from 1973 onwards, resulted in a different style of research collaboration and changes in research focus by institutes and their staff. It became fashionable for institutes to have mission statements and these were changed frequently by directors due to the need to respond to funding possibilities. Successive severe and progressive reductions in staffing and, inevitably, outputs culminated in the complete cessation of nematology research at Rothamsted in 2013, even though cutting edge work on biological control and molecular interactions between nematodes and their plant hosts was still being carried out.     

Invasion of HeLa cells by Mycoplasma penetrans and the induction of tyrosine phosphorylation of a 145-kDa host cell protein

Abstract The ability of Mycoplasma penetrans to invade eukaryotic cells was studied using a HeLa cell line. The bactericidal antibiotic, gentamicin, in combination with low concentrations of Triton X-100, was utilized to kill mycoplasmas that had not entered the cells, allowing the quantitation of internalized organisms. The intracellular location of the mycoplasma was also documented by transmission electron microscopy. The actin polymerization inhibitor cytochalasin-D markedly inhibited the internalization process, whereas the tyrosine phosphorylation inhibitors, staurosporin and genistein had only a slight effect. As against the invasion of enteropathogenic Escherichia coli which depends on tyrosine phosphorylation of a 90-kDa (Hp90) HeLa cell protein, internalization of M. penetrans by HeLa cells was independent of the phosphorylation of Hp90. Nonetheless, tyrosine phosphorylation of a 145-kDa HeLa cell protein was found to be associated with the interaction of M. penetrans with HeLa cells.     

Multiple-strand displacement and identification of single nucleotide polymorphisms as markers of genotypic variation of Pasteuria penetrans biotypes infecting root-knot nematodes

Pasteuria species are endospore-forming obligate bacterial parasites of soil-inhabiting nematodes and water-inhabiting cladocerans, e.g. water fleas, and are closely related to Bacillus spp. by 16S rRNA gene sequence. As naturally occurring bacteria, biotypes of Pasteuria penetrans are attractive candidates for the biocontrol of various Meloidogyne spp. (root-knot nematodes). Failure to culture these bacteria outside their hosts has prevented isolation of genomic DNA in quantities sufficient for identification of genes associated with host recognition and virulence. We have applied multiple-strand displacement amplification (MDA) to generate DNA for comparative genomics of biotypes exhibiting different host preferences. Using the genome of Bacillus subtilis as a paradigm, MDA allowed quantitative detection and sequencing of 12 marker genes from 2000 cells. Meloidogyne spp. infected with P. penetrans P20 or B4 contained single nucleotide polymorphisms (SNPs) in the spoIIAB gene that did not change the amino acid sequence, or that substituted amino acids with similar chemical properties. Individual nematodes infected with P. penetrans P20 or B4 contained SNPs in the spoIIAB gene sequenced in MDA-generated products. Detection of SNPs in the spoIIAB gene in a nematode indicates infection by more than one genotype, supporting the need to sequence genomes of Pasteuria spp. derived from single spore isolates.     

A Strain of Bacillus thuringiensis for the Control of Plant‐parasitic Nematodes

An isolate of Bacillus thuringiensis, designated CR‐371, was evaluated for efficacy in controlling plant‐parasitic nematodes. This isolate was first shown to be nematicidal to Caenorhabditis elegans in an in vitro laboratory assay. Treatment resulted in a significant reduction in galls due to root‐knot nematode on tomato in a greenhouse trial. In two field trials in Puerto Rico, CR‐371‐treated tomatoes and pepper had significantly fewer root galls due to Meloidogyne incognita than untreated controls, and populations of Rotylenchulus reniformis were smaller. In one experiment, CR‐371 treatment was associated with significant increases in pepper yields, while in the second trial small yield increases of pepper and tomato occurred. In a greenhouse trial, incorporation of CR‐371 into a methyl cellulose seed coat gave similar control of root‐knot nematode on tomato as compared to CR‐371 applied as a drench. CR‐371‐treated strawberry plants also had smaller populations of Pratylenchus penetrans in roots in a greenhouse trial in Massachusetts.     

Estimating Incidence of Attachment of Pasteuria penetrans Endospores to Meloidogyne spp. with Tally Thresholds

Pasteuria penetrans has .been identified as an important biological control agent of root-knot nematodes. In this study the use of tally thresholds was evaluated for estimating P. penetrans endospore attachment to second-stage juveniles (J2) of Meloidogyne spp. A tally threshold (T) is defined as the maximum number of individuals in a sample unit that may be treated as absent based on binomial sampling. Three different data sets that originated from centrifugal bioassay, incubation bioassay, and field experiments were investigated. The data sets each contained 70, 33, and 111 estimates of the mean number of endospores attached per J2 (m), respectively. Empirical relationships between m and proportions of J2 with ≤T endospores attached (P_T) were developed using parameters from the linear regression of ln(m) on P_T (0 < P_T < 1): ln(m) = a + b P_T, T was set to 0, 1, 2, 3, 4, 5, 8, and 10 endospores/J2. The results indicated that the variances of linear equations tended to decrease with increasing T values for all three data sets. T values of 0, 1, 8, and 10 endospores/J2 for centrifugal bioassay and incubation bioassay, and of 0, 1, 2, and 3 endospores/J2 for field experiments were associated with an r² of >= 0.8. These T values were robust for estimating m from P_T, reducing the variability as well as the time and effort spent in estimating the mean number of endospores attached per J2.     

Genetic variation for maternal effects on parasite susceptibility

The expression of infectious disease is increasingly recognized to be impacted by maternal effects, where the environmental conditions experienced by mothers alter resistance to infection in offspring, independent of heritability. Here, we studied how maternal effects (high or low food availability to mothers) mediated the resistance of the crustacean Daphnia magna to its bacterial parasite Pasteuria ramosa. We sought to disentangle maternal effects from the effects of host genetic background by studying how maternal effects varied across 24 host genotypes sampled from a natural population. Under low‐food conditions, females produced offspring that were relatively resistant, but this maternal effect varied strikingly between host genotypes, i.e. there were genotype by maternal environment interactions. As infection with P. ramosa causes a substantial reduction in host fecundity, this maternal effect had a large effect on host fitness. Maternal effects were also shown to impact parasite fitness, both because they prevented the establishment of the parasites and because even when parasites did establish in the offspring of poorly fed mothers, and they tended to grow more slowly. These effects indicate that food stress in the maternal generation can greatly influence parasite susceptibility and thus perhaps the evolution and coevolution of host–parasite interactions.     

Persistence and Suppressiveness of Pasteuria penetrans to Meloidogyne arenaria Race

The long-term persistence and suppressiveness of Pasteuria penetrans against Meloidogyne arenaria race 1 were investigated in a formerly root-knot nematode suppressive site following 9 years of continuous cultivation of three treatments and 4 years of continuous peanut. The three treatments were two M. arenaria race 1 nonhost crops, bahiagrass (Paspalum notatum cv. Pensacola var. Tifton 9), rhizomal peanut (Arachis glabrata cv. Florigraze), and weed fallow. Two root-knot nematode susceptible weeds commonly observed in weed fallow plots were hairy indigo (Indigofera hirsuta) and alyce clover (Alysicarpus vaginalis). The percentage of J2 with endospores attached reached the highest level of 87% in 2000 in weed fallow, and 63% and 53% in 2002 in bahiagrass and rhizomal peanut, respectively. The percentage of endospore-filled females extracted from peanut roots grown in weed fallow plots increased from nondetectable in 1999 to 56% in 2002, whereas the percentages in bahiagrass and rhizomal peanut plots were 41% and 16%, respectively. Over 4 years, however, there was no strong evidence that endospores densities reached suppressive levels because peanut roots, pods, and pegs were heavily galled, and yields were suppressed. This might be attributed to the discovery of M. javanica infecting peanut in this field in early autumn 2001. A laboratory test confirmed that although the P. penetrans isolate specific to M. arenaria attached to M. javanica J2, no development occurred. In summary, P. penetrans increased on M. arenaria over a 4-year period, but apparently because of infection of M. javanica on peanut at the field site root-knot disease was not suppressed. This was confirmed by a suppressive soil test that showed a higher level of soil suppressiveness than occurred in the field (P ≤ 0.01).     

Host sexual dimorphism affects the outcome of within‐host pathogen competition

Natural infections often consist of multiple pathogens of the same or different species. When coinfections occur, pathogens compete for access to host resources and fitness is determined by how well a pathogen can reproduce compared to its competitors. Yet not all hosts provide the same resource pool. Males and females, in particular, commonly vary in both their acquisition of resources and investment in immunity, but their ability to modify any competition between different pathogens remains unknown. Using the Daphnia magna–Pasteuria ramosa model system, we exposed male and female hosts to either a single genotype infection or coinfections consisting of two pathogen genotypes of varying levels of virulence. We found that coinfections within females favored the transmission of the more virulent pathogen genotype, whereas coinfections within male hosts resulted in equal transmission of competing pathogen genotypes. This contrast became less pronounced when the least virulent pathogen was able to establish an infection first, suggesting that the influence of host sex is shaped by priority effects. We suggest that sex is a form of host heterogeneity that may influence the evolution of virulence within coinfection contexts and that one sex may be a reservoir for pathogen genetic diversity in nature.