Application of alginate gel for protection of wounds against crown gall (Agrobacterium tumefaciens)

In glasshouse and laboratory experiments, calcium alginate gel, produced in situ on wounds by successive dipping in solutions of sodium alginate and CaCl₂ gave significant protection against infection by pathogenic isolates of Agrobacterium tumefaciens on carrot discs, tomato cuttings and chrysanthemum cuttings. Gel protection was sometimes equal to that given by strain K84 of A. radiobacter and also operated against a pathogenic isolate insensitive to control by strain K84. Sodium alginate or CaCl₂ alone gave little or no protection, suggesting that calcium alginate acted by physically excluding the pathogen from wound surfaces. The use of the method described is discussed for delivery of biological or chemical control agents to plant surfaces whilst also providing protection in its own right.     

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.     

Biological control of crown gall in rose nursery stock

Rooted plants and cuttings ofRosa indica were dipped or sprayed withAgrobacterium radiobacter strain 84 and planted in a commercial rose nursery. Crown gall was controlled effectively and equally well when the protectant inoculum was grown in broth or on agar and used for treating plants which were immediately planted in naturally infested soil (mean disease control was 91%). Untreated rooted-plant stock and unrooted cuttings had 20% and 0.7% galled plants, respectively, at the end of 8 months in the field; plants and cuttings dipped inA. radiobacter} inoculum had 0.6% and 0.1% galled plants, respectively. Spraying with inoculum also gave effective control of the disease. Fewer plants of the untreated nursery stock survived the 8 months between planting and harvesting than treated stock.     

A short‐length single chimeric transgene induces simultaneous silencing of Agrobacterium tumefaciens oncogenes and resistance to crown gall

Transformation with self‐complementary oncogene sequences was used to silence the Agrobacterium tumefaciens oncogenes ipt and iaaM. The silencing response was triggered by using a very short chimeric sequence where conserved fragments from both oncogenes were fused in one unique transgene. Most T₀ transgenic tobacco lines and T₁ seedlings evaluated in vitro had intermediate or very low susceptibility to A. tumefaciens as compared with the wildtype plants. A greenhouse evaluation of whole plants confirmed the lines that were resistant. Low levels of transgene hairpin RNA (hpRNA) coupled with small interfering RNA (siRNA) accumulation correlated with oncogene silencing and, therefore, resistance to crown gall. After infection with the oncogenic strain, much lower levels of the oncogenes’ mRNA were found in resistant lines than in wildtype plants. The frequency of resistant lines, with few or no symptoms, produced with the chimeric construct was similar to the highest reported efficiencies obtained by using sense and antisense whole oncogene sequences.     

Biological control of crown gall of grapevine, rose, and tomato by nonpathogenic Agrobacterium vitis strain VAR03-1

A nonpathogenic strain of Agrobacterium vitis VAR03-1 was tested as a biological control agent for crown gall of grapevine (Vitis vinifera). When roots of grapevine, rose (Rose multiflora), and tomato (Lycopersicon esculentum) were soaked in a cell suspension of antagonists before planting in soil infested with tumorigenic A. vitis, A. rhizogenes, and A. tumefaciens, respectively, treatment with VAR03-1 significantly reduced the number of plants with tumors and disease severity in the three plant species. The inhibitory effects of treatment with VAR03-1 and the nonpathogenic A. rhizogenes strain K84 on crown gall of rose and tomato were almost identical, and the inhibitory effect of VAR03-1 on grapevine was superior to that of K84. Moreover, VAR03-1 greatly controlled crown gall of grapevine due to tumorigenic A. vitis in the field. VAR03-1 established populations averaging 10(6) colony forming units (CFU)/g of root in the rhizosphere of grapevine and persisted on roots for 2 years. VAR03-1 was bacteriocinogenic, producing a halo of inhibition against those three species of Agrobacterium. This is the first report that a nonpathogenic strain, VAR03-1, can effectively control crown gall caused by tumorigenic A. vitis, A. rhizogenes, and A. tumefaciens.     

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.     

The presence of crown gall of grape incited byagrobacterium tumefaciens biovar 3 in Israel

Crown gall was previously reported on grape in Israel but the pathogen was not isolated and characterized. The three recognized biovars ofAgrobacterium tumefaciens can be tumorigenic on grape, but biovar 3 is the most important world wide. A single occurrence of tumors in a vineyard yielded bacteria which incited galls on grape,Nicotiana glauca and tomato, but not on bryophyllum. The bacteria were confirmed asA. tumefaciens because they contained DNA which hybridized with T-DNA from a Ti plasmid. Biochemical and physiological tests, octopine production and utilization, and agrocin 84 insensitivity conformed with those of bv. 3. Subsequent occurrences of the grape disease have not been found, but the presence ofA. tumefaciens bv. 3 in Israel is a potential threat to nurseries and vineyards.     

Inhibition of crown gall formation by Agrobacterium radiobacter biovar 3 strains isolated from grapevine

Graft unions of nursery stock of grapevine (Vitis vinifera L.) collected in Japan yielded pathogenic and nonpathogenic strains of Agrobacterium. On the basis of classical diagnostic tests, a sequence analysis, and a multiplex polymerase chain reaction method previously reported, the pathogenic strain was identified as Agrobacterium tumefaciens biovar 3, whereas the nonpathogenic strains were assigned to Agrobacterium radiobacter biovar 3. Stems of tomato (Lycopersicon esculentum Mill.) seedlings were inoculated with both A. tumefaciens biovar 3 strain G-Ag-27 as a pathogen and one of the control strains isolated from grapevine or A. radiobacter biovar 2 strain K84 as competitors to assay the suppression of gall formation caused by the pathogen. In a test with a 1 : 1 pathogen/nonpathogen cell ratio, all A. radiobacter biovar 3 strains reduced gall incidence and size compared to that of the positive control inoculated only with the pathogen. Strain VAR03-1 was especially effective in reducing the incidence of gall formation on grapevine and reduced gall size by 84%–100% of those on the positive control. Many tested nonpathogenic biovar 3 strains were bacteriocinogenic, causing an inhibition zone against A. tumefaciens biovar 3 strains on YMA medium. Strain VAR03-1 was the most effective against indicator strains and appears to be a promising agent for controlling crown gall of grapevine.     

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.     

Inhibitory effect of ACC deaminase‐producing bacteria on crown gall formation in tomato plants infected by Agrobacterium tumefaciens or A. vitis

This study showed that various rhizosphere bacteria producing the enzyme 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase (ACCD), which can degrade ACC, the immediate precursor of ethylene in plants, and thereby lower plant ethylene levels, can act as promising biocontrol agents of pathogenic strains of Agrobacterium tumefaciens and A. vitis. Soaking the roots of tomato (Solanum lycopersicum) seedlings in a suspension of the ACCD‐producing Pseudomonas putida UW4, Burkholderia phytofirmans PsJN or Azospirillum brasilense Cd1843 transformed by plasmid pRKTACC carrying the ACCD‐encoding gene acdS from UW4, significantly reduced the development of tumours on tomato plants injected 4–5 days later with pathogenic Agrobacterium strains via wounds on the plant stem. The fresh mass of tumours formed by plants pretreated with ACCD‐producing strains was typically four‐ to fivefold less than that of tumours formed on control plants inoculated only with a pathogenic Agrobacterium strain. Simultaneously, the level of ethylene evolution per amount of tumour mass on plants pretreated with ACCD‐producing bacteria decreased four to eight times compared with that from tumours formed on control plants or plants pretreated with bacteria deficient in ACCD production. Moreover, transgenic tomato plants expressing a bacterial ACCD were found to be highly resistant to crown gall formation relative to the parental, non‐transformed tomato plants. The results support the hypothesis that ethylene is a crucial factor in Agrobacterium tumour formation, and that ACCD‐produced rhizosphere bacteria may protect plants infected by pathogenic Agrobacteria from crown gall disease.     

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.     

葡萄根癌病生防菌株E26中可接合转移的遗传因子的检测及功能初步分析

葡萄土壤杆菌E26是一株效果良好的葡萄根癌病生物防治菌株。本研究结合利用Tn5转座子插入突变与生物化学检测手段对E26菌株中的可接合转移的遗传因子进行了检测和功能初步分析。结果显示:野生菌株E26中携带有能够接合转移到其近缘细菌菌株Agrobacterium tumefaciens NTL4的遗传因子,该遗传因子可能是一个功能未知的质粒,该质粒含有可以分解5-溴-4-氯-3-吲哚-β-半乳糖苷(X-gal)的基因。     

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     

Mutations affecting chemotaxis of Agrobacterium vitis strain E26 also alter attachment to grapevine roots and biocontrol of crown gall disease

Crown gall in grapevine, caused by tumorigenic Agrobacterium vitis strains, can cause severe losses in most viticulture regions worldwide. The only effective means of control is through cultivation practices. One non-tumorigenic A. vitis strain, E26, can prevent crown gall infection when applied to wounds on grapevine prior to or simultaneous with inoculation of tumorigenic strains. ME19, a Tn5 mutant of strain E26, was impaired in terms of its ability to be chemo-attracted by grapevine root tissue extracts and its attachment to grapevine roots, and had reduced biological control activity; it did not significantly differ from the wild-type strain of E26 in phenotypes of agrocin production, growth in minimum medium or swarming activity. Complementation of ME19 with the cosmid clone of CP1543 from an E26 DNA library restored the chemotaxis, attachment, and biocontrol phenotypes. A 7·3-kb Kpn I fragment from CP1543 was cloned and sequenced, and sequence analysis revealed that the Tn 5 insertion occurred in a region that shares a significant homology with genes coding for methyl-accepting chemotaxis proteins (MCPs) in many bacteria. Complementation of the mcp gene mutants restored the affected phenotypes to the level of wild-type E26. An in-frame deletion mutant of the mcp gene was generated and was determined to have the same phenotypes as the original Tn5 mutant.