烟草疫霉拮抗菌枯草芽孢杆菌21b菌株的分离鉴定及其生物学特性研究

【目的】为了控制烟草疫霉(Phytophthora nicotianae)引起的烟草黑胫病对烟草生产造成的危害。【方法】采用稀释平板法从贵州省毕节地区烟草根际土壤中分离筛选拮抗烟草疫霉的细菌菌株,然后经形态观察、Biolog鉴定及16S rRNA基因序列分析,对分离菌株进行鉴定,同时测定抗菌谱,单因子变量分析、优化生长条件。【结果】共分离得到44株拮抗烟草疫霉的细菌菌株,其中菌株21b对烟草黑胫病菌菌丝生长的抑制率达78.33%,鉴定为枯草芽孢杆菌(Bacillus subtilis)。该菌株对烟草青枯病菌(Ralstonia solanacearum)、烟草灰霉病菌(Botrytis cinerea)、烟草赤星病菌(Alternaria alternate)和烟草炭疽病菌(Colletotrichum destructivum)均具有拮抗作用,抑菌圈大小分别为19.5、18.2、14.6和13.4 mm,最佳的发酵条件为:温度30°C、p H 7.0–8.0、装液量12%、盐浓度0.5%。【结论】分离筛选到一株对烟草寄生疫霉有较强拮抗活性的细菌菌株,为进一步开发烟草黑胫病的生防菌剂提供了菌种资源。     

Highly virulent strains of Pseudomonas solanacearum that are defective in extracellular-polysaccharide production

Extracellular polysaccharide (EPS) has long been regarded as one of the most important factors involved in wilting of plants by Pseudomonas solanacearum. By means of transposon Tn5 mutagenesis, we have isolated a class of mutants that have an afluidal colony morphology but retain the ability to cause severe wilting and death of tobacco plants. One such mutant, KD700, was studied in detail. By marker exchange mutagenesis, the altered colony morphology was shown to be the result of a single Tn5 insertion in a 14.3-kilobase EcoRI fragment. This defect could be corrected by introducing a homologous clone from a cosmid library of the wild-type, parental strain K60. The Tn5-containing fragment was introduced into other P. solanacearum wild-type strains by marker exchange, and these altered strains had the same afluidal phenotype as KD700. N-Acetylgalactosamine (GalNac), the major constituent of EPS of all wild-type strains of P. solanacearum, was not detected by gas chromatography-mass spectrometry analysis of vascular fluids from wilting plants infected by KD700. In contrast, GalNac was readily detected in similar fluids of plants infected by K60. Polysaccharides extracted from culture filtrates of KD700 contained approximately one-fifth of the GalNac present in polysaccharides from K60. No differences in growth rates in culture or in planta between the mutant and the parental strains were observed. Since strains that are deficient in EPS production can remain highly virulent to tobacco, we conclude that EPS, or at least its GalNac-containing component, may not be required for disease development by P. solanacearum.     

Relationship between avirulence gene (avrA) diversity in Ralstonia solanacearum and bacterial wilt incidence

Bacterial wilt, caused by Ralstonia solanacearum, is a serious disease of tobacco in North and South Carolina. In contrast, the disease rarely occurs on tobacco in Georgia and Florida, although bacterial wilt is a common problem on tomato. We investigated whether this difference in disease incidence could be explained by qualitative characteristics of avirulence gene avrA in the R. solanacearum population in the southeastern United States. Sequence analysis established that wild-type avrA has a 792-bp open reading frame. Polymerase chain reaction (PCR) amplification of avrA from 139 R. solanacearum strains generated either 792-bp or approximately 960-bp DNA fragments. Strains that elicited a hypersensitive reaction (HR) on tobacco contained the 792-bp allele, and were pathogenic on tomato and avirulent on tobacco. All HR-negative strains generated a approximately 960-bp DNA fragment, and wilted both tomato and tobacco. The DNA sequence of avrA in six HR-negative strains revealed the presence of one of two putative miniature inverted-repeat transposable elements (MITEs): a 152-bp MITE between nucleotides 542 and 543, or a 170-bp MITE between nucleotides 461 and 462 or 574 and 575. Southern analysis suggested that the 170-bp MITE is unique to strains from the southeastern United States and the Caribbean. Mutated avrA alleles were present in strains from 96 and 75% of North and South Carolina sites, respectively, and only in 13 and 0% of the sites in Georgia and Florida, respectively. Introduction of the wildtype allele on a plasmid into four HR-negative strains reduced their virulence on both tobacco and tomato. Inactivation of avrA in an HR-positive, avirulent strain, resulted in a mutant that was weakly virulent on tobacco. Thus, the incidence of bacterial wilt of tobacco in the southeastern United States is partially explained by which avrA allele dominates the local R. solanacearum population.     

Natural transformation in the Ralstonia solanacearum species complex: number and size of DNA that can be transferred

Ralstonia solanacearum is a widely distributed phytopathogenic bacterium that is known to invade more than 200 host species, mainly in tropical areas. Reference strain GMI1000 is naturally transformable at in vitro and also in planta conditions and thus has the ability to acquire free exogenous DNA. We tested the ubiquity and variability of natural transformation in the four phylotypes of this species complex using 55 strains isolated from different hosts and geographical regions. Eighty per cent of strains distributed in all the phylotypes were naturally transformable by plasmids and/or genomic DNA. Transformability can be considered as a ubiquitous physiological trait in the R. solanacearum species complex. Transformation performed with two independent DNA donors showed that multiple integration events occurred simultaneously in two distant genomic regions. We also engineered a fourfold-resistant R. solanacearum GMI1000 mutant RS28 to evaluate the size of DNA exchanged during natural transformation. The results demonstrated that this bacterium was able to exchange large DNA fragments ranging from 30 to 90 kb by DNA replacement. The combination of these findings indicated that the natural transformation mechanism could be the main driving force of genetic diversification of the R. solanacearum species complex.     

Bacteriophage P4282, a parasite of Ralstonia solanacearum, encodes a bacteriolytic protein important for lytic infection of its host

To enhance bacterial wilt resistance in tobacco expressing a foreign protein, we isolated the bacteriolytic gene from a bacteriophage that infects Ralstonia solanacearum. The bacteriolytic protein of phage P4282 isolated in Tochigi Prefecture was purified from a lysate of R. solanacearum M4S cells infected with the phage, and its bacteriolytic activity was assayed by following the decrease in the turbidity of suspensions of R. solancacearum M4S cells. The molecular weight of the bacteriolytic protein was approximately 71 kDa, and the sequence of the N-terminal 13 amino acids was determined. We used oligonucleotide probes based on this amino acid sequence to isolate the bacteriolytic gene from phage P4282 DNA. This gene of 2061 bp encodes a product of 687 amino acids, whose calaculated molecular weight was 70.12 kDa. The bacteriolytic gene was placed under the control of an inducible promoter. and the plasmid was transformed into Escherichia coli NM522. The soluble proteins extracted from E.coli NM522 cells harboring the plasmid with the bacteriolytic gene showed obvious bacteriolytic activities against several strains of R. solanacearum isolated in various districts in Japan. DNA fragments from five phages, isolated in Niigata, Aomori, Okinawa, Fukushima and Yamaguchi Prefectures, hybridized to the bacteriolytic gene of phage P4282. These observations indicate that the bacteriolytic protein shows nonspecific activity against R. solanacearum strains, and a sequence similar to that of the bacteriolytic gene is conserved in the DNA of other bacteriophages. These results indicate that the generation of transgenic (tobacco) plants expressing the bacteriolytic gene of phage P4282 might result in enhanced resistance to bacterial wilt in tobacco.     

烟草青枯病劳尔氏菌与拮抗菌对根系分泌物的竞争作用

[目的]研究青枯病病原菌与拮抗菌的营养特性及其对烟草根系分泌物的响应差异,对提高拮抗菌定殖能力、有效生物防控烟草青枯病具有非常重要的意义。[方法]本研究通过筛选与鉴定贵州烟区青枯病病原菌株及拮抗菌株,通过Biolog表型芯片技术分别检测病原菌与拮抗菌的特征性碳、氮源,利用气质联用(GC-MS)检测烟草主栽品种K326根系分泌物的主要物质,在此基础上进行病原菌与拮抗菌对其利用能力、利用强度以及共培养的研究。[结果]经鉴定,分离、筛选到的病原菌株和拮抗菌株分别为青枯劳尔氏菌(Ralstonia solawacearum)和枯草芽孢杆菌(Bacillus subtilis);在含量为0.01μg/mL以上的根系分泌物中,12种物质的含量从高到低排序为:果胶>葡萄糖>木糖>阿拉伯糖>半乳糖>核糖>蔗糖>苯甲酸>果糖=D-甘露醇>棕榈酸>富马酸,果胶含量最高且明显高于其他物质;拮抗菌(LX4)对碳源利用能力高于病原菌(Rs)的碳源有阿拉伯糖、木糖和核糖,分别是病原菌利用能力的1.22、1.95和2.17倍;前12 h拮抗菌利用果糖强度高于病原菌,不同碳源共培养24 h后LX4对gfp-Rs(绿色荧光蛋白标记后的青枯病病原菌)抑制率为18.34%(阿拉伯糖)、53.23%(木糖)、63.53%(核糖)和52.09%(果糖)。[结论]拮抗菌对烟草根系分泌物的利用不及病原菌,但在特定碳源条件下拮抗菌能够利用根系分泌物中的某些碳源产生某种拮抗物质抑制病原菌,拮抗菌与病原菌之间同时存在利用性竞争和干扰性竞争关系,研究结果为进一步研究烟草青枯病的生物防控提供了新的理论依据。     

Ralstonia syzygii, the Blood Disease Bacterium and some Asian R. solanacearum strains form a single genomic species despite divergent lifestyles

The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical evolution than to the acquisition of DNA by horizontal transfer.     

无致病力青枯雷尔氏菌对烟草根系土壤微生物脂肪酸生态学特性的影响

为了探讨无致病力青枯雷尔氏菌对烟草根系土壤微生物群落结构的影响,测定了接种无致病力青枯雷尔氏菌RS-1403和清水对照的烟草根系土壤的磷脂脂肪酸(Phospholipid Fatty Acids,PLFAs),比较分析两种处理下的烟草根系土壤微生物PLFAs组成、含量、微生物群落结构及多样性的差异,以期从微生物群落水平解析RS-1403菌株胁迫对烟草青枯病的免疫抗病机制。结果表明,与对照相比,RS-1403菌株胁迫下烟草根系土壤微生物PLFAs的组成及含量发生了变化,分为5种变化类型,分别为下降型、无变化型、增加50%以下类型、增加50%—100%类型,增加100%以上类型,其中指示放线菌的10Me16∶0含量降低,为下降型,增加100%以上类型的PLFAs均指示革兰氏阴性菌。进一步分析表明,接种RS-1403菌株能改变烟草根系土壤微生物群落结构,促进细菌和真菌的生长,抑制放线菌的生长。接种RS-1403菌株能提高烟草根系土壤的群落优势度Simpson指数、群落丰富度Shannon指数和均匀度Pielou指数,增加土壤的微生物群落多样性。对RS-1403菌株胁迫下烟草根系土壤微生物亚群落分化的比较分析,显示RS-1403菌株处理组与对照组亚群落分化不同,当兰氏距离为2.56时,可将处理组和对照组均分为4个亚群落,但它们的各亚群落组成及特征不同。聚类分析表明,基本上可将取样期内的RS-1403菌株胁迫处理和清水对照的烟草根系土壤分别聚在两个不同的类群中,说明RS-1403菌株能明显改变烟草根系土壤微生物群落结构。     

Isolation of an insertion sequence from Ralstonia solanacearum race 1 and its potential use for strain characterization and detection

A new insertion sequence (IS), IS1405, was isolated and characterized from a Ralstonia solanacearum race 1 strain by the method of insertional inactivation of the sacB gene. Sequence analysis indicated that the IS is closely related to the members of IS5 family, but the extent of nucleotide sequence identity in 5' and 3' noncoding regions between IS1405 and other members of IS5 family is only 23 to 31%. Nucleotide sequences of these regions were used to design specific oligonucleotide primers for detection of race 1 strains by PCR. The PCR amplified a specific DNA fragment for all R. solanacearum race 1 strains tested, and no amplification was observed with some other plant-pathogenic bacteria. Analysis of nucleotide sequences flanking IS1405 and additional five endogenous IS1405s that reside in the chromosome of R. solanacearum race 1 strains indicated that IS1405 prefers a target site of CTAR and has two different insertional orientations with respect to this target site. Restriction fragment length polymorphism (RFLP) pattern analysis using IS1405 as a probe revealed extensive genetic variation among strains of R. solanacearum race 1 isolated from eight different host plants in Taiwan. The RFLP patterns were then used to subdivide the race 1 strains into two groups and several subgroups, which allowed for tracking different subgroup strains of R. solanacearum through a host plant community. Furthermore, specific insertion sites of IS1405 in certain subgroups were used as a genetic marker to develop subgroup-specific primers for detection of R. solanacearum, and thus, the subgroup strains can be easily identified through a rapid PCR assay rather than RFLP analysis.     

Ralstonia solanacearum strains from Martinique (French West Indies) exhibiting a new pathogenic potential

We investigated a destructive pathogenic variant of the plant pathogen Ralstonia solanacearum that was consistently isolated in Martinique (French West Indies). Since the 1960s, bacterial wilt of solanaceous crops in Martinique has been caused primarily by strains of R. solanacearum that belong to either phylotype I or phylotype II. Since 1999, anthurium shade houses have been dramatically affected by uncharacterized phylotype II strains that also affected a wide range of species, such as Heliconia caribea, cucurbitaceous crops, and weeds. From 1989 to 2003, a total of 224 R. solanacearum isolates were collected and compared to 6 strains isolated in Martinique in the 1980s. The genetic diversity and phylogenetic position of selected strains from Martinique were assessed (multiplex PCRs, mutS and egl DNA sequence analysis) and compared to the genetic diversity and phylogenetic position of 32 reference strains covering the known diversity within the R. solanacearum species complex. Twenty-four representative isolates were tested for pathogenicity to Musa species (banana) and tomato, eggplant, and sweet pepper. Based upon both PCR and sequence analysis, 119 Martinique isolates from anthurium, members of the family Cucurbitaceae, Heliconia, and tomato, were determined to belong to a group termed phylotype II/sequevar 4 (II/4). While these strains cluster with the Moko disease-causing strains, they were not pathogenic to banana (NPB). The strains belonging to phylotype II/4NPB were highly pathogenic to tomato, eggplant, and pepper, were able to wilt the resistant tomato variety Hawaii7996, and may latently infect cooking banana. Phylotype II/4NPB constitutes a new pathogenic variant of R. solanacearum that has recently appeared in Martinique and may be latently prevalent throughout Caribbean and Central/South America.     

Mutants of Ralstonia (Pseudomonas) solanacearum sensitive to antimicrobial peptides are altered in their lipopolysaccharide structure and are avirulent in tobacco.

Ralstonia solanacearum K60 was mutagenized with the transposon Tn5, and two mutants, M2 and M88, were isolated. Both mutants were selected based on their increased sensitivity to thionins, and they had the Tn5 insertion in the same gene, 34 bp apart. Sequence analysis of the interrupted gene showed clear homology with the rfaF gene from Escherichia coli and Salmonella typhimurium (66% similarity), which encodes a heptosyltransferase involved in the synthesis of the lipopolysaccharide (LPS) core. Mutants M2 and M88 had an altered LPS electrophoretic pattern, consistent with synthesis of incomplete LPS cores. For these reasons, the R. solanacearum gene was designated rfaF. The mutants were also sensitive to purified lipid transfer proteins (LTPs) and to an LTP-enriched, cell wall extract from tobacco leaves. Mutants M2 and M88 died rapidly in planta and failed to produce necrosis when infiltrated in tobacco leaves or to cause wilting when injected in tobacco stems. Complemented strains M2* and M88* were respectively obtained from mutants M2 and M88 by transformation with a DNA fragment harboring gene rfaF. They had a different degree of wild-type reconstituted phenotype. Both strains retained the rough phenotype of the mutants, and their LPS electrophoretic patterns were intermediate between those of the wild type and those of the mutants.     

番茄青枯病拮抗菌的筛选

利用点接法从山东寿光和苍山不同种植年限的蔬菜大棚中分离得到的45株菌株以及实验室保存的19株菌株中,筛选到14株对番茄青枯病病原细菌有拮抗作用的菌株,然后通过牛津杯法复筛得到6株抑菌效果较好的细菌株。通过温室盆栽试验表明拮抗菌X10的防治效果最好,液体菌剂防治效果达到了81.8%,固体菌剂防治效果达到了65.4%,具有良好的应用前景。对菌株X10进行了培养特征、形态特征和生理生化特征测定,鉴定为侧胞短杆芽胞杆菌(Brevi Bacillus laterosporus)。     

Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments

Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. A specific and sensitive PCR detection method that uses primers targeting the gene coding for the flagella subunit, fliC, was established. Based on the first fliC gene sequence of R. solanacearum strain K60 available at GenBank, the Ral_fliC PCR primer system was designed; this system yielded a single 724-bp product with the DNAs of all of the R. solanacearum strains tested. However, R. pickettii and four environmental Ralstonia isolates also yielded amplicons. The Ral_fliC PCR products obtained with 12 strains (R. solanacearum, R. pickettii, and environmental isolates) were sequenced. By sequence alignment, Rsol_fliC primers specific for R. solanacearum were designed. With this primer system, a specific 400-bp PCR product was obtained from all 82 strains of R. solanacearum tested. Six strains of R. pickettii and several closely related environmental isolates yielded no PCR product; however, a product was obtained with one Pseudomonas syzygii strain. A GC-clamped 400-bp fliC product could be separated in denaturing gradient gels and allowed us to distinguish P. syzygii from R. solanacearum. The Rsol_fliC PCR system was applied to detect R. solanacearum in soil. PCR amplification, followed by Southern blot hybridization, allowed us to detect about one target DNA molecule per PCR, which is equivalent to 10(3) CFU g of bulk soil(-1). The system was applied to survey soils from different geographic origins for the presence of R. solanacearum.     

Serological Strains of Tobacco Ringspot Virus Transmitted by Xiphinema americanum

Five serological strains of tobacco ringspot virus isolated from naturally infected tobacco in North Carolina, and a strain isolated from watermelon in the Rio Grande Valley of Texas were transmitted from cucumber to cucumber by mass-screened and handpicked Xiphinerna americanum from North Carolina. The Eucharis mottle strain from Peru was not transmitted, indicating that a specific strain-vector relationship may exist between the geographically isolated strains from North and South America.     

Lipopolysaccharide Composition of the Wilt Pathogen, Pseudomonas solanacearum: CORRELATION WITH THE HYPERSENSITIVE RESPONSE IN TOBACCO

In the induction of the hypersensitive response (HR) in tobacco by Pseudomonas solanacearum, the recognition between host and pathogen is thought to involve an interaction between plant lectins and bacterial lipopolysaccharide (LPS). The LPS of a series of strains of P. solanacearum were examined to determine if there are structural differences that might account for the ability or inability of these strains to induce the hypersensitive response. Analysis of the components of LPS by gas chromatography indicates a clear difference in sugar composition between the HR-inducing and non-HR-inducing strains, especially in terms of the percentage of glucose, xylose and rhamnose. Sodium dodecyl sulfate polyacrylamide gel electrophoresis shows there are two distinct kinds of LPS, differing greatly in size, which correspond to rough and smooth LPS in other systems. In addition, a phage, CH154, was isolated which lyses non-HR-inducing bacteria and which is inactivated by LPS from these bacterial strains. Therefore, differences in LPS structure correlate strongly with host recognition of Pseudomonas solanacearum.     

Partial sequencing of the hrpB and endoglucanase genes confirms and expands the known diversity within the Ralstonia solanacearum species complex

We determined partial hrpB and endoglucanase genes sequences for 30 strains of Ralstonia solanacearum and one strain of the blood disease bacterium (BDB), a close relative of Ralstonia solanacearum. Sequence comparisons showed high levels of variability within these two regions of the genome involved in pathogenicity. Phylogenetic analysis based upon sequence comparisons of these two regions revealed three major clusters comprising all Ralstonia solanacearum isolates, the BDB strain constituted a phylogenetically distinct entity. Cluster 1 and cluster 2 corresponded to the previously defined divisions 1 and 2 of Ralstonia solanacearum. Moreover, two subclusters could be identified within cluster 2. The last cluster, designated cluster 3 in this study, included biovar 1 and N2 strains originating from Africa. This recently described group of strains was confirmed to be clearly different from the other strains suggesting a separate evolution from those of both divisions 1 and 2.     

Genetic diversity of Ralstonia solanacearum strains from China assessed by PCR-based fingerprints to unravel host plant- and site-dependent distribution patterns

Bacterial wilt caused by Ralstonia solanacearum is a serious threat to crop production in China. A collection of 319 R. solanacearum strains isolated from 14 different diseased host plants collected in 15 Chinese provinces was investigated by BOX fingerprints in order to test the influence of the site and the host plant on their genetic diversity. Phylotype, fliC-RFLP patterns and biovar were determined for all strains and the sequevar for 39 representative strains. The majority of strains belonged to the Asian phylotype I, shared identical fliC-RFLP patterns and were assigned to four biovars (bv3:123; bv4:162; bv5:3; and bv6:11). Twenty strains were phylotype II, assigned to biovar 2, and had distinct fliC-RFLP patterns. BOX-PCR fingerprints generated from the genomic DNA of each strain revealed a high diversity of the phylotype I strains, where 28 types of BOX fingerprints could be distinguished. While many BOX clusters comprised isolates from different provinces and several host plants, some groups contained isolates that were plant or site specific. All phylotype II isolates originating from 10 provinces belonged to sequevar 1 and displayed identical BOX patterns as the potato brown rot strains from various regions of the world.     

A polyphasic approach for studying the interaction between Ralstonia solanacearum and potential control agents in the tomato phytosphere.

Ralstonia solanacearum biovar 2, the causative agent of brown rot in potato, has been responsible for large crop losses in Northwest Europe during the last decade. Knowledge on the ecological behaviour of R. solanacearum and its antagonists is required to develop sound procedures for its control and eradication in infested fields.A polyphasic approach was used to study the invasion of plants by a selected R. solanacearum biovar 2 strain, denoted 1609, either or not in combination with the antagonistic strains Pseudomonas corrugata IDV1 and P. fluorescens UA5-40. Thus, this study combined plating (spread and drop plate methods), reporter gene technology (gfp mutants) and serological (imunofluorescence colony staining [IFC]) and molecular techniques (fluorescent in situ hybridization [FISH], PCR with R. solanacearum specific primers and PCR-DGGE on plant DNA extracts). The behaviour of R. solanacearum 1609 and the two control strains was studied in bulk and (tomato) rhizosphere soil and the rhizoplane and stems of tomato plants.The results showed that an interaction between the pathogen and the control strains at the root surface was likely. In particular, R. solanacearum 1609 CFU numbers were significantly reduced on tomato roots treated with P. corrugata IDV1(chr:gfp1) cells as compared to those on untreated roots. Concomitant with the presence of P. corrugata IDV1(chr:gfp1), plant invasion by the pathogen was hampered, but not abolished.PCR-DGGE analyses of the tomato rhizoplane supported the evidence for antagonistic activity against the pathogen; as only weak R. solanacearum 1609 specific bands were detected in profiles derived from mixed systems versus strong bands in profiles from systems containing only the pathogen. Using FISH, a difference in root colonization was demonstrated between the pathogen and one of the two antagonists, i.e. P. corrugata IDV1(chr:gfp1); R. solanacearum strain 1609 was clearly detected in the vascular cylinder of tomato plants, whereas strain IDV1 was absent.R. solanacearum 1609 cells were also detected in stems of plants that had developed in soils treated with this strain, even in cases in which disease symptoms were absent, indicating the occurrence of symptomless infection. In contrast, strain 1609 cells were not found in stems of several plants treated with either one of the two antagonists.The polyphasic analysis is valuable for testing antagonistic strains for approval as biocontrol agents in agricultural practice.     

Ralstonia solanacearum virulence increased following large interstrain gene transfers by natural transformation

Horizontal gene transfer (HGT) is a major driving force of evolution and is also likely to play an important role in the threatening emergence of novel pathogens, especially if it involves distantly related strains with substantially different pathogenicity. In this study, the impact of natural transformation on pathogenicity in six strains belonging to the four phylotypes of the plant-pathogenic bacterium Ralstonia solanacearum was investigated. The study focused on genomic regions that vary between donor and recipient strains and that carry genes involved in pathogenicity such as type III effectors. First, strains from R. solanacearum species complex were naturally transformed with heterologous genomic DNA. Transferred DNA regions were then determined by comparative genomic hybridization and polymerase chain reaction sequencing. We identified three transformant strains that acquired large DNA regions of up to 80 kb. In one case, strain Psi07 (phylotype IV tomato isolate) acquired 39.4 kb from GMI1000 (phylotype I tomato isolate). Investigations revealed that i) 24.4 kb of the acquired region contained 20 new genes, ii) an allelic exchange of 12 genes occurred, and iii) 27 genes (33.4 kb) formerly present in Psi07 were lost. Virulence tests with the three transformants revealed a significant increase in the aggressiveness of BCG20 over its Psi07 parent on tomato. These findings demonstrate the potential importance of HGT in the pathogenic evolution of R. solanacearum strains and open new avenues for studying pathogen emergence.     

广藿香内生真菌多样性及其对青枯菌的拮抗活性

【目的】对广藿香各部位内生真菌的类群结构及多样性进行系统分析,筛选出对青枯菌具拮抗活性的菌株。【方法】采用组织块分离法从广藿香健康植株的根、茎、叶中分离内生真菌,结合形态学和基于ITS-r DNA序列分析鉴定方法分析广藿香内生真菌的类群结构及多样性,并利用双层平板拮抗法筛选对青枯菌具有抑制活性的菌株。【结果】从广藿香中共分离获得313株内生真菌,隶属于30个属,其中链格孢属(占28.75%)、拟茎点霉属(占23.00%)和炭疽菌属(11.82%)为优势类群,此外还分离到炭皮菌属、弯孢聚壳属、Gibellulopsis、新萨托菌属、葡萄座腔菌属、篮状菌属等较为少见的类群。茎、叶内生真菌的定殖率和分离率明显高于根,而根部多样性指数为2.64,要高于茎(2.00)和叶(1.97);广藿香茎与叶、根与茎、根与叶之间的内生真菌相似性指数分别为0.35、0.20、0.19,均小于0.5,显示各部位之间的内生真菌组成不相似程度高。从313株广藿香内生真菌中通过拮抗实验筛选到16株对青枯菌有拮抗活性的菌株,其中GHXR07、GHXR27和GHXR29的拮抗活性尤为显著,分别被鉴定为Talaromyces sp.、Myrothecium roridum Tode和Talaromyces wortmannii(Kl?cker)Benjamin。【结论】广藿香内生真菌具有丰富的物种多样性且其类群分布具有一定的组织特异性,其中部分菌株对青枯菌具有明显的拮抗活性。