应用分子生物学方法快速检测根癌土壤杆菌

根据根癌土壤杆菌（Agrobacterium tumefaciens）VirC2基因序列,设计并合成了特异性PCR检测引物,对根癌土壤杆菌及感病植物的肿瘤组织进行了PCR扩增反应.结果表明,根癌土壤杆菌及植物肿瘤组织的PCR产物均出现500bp的特异性扩增条带,而非根癌土壤杆菌均未出现扩增条带,证明这对引物具有根癌土壤杆菌鉴定特异性.将分离的根癌土壤杆菌做梯度稀释,测定该检测体系的敏感度.结果表明,此体系可检出105cfu/mL根癌土壤杆菌菌液提取的模板.研究表明,分子生物学方法是一种特异、敏感、快速的根癌土壤杆菌检测方法.     

Induction of systemic resistance to Agrobacterium tumefaciens by endophytic bacteria in grapevine

Crown gall disease of grapevine, caused by Agrobacterium tumefaciens, often results in severe economic loss to grape production worldwide. This study demonstrated the ability of the endophytic bacteria Pseudomonas sp. Sn48 and Pantoea sp. Sa14 isolated from domesticated and wild grapevines to induce resistance in both above- and belowground tissues of grapevines infected with A. tumefaciens. Our results provide evidence that both strains can colonize roots and/or shoots. We showed that the strains Pseudomonas sp. Sn48 and Pantoea sp. Sa14 are capable of inducing stilbenic phytoalexin production in grapevine tissues and to further prime plantlets for enhanced phytoalexin production after A. tumefaciens inoculation. We also showed that in the majority of treatments, polyamine accumulation remained unchanged or slightly increased in plantlets treated with Pseudomonas sp. Sn48 and Pantoea sp. Sa14 compared with the control. Our findings indicated that the levels of polyamines remain unchanged or significantly decrease in plantlets treated with endophytic bacteria after A. tumefaciens challenge compared to the control and plantlets treated with individual endophytic bacterial strains. PR1, PR2, and PR4 gene expression levels of plantlets treated with Pseudomonas sp. Sn48 and Pantoea sp. Sa14 significantly increased after A. tumefaciens inoculation. The findings revealed the efficacy of the selected endophytic bacteria in triggering grapevine resistance against A. tumefaciens and the possible use of these strains as an alternative to chemical control methods in grapevine crown gall disease management.     

Detection of Xylophilus ampelinus in grapevine cuttings using a nested polymerase chain reaction

A sensitive and specific assay was developed to detect bacterial blight of grapevine caused by Xylophilus ampelinus (Panagopoulos, 1969) comb. nov. in grapevine cuttings. The 16S−23S rDNA intergenic spacer region of X. ampelinus was sequenced and pathogen-specific primers were designed from a region in the spacer between the tRNA (Ala) and the 23S genes. A nested PCR (n-PCR) reaction was applied with a first-stage PCR using universal primers within the ends of the 16S and 23S genes, followed by a second-stage PCR with nested primers specific to the X. ampelinus spacer region. A 277-bp fragment was amplified from 38 Xylophilus strains tested, but not from saprophytes associated with grapevine or phylogenetically related phytobacteria. The 277-bp product was shown to be derived from the X. ampelinus spacer region by restriction with Dra I, Sau 3AI, Taq I and Msp I, Southern hybridization and genomic DNA dot blots. When the (n-PCR) procedure was applied in the absence of nontarget DNA, the limit of detection was less than 10 colony-forming units (CFU) per µ L. The same number of  X. ampelinus CFU could be detected in the presence of 1·5 × 10⁵ CFU  µ L⁻¹ of Erwinia herbicola cells using the n-PCR procedure.     

Susceptibility of stone-fruit rootstocks, rose and grapevine to Agrobacterium radiobacter var. tumefaciens in Arab Mediterranean countries

Crown gall was found in all fruit-growing areas in Algeria, Jordan, Morocco and Tunisia. In the last three countries, shoot and/or root inoculation of stone fruits, rose and/or grapevine (according to country) with Agrobacterium radiobacter var. tumefaciens showed that all species were susceptible independently of country and origin of bacterial strains, with the exception of the Beldi land race of apricot in Morocco, which was tolerant. The level of susceptibility varied according to the strains used.     

Problems of Agrobacterium tumefaciens in planting material and its control1

Crown gall caused by Agrobacterium tumefaciens is a problem in many European nurseries especially on stone fruits, apples and some herbaceous hosts (biovars 1 and 2) and on grapevines (biovar 3). Nursery infection can arise from land infested with the pathogen. Disease incidence can be reduced to some extent by cultural methods such as crop rotation, minimizing wounding and selection of less susceptible cultivars. The best control of infection is the use of a pre-planting dip in the antagonist A. radiobacter strain 84, which gives excellent control of strains of the pathogen harbouring the nopaline-type Ti plasmid. The antagonist is not yet cleared for use in all European countries. This biological control is sometimes ineffective because of the presence of mixed populations of sensitive and resistant strains of the pathogen, use of too low inoculum doses of the antagonist or because the host plant affects the antagonist action. All controls are preventive and not curative, so latent infections in layer or stool beds cannot be prevented from developing. Control of the strains of the pathogen insensitive to strain 84 may be possible in future by the use of other antagonistic organisms or by genetic manipulation of strain 84. Until such a new development becomes available, growers must make careful selection of propagating material to minimize the risk of spreading infection.     

Results of seven years of biological control of Agrobacterium tumefaciens in Spain1

Biological control of Agrobacterium tumefaciens using the K84 strain of A. radiobacter has been practised in Spain since 1979. Strain K84 is produced in Sevilla by the Agricultural Research Service and is mixed with peat. This formulation is authorized for use on stone fruits and roses, since results of various trials on rootstocks of these crops have shown that K84 is effective in controlling the disease in naturally infected nursery soil, as well as in experimental tests carried out on artificially inoculated soil. Results of biological control trials have shown that K84 has some effect on A. tumefaciens strains resistant to agrocin 84. Factors other than susceptibility to agrocin 84 influence the efficacy of biological control. This was demonstrated by using a K84 agrocin non-producer strain which proved effective against both resistant and susceptible strains. Biological control on apple or pear is not yet authorized in Spain due to a paucity of data on field trials. However, initial pot tests showed good K84 activity on these hosts, against Spanish strains of A. tumefaciens. On grapevine, most of the A. tumefaciens strains that were isolated belong to biotype 3 and are resistant to agrocin 84. Because the disease is systemic on this species the use of K84 is not authorized.     

Crown gall of hop caused by Agrobacterium tumefaciens biovar 1 in South Africa

Crown gall of hop caused by Agrobacterium tumefaciens biovar 1 is reported for the first time from South Africa. The causal organism was inhibited in vitro by the agrocin of A. radiobacter strain D286 but not by that of the control strain K84. Nevertheless, control was achieved on hop stems in glasshouse inoculations by both biological control strains.     

桃树根癌病防治技术探讨

桃树根癌病是公认最难防治的土传细菌病害之一。在对该病进行较系统研究的基础上,采用多种抗菌药剂、多种方法进行了室内、外药敏测定与防治试验,显示出异菌氰、次氯酸钠、乙蒜素、妥布霉素、环丙沙星、庆大霉素及其混用对防治桃树根癌病菌有明显防效。     

Opine utilization by Spanish isolates of Agrobacterium tumefaciens

Among 115 Spanish isolates of Agrobacterium tumefaciens from 13 hosts and 47 locations, biotypes 1, 2, and 3 were identified. Most isolates were of biotype 2. Biotype 3 isolates were obtained only from grapevine, and all were resistant to agrocin 84. Nopaline was utilized by 84% of isolates. Only three isolates did not utilize either octopine, nopaline or mannopinic acid.
Isolates from fruit trees were mostly biotype 2 and utilized only nopaline; some isolates from a peach x almond hybrid utilized mannopinic acid. Rose and osier isolates were biotype 1 and utilized only nopaline. Most grapevine isolates were biotype 3, but varied widely in opine utilization.
With one exception all isolates sensitive to agrocin 84 catabolized nopaline; no biotype 3 strains catabolized mannopinic acid and no biotype 1 or 2 strains catabolized octopine.     

葡萄带根嫁接苗的生产及主要病毒的检测方法

葡萄主要分布在北半球。按地理分布和生态特点 ,一般把葡萄属各个种划分为 3大种群 ,即欧洲种群 ,北美种群和东亚种群。欧洲种群葡萄里仅有一种Vitisvinifera ,即我们通常所称的欧洲葡萄 ,世界许多著名的葡萄鲜食和加工品种大多属本种 ;北美种群的葡萄在改良葡萄的品质和增强抗逆性方面起着重要的作用 ,常用作砧木 ;亚洲种群的葡萄主要用于培育一些抗寒的杂交品种[1] 。近几年 ,我国葡萄酿酒业发展很快 ,为满足酿酒的需求 ,从国外引进了多种优良的酿酒葡萄品种 ,其中从法国引进的酿酒葡萄品种中 ,就有带根的嫁接葡萄苗 ,这种葡萄苗易于成活…     

Evidence of Migration and Endophytic Presence of Agrobacterium tumefaciens in Rose Plants

Agrobacterium tumefaciens was isolated from stem tumors of several rose cultivars showing that the bacterium is the causal agent of aerial galls in rose plants. No differences were observed in the characteristics of the Agrobacterium isolates from crown or aerial galls. Stem inoculation of ten rose cultivars showed that all of them were susceptible to A. tumefaciens but differences in the size of the resulting tumors were observed. The movement of A. tumefaciens in rose plants was demonstrated using two wild type strains and two antibiotic resistant mutants. Three months after inoculation, the inoculated strains were recovered in the roots, crown and below and above the inoculation site but low numbers of pathogenic Agrobacterium cells were isolated. New tumors appeared in 5% of the noninoculated wounds. A. tumefaciens was isolated from the stem at different distances from the tumor in naturally infected plants. In symptomless commercial plants, the isolation from the roots, crown and at different stem levels demonstrated the existence of systemic and latent infections in rose. Direct isolation using a nonselective and selective media with or without a previous enrichment step were efficient methods for isolating tumorigenic Agrobacterium from the different parts of rose plants.     

Resistance of rose rootstocks to crown gall (Agrobacterium tumefaciens)

Various types of rose rootstocks were tested for their resistance to crown gall. The rootstock Iowa State University (ISU) 60–5 was the most resistant, followed by Brooks 48, Clarke 1957 and Welch. Rosa multiflora, R. noisettiana (Manetti) and Basye No. 3 were very susceptible. The inoculations were made with four isolates ofAgrobacterium tumefaciens (Smith et Townsend) Conn, respectively from aDahlia sp.,Rosa spp. andPrunus persica. It was found that the isolate fromDahlia was a different race to the isolates fromRosa andPrunus spp.Samenvatting Bij onderstammen van rozen, kunstmatig geïnoculeerd metAgrobacterium tumefaciens (Smith et Townsend) Conn., werden verschillen in resistentie tegen wortelknobbel gevonden. Het meest resistent was Iowa State University (ISU) 60–5, gevolgd door Brooks 48, Clarke 1957 en Welch, Zeer vatbaar warenRosa multiflora, R. noisettiana (Manetti) en Basye No. 3. De vier isolaten vanA. tumefaciens, gebruikt voor de inoculaties, waren respectievelijk afkomstig van eenDahlia sp.,Rosa spp. enPrunus persica. Het isolaat vanDahlia en de isolaten vanRosa enPrunus spp. behoorden tot twee verschillende fysiologische rassen. De vorming van tumoren was in sommige gevallen afhankelijk van de methode van inoculatie; inoculaties bij de stambasis waren meer succesvol dan in het midden van de stam.     

Eucalyptus occidentalis plantlets are naturally infected by pathogenic Agrobacterium tumefaciens

In the Sidi M’djahed nursery (Algeria), over 60,000 eucalyptus (Eucalyptus occidentalis) plantlets exhibited tumour-like growths localized at the crown of the plants that resembled crown galls caused by Agrobacterium tumefaciens. Bacteria colonizing the galls were isolated and purified. Most (22 out of 24) of the isolates had cultural and biochemical characteristics similar to those of strains of the biovar 1 of A. tumefaciens. Twenty out of 22 Agrobacterium isolates induced tumour formation on various test plants. In PCR experiments, DNA extracted from these virulent strains yielded an amplification signal corresponding to a 247-bp fragment located within the virulence region of nopaline type Ti plasmid. Consistent with this, the opine nopaline was detected in the tumours induced on test plants – but not on eucalyptus plants. Nopaline was degraded by the 20 pathogenic isolates that were also sensitive to agrocin 84, indicating the presence of a nopaline-type pTi in these strains. The chromosomal region encoding the 16S rRNA was analyzed in a sub-population of the pathogenic agrobacterial isolates. The analyzed strains were found to belong to the ribogroup of the reference strain B6. Interestingly, Eucalyptus camaldulensis and Eucalyptus cladocalyx grown in the same nursery and in the same soil substrate developed no galls.     

50%氯溴异氰尿酸防治月季根癌病

田间药效试验表明,50％氯澳异氰尿酸水溶性粉剂1000倍液,采用灌根方法施药,防治月季根癌病效果为66.67％-85.71％,持效期为15 d,防效显著高于72％农用链霉素水溶粉剂1000倍液的50.00％-64.29％,同时具有促进月季生长的作用,可使月季增高6.79％、花朵数增加10.26％、花朵直径增大5.36％。     

Isolation and Characterization of a New Virulence Gene (abvA) of Agrobacterium tumefaciens

A mutant (M-1) was isolated by transposon (Tn5) insertion mutagenesis of Agrobacterium tumefaciens (strain A-208, C58 chromosome, nopaline type T37 pTi, virulent). The M-1 mutant exhibited a complete avirulent phenotype on Kalanchoe daigremontiana leaf and Kalanchoe pinnata stem but a very attenuated virulent phenotype on root of Daucus carota. The mutant had one insertion of Tn5 in pTi. A wild-type target segment (2.3 kb) that included the site of Tn5 insertion in M-1 mutant was cloned. Introducing the 2.3 kb segment into M-1 complemented completely the avirulent phenotype, producing galls as big as strain A-208. The 2.3 kb segment was sequenced, identifying three open reading frames, ORF 1 (354 bp), ORF 2 (261 bp) and ORF 3 (801 bp) in the segment. A Tn5 was inserted between the third and fourth nucleotide of ORF 1 in M-1. The ORF 1 had no homology to any reported genes and thus was named the abvA gene. The ORF 3 had the high homology (identities 44%, positive 68%) to the gene of the sarcosine oxidase β subunit (accession no. sp/P40875). Introduction of the DNA segment (743 bp) containing the abvA gene and its promoter region into M-1 partially complemented the avirulent phenotype of the mutant, producing galls smaller than strain A-208. The abvA gene was distributed not only on nopaline-type pTi (T37) but also on octopine-type pTi (A6NC) and chromosome (C58) of A. tumefaciens. M-1, being avirulent on K. daigremontiana and K. pinnata, had a Tn5 insertion only in the abvA gene on pTi but not in the abvA gene on the chromosome, implying that the abvA gene on the chromosome in strain A-208 is not functional. A binary vector, pIG121-Hm, containing the β -glucuronidase (GUS) gene with an intron was introduced into M-1, which was then applied to leaves of K. daigremontiana to assay GUS activity for monitoring T-DNA transfer to the host nucleus. High GUS activity comparable to that in strain A-208 was detected in M-1 in spite of its inability to induce galls, suggesting that M-1 can transfer T-DNA into the host nucleus, but cannot integrate it into the chromosome. Received 25 October 2000/ Accepted in revised form 28 December 2000     

根癌病生防菌——葡萄土壤杆菌E26菌株在葡萄植株的定殖研究

 利用绿色荧光蛋白基因(gfp)标记示踪,研究了葡萄根癌病生防菌葡萄土壤杆菌E26菌株应用到田间后在玫瑰香葡萄(Vitis vinifera cv. Muscat Humbug)根表面和根际土壤中的群体数量变化,比较了E26菌株与葡萄根癌病原菌K308菌株室内人工接种后在玫瑰香葡萄苗茎和根外植体伤口部位的附着情况.在田间自然状况下,E26菌株可以在葡萄根表面和根际土壤中存活定殖.接种5个月后,E26在根表面的平均数量为10⁴cfu/g根(鲜重),在根际土壤中的平均数量为10⁴cfu/g土壤(干重).在室内,E26菌株和K308菌株分别单独接种时均能以相似水平附着在葡萄茎和根的伤口;E26和K308以相同数量同时接种时,附着在葡萄伤口细胞的K308的数量显著低于K308单独接种时所附着在葡萄伤口细胞的数量.扫描电镜显微观察证实E26菌株能够和病菌K308菌株一样附着于葡萄根部伤口处.