Bisanthraquinones,Inhibitors of Plant Transformation

A potato tuber disk assay of culture broths which had been confirmed to show neither antibacterial nor phytotoxic activity resulted in the discovery of a novel type of crown gall formation inhibitors, julimycin B-II and julichrome Q_1.3 These two bisanthraquinones, products of Streptomyces sp. TM-71, inhibited crown gall formation on potato tuber disks at a minimum inhibitory dose of 1.6 μg/disk without affecting the growth of Agrobacterium tumefaciens or the germination of alfalfa seeds.     

The Effect of Bacillus subtilis on in vitro Growth and Pathogenicity of Agrobacterium tumefaciens

Five isolates of Bacillus subtilis isolated from the soil, were found to be antagonistic to 6 isolates of Agrobacterium tumefaciens in vitro. Inoculation of B. subtilis in wounded castor bean plants 30 min before or simultaneous inoculation with A. tumefaciens resulted in excellent control of the crown gall symptoms on the host within 50 days of inoculation. Application of B. subtilis 30 min after inoculation with A. tumefaciens did not result in appreciable disease reduction. Treatment of the tested plants by B. subtilis did not induce any phytotoxic injury or growth retarding side effects. It appears that B. subtilis could potentially be incorporated for crown gall control. However, further tests are needed to test this biological control agent with other plant species especially fruits, nuts and vine nursery stock.     

Evaluation of Biological and Chemical Treatments for Control of Crown Gall on Young Apple Trees in the Kootenay Valley of British Columbia

Field trials at Creston in 1988, 1989, and 1990 on silty loam soil naturally infested with Agrobacterium tumefaciens showed that Agrobacterium radiobacter strain K84 did not control crown gall on young Antonovka apple trees when apphed as a root-dip treatment. Copper oxychloride applied as a root-dip treatment at 2.5 and 5.0 g ai/1 and sewage sludge applied at 260 g per tree as broadcast were effective in reducing crown gall infection but these treatments were toxic to young apple trees in 1989. Lower rates of copper oxychloride, 0.3, 0.6 and 0.9 g ai/1, and sewage sludge at 130 g per tree, did not control crown gall in the 1990 field trial. The biological treatment with the strain AB8 of Bacillus subtilis applied as a root-dip effectively controlled crown gall and was not phytotoxic to young Antonovka apple trees. These results suggest that strain AB8 of B. subtilis has the potential to control crown gall on young apple trees under field conditions.     

Antibacterial Activity of Synthetic Peptides Against Plant Pathogenic Pectobacterium Species

The aim of the work was to check the antibacterial activity of three synthetic peptides: CAMEL, Iseganan and Pexiganan as well as their possible application against plant pathogenic bacteria from the species Pectobacterium carotovorum (Pc) and Pectobacterium chrysanthemi (Pch). The antibacterial activity of the three chosen synthetic peptides was evaluated with the use of two tests: minimal inhibitory concentration and minimal bactericidal concentration. The CAMEL proved to be the most effective peptide, inhibiting the growth of different species of Pectobacterium in concentrations ranging from 2 to 8 μg/ml. Iseganan and Pexiganan also demonstrated activity against Pectobacterium sp., but it was lower than CAMEL. The CAMEL was able to inhibit Pc and Pch bacterial growth and tissue maceration in pathogenicity tests performed on potato tuber slices.     

Modulation of the venation pattern of cotyledons of transgenic tobacco for the tumorigenic <Emphasis Type="Italic">6b</Emphasis> gene of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> AKE10

Neoplastic plant-tissue formation, termed crown gall disease, is induced on infection with Agrobacterium tumefaciens. The tumorous tissues develop an extensive vascular system, with a venation pattern distinct from that of native host plants. We report here that the plant-tumorigenic 6b gene of the A. tumefaciens strain AKE10 is capable of inducing extensive vein formation in transgenic tobacco seedlings with distinct pattern formation. Unlike the wild-type cotyledons, transgenic cotyledons had wavy and striate veins depending on the extent of severity of leaf morphology. Graph analysis of the transgenic cotyledonous vein patterns revealed an increase in the number of branch points of veins, end-points of veins, and areas surrounded by the veins. Histological analysis showed abnormal tissue growth on the abaxial side of the cotyledon blades and continual formation of adventitious veins. These adventitiously formed veins included inverted dorso-ventrality and formation of a radial axis.     

Nigrosporins A and B,New Phytotoxic and Antibacterial Metabolites Produced by a Fungus Nigrospora oryzae

Nigrosporin A and B, two new phytotoxic and antibacterial metabolites were isolated from a culture filtrate of Nigrospora oryzae.

The active principles were absorbed on XAD-2 resin and purified by successive ODS-HPLC. The structures were identified by spectroscopic and derivatization analysis as naphthoquinone derivatives. The substances showed phytotoxic activities, such as root elongation inhibition, necrotic effects, oxygen evolution inhibition, starch synthesis inhibition, and CO₂ fixation inhibition at concentrations of 10–100 ppm. They also showed growth inhibition activity against Bacillus subtilis in a disc diffusion assay as well as when compared with streptomycin.     

Evaluation of 12 β-lactam antibiotics for Agrobacterium-mediated transformation through in planta antibacterial activities and phytotoxicities

The antibacterial activities of 12 beta-lactam antibiotics against Agrobacterium tumefaciens strains LBA4404 and EHA101 living in tobacco (Nicotiana tabacum L.) leaf tissues, and their phytotoxicities to tobacco leaf tissues were evaluated. All beta-lactams at minimum bactericidal concentration (MBC) or higher showed weak bactericidal activities against agrobacteria persisting in tobacco leaf tissues. The beta-lactams evaluated were classified into two major groups according to their inhibitory effect on shoot regeneration of tobacco leaf tissues: (1) highly phytotoxic drugs, and (2) moderately phytotoxic drugs. According to these results, suitable kind and concentration of beta-lactam antibiotics were evaluated for Agrobacterium-mediated transformation.     

Thyme and suico essential oils: promising natural tools for potato common scab control

• Potato common scab is a worldwide disease mainly caused by Streptomyces scabiei. It seriously affects potato crops by decreasing tuber quality. Essential oils (EO) are natural products with recognised antimicrobial properties. In this research, the antibacterial activities of thyme, oregano, suico and mint EO against S. scabiei were analysed.
• Infected tubers and soil samples were used for bacterial isolation; the obtained isolates were genetically identified. The chemical composition of the EO was determined by GC‐MS. The broth microdilution method was used to analyse antibacterial properties of EO.
• Thirty‐one bacterial isolates were obtained. The isolate chosen for antibacterial assays was morpho‐physiologically and genetically identified as S. scabiei. Thyme EO was mainly composed of thymol and o‐cymene; suico EO of dihydrotagetone, trans‐tagetone and verbenone; oregano EO of trans‐sabinene hydrate, thymol and ?‐terpinene; and mint EO of menthone and menthol. All the EO tested were effective against S. scabiei, but thyme and suico EO were the most successful, with a minimum inhibitory concentration of 0.068 g·l^?1 and 0.147 g·l^?1, respectively, and a minimum bactericidal concentration of 0.137 g·l^?1 and 0.147 g·l^?1, respectively. Scanning electron microscopy showed similar damage caused by both thyme and suico EO to the bacterial envelope. Total phenolic content of EO was not related to their antibacterial activity.
• Thyme and suico EO are effective antibacterial agents against S. scabiei, impeding bacterial viability and disturbing the bacterial cell envelope. These EO are promising tools for control of potato common scab.

     

Studies on superoxide dismutase activities in virulent and avirulent strains of Agrobacterium tumefaciens and also in normal and crown gall tumor cells of Bryophyllum calycinum

Superoxide dismutase activity in virulent strains of Agrobacterium tumefaciens was found to be higher than that in avirulent strains. Polyacrylamide gel electrophoresis revealed two isoenzymes in both these strains. These isoenzymes are suggested to be iron and manganese containing superoxide dismutases. Crown gall tumor cells of the plant Bryophyllum calycinum were found to have higher superoxide dismutase activity than the normal plant cells. Polyacrylamide gel electrophoresis revealed two isoenzymes in both normal and crown gall tumor cells. Advantages of the higher superoxide dismutase activities in respect of the survival of virulent strains of A. tumefaciens and crown gall tumor growth have been discussed.     

DNA from Agrobacterium rhizogenes in transferred to and expressed in axenic hairy root plant tissues

Summary Axenic root tissue cultures were established from primary hairy roots induced on carrot and potato by Agrobacterium rhizogenes strain 15834. cDNA made towards poly-A⁺ RNA isolated from these tissues, hybridized with a limited number of well-defined fragments of the plasmid DNA present in the inciting A. rhizogenes strain. These data therefore demonstrate that at least part of the rootinducing (Ri) plasmid of Agrobacterium rhizogenes is transferred, stably maintained and expressed in hairy-root plant tissues and confirm that hairy roots are a special type of crown gall. The T-DNA in hairy-root cells appears to have several regions which are related in terms of sequence homology and probably also function to the T-DNA in octopine and nopaline crown gall tumours.     

ACC deaminase from plant growth-promoting bacteria affects crown gall development

In addition to the well-known roles of indoleacetic acid and cytokinin in crown gall formation, the plant hormone ethylene also plays an important role in this process. Many plant growth-promoting bacteria (PGPB) encode the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which can degrade ACC, the immediate precursor of ethylene in plants, to alpha-ketobutyrate and ammonia and thereby lower plant ethylene levels. To study the effect of ACC deaminase on crown gall development, an ACC deaminase gene from the PGPB Pseudomonas putida UW4 was introduced into Agrobacterium tumefaciens C58, so that the effect of ACC deaminase activity on tumour formation in tomato and castor bean plants could be assessed. Plants were also coinoculated with A. tumefaciens C58 and P. putida UW4 or P. putida UW4-acdS- (an ACC deaminase minus mutant strain). In both types of experiments, it was observed that the presence of ACC deaminase generally inhibited tumour development on both tomato and castor bean plants.     

Biochemical and genetic characterisation of Agrobacterium tumefaciens the causal agent of walnut crown gall disease in Iran

In 2009–2010, crown tumours were collected from walnut (Juglans regia L.) trees in northern Iran. Gram-negative, rod shaped and aerobic bacteria with circular, convex and white-coloured colonies on potato dextrose agar plus CaCO3 medium were isolated from galls. In pathogenicity tests, tomato seedlings were inoculated with all strains and tumours started to appear three weeks after inoculation. Strains yielded a 224?bp amplicon from the virD2 gene in PCR. When the 16S rRNA gene sequence of strains was compared by BLASTn with nucleotide sequences from GenBank, it showed 99.6% identity with the 16S rRNA sequence of Agrobacterium tumefaciens ATCC 33970. Based on phenotypic and genotypic properties, the bacterium that causes crown gall of walnut trees was identified as A. tumefaciens.     

Biological Control of Crown Gall: Field Measurements and Glasshouse Experiments

S ummary : An ecological study showed that the pathogeniec form of Agrobacterium radiobacter (A.r. var. tumefaciens ), biotype 2 was present in soil near galled almond seedlings, where it formed a significant proportion of the total biotype 2 population; the pathogen was not detected near healthy plants. One nonpathogenic biotype 2 isolate was able, when present in numbers approximately equal to the pathogen, to prevent crown gall induction in tomato and peach seedlings. This isolate gave complete biological control of crown gall in a glasshouse test. Six other nonpathogenic isolates did not significantly inhibit gall induction by equal numbers of the pathogen. In the field, gall induction probably depends on the size of the population of the pathogen (both absolutely and relative to that of the nonpathogen) at a wound site and on the inhibitory efficiency of the nonpathogen.     

Quorum quenching by Bacillus cereus U92: a double-edged sword in biological control of plant diseases

In the present survey, quorum quenching activity was examined from a biocontrol point of view. Acyl-homoserine lactone (AHL) degrading bacteria were isolated from tomato rhizosphere using two standard bioreporter strains and different synthetic AHLs and then identified according to 16S rDNA sequences. Five isolates capable of inactivating both short and long 3oxo-substituted AHLs showed high similarity with the genera Bacillus, Microbacterium and Arthrobacter, and thereby Bacillus cereus U92 was determined as the most efficient quorum quencher strain. In the quantitative experiments, this strain remarkably inactivated all synthetic AHLs up to 80%. In the laboratory co-cultures, B. cereus U92 efficiently quenched QS-regulated phenotypes in Agrobacterium tumefaciens, Pseudomonas aeruginosa, Pseudomonas chlororaphis and Chromobacterium violaceum. The strain successfully reduced the frequency of Ti-plasmid conjugal transfer in A. tumefaciens by about 99% in the binary cultures. Meanwhile, in a more natural environment, this strain acted as a biocontrol agent, efficient in alleviating QS-regulated crown gall incidence on tomato roots (up to 90%) as well as attenuating Pectobacterium soft rot on potato tubers (up to 60%). On the other hand, reducing phenazine production in P. chlororaphis operated as a suppressor of its QS-regulated biocontrol activity and also inhibited pyocyanin production in P. aeruginosa, a plant growth-promoting bacterium, by 75%. In general, B. cereus U92 seems very promising in the biological control of pathogenic bacteria; however, its broad AHL-degrading activity has a detrimental role on beneficial microbes which should not be neglected.     

New Antagonistic Strains of Non‐Pathogenic Agrobacterium vitis to Control Grapevine Crown Gall

Graft unions of nursery stock of grapevine (Vitis vinifera L.) collected in Japan yielded non‐pathogenic strains of Agrobacterium. On the basis of classic diagnostic tests, a sequence analysis and a previously reported multiplex PCR method, the non‐pathogenic strains ARK‐1, ARK‐2 and ARK‐3 were identified as Agrobacterium vitis. Stems of grapevine seedlings were inoculated with both a cell suspension of seven mixed strains of A. vitis (Ti) as a pathogen and one of a new strain or A. vitis strain VAR03‐1, one of the biological control agents against crown gall previously reported, as competitors to assay the suppression of tumour formation caused by the pathogen. In a test with a 1:1 cell ratio of pathogen/nonpathogen, strains ARK‐1, ARK‐2 and ARK‐3 reduced the tumour incidence.. In particular, strain ARK‐1 was strongest at inhibiting tumour formation in this study. Strain ARK‐1 established populations on roots of grapevine tree rootstock and persisted on roots for a year. ARK‐1, ARK‐2 and ARK‐3 did not produce a halo of inhibition against A. vitis (Ti) strain on YMA medium. Moreover, strain ARK‐1 did not reduce tumour incidence on the stems of grapevine when ARK‐1 was dead or only culture filtrate was used. This result indicates the possibility that these new strains inhibit grapevine crown gall in planta by a different mechanism other than VAR03‐1. In particular, one of the new strains, named ARK‐1, was most effective in inhibiting tumour formation on grapevine and appears to be a promising new agent to control grapevine crown gall.     

Immunolocalisation of two tropinone reductases in potato (Solanum tuberosum L.) root, stolon, and tuber sprouts

Tropinone reductases (TRs) are essential enzymes in the tropane alkaloid biosynthesis, providing either tropine for hyoscyamine and scopolamine formation or providing pseudotropine for calystegines. Two cDNAs coding for TRs were isolated from potato (Solanum tuberosum L.) tuber sprouts and expressed in E. coli. One reductase formed pseudotropine, the other formed tropine and showed kinetic properties typical for tropine-forming tropinone reductases (TRI) involved in hyoscyamine formation. Hyoscyamine and tropine are not found in S. tuberosum plants. Potatoes contain calystegines as the only products of the tropane alkaloid pathway. Polyclonal antibodies raised against both enzymes were purified to exclude cross reactions and were used for Western-blot analysis and immunolocalisation. The TRI (EC 1.1.1.206) was detected in protein extracts of tuber tissues, but mostly in levels too low to be localised in individual cells. The function of this enzyme in potato that does not form hyoscyamine is not clear. The pseudotropine-forming tropinone reductase (EC 1.1.1.236) was detected in potato roots, stolons, and tuber sprouts. Cortex cells of root and stolon contained the protein; additional strong immuno-labelling was located in phloem parenchyma. In tuber spouts, however, the protein was detected in companion cells.     

Needle in a haystack: Antibacterial activity-guided fractionation of a potato wound tissue extract

Erwinia carotovora is a major cause of potato tuber infection, which results in disastrous failures of this important food crop. There is currently no effective antibiotic treatment against E. carotovora. Recently we reported antibacterial assays of wound tissue extracts from four potato cultivars that exhibit a gradient of russeting character, finding the highest potency against this pathogen for a polar extract from the tissue formed immediately after wounding by an Atlantic cultivar. In the current investigation, antibacterial activity-guided fractions of this extract were analyzed by liquid chromatography-mass spectrometry (LC-MS) utilizing a quadrupole-time-of-flight (QTOF) mass spectrometer. The most active chemical compounds identified against E. carotovora were: 6-O-nonyl glucitol, Lyratol C, n-[2-(4-Hydroxyphenyl)] ethyldecanamide, α-chaconine and α-solanine. Interactions among the three compounds, ferulic acid, feruloyl putrescine, and α-chaconine, representing metabolite classes upregulated during initial stages of wound healing, were also evaluated, offering possible explanations for the burst in antibacterial activity after tuber wounding and a chemical rationale for the temporal resistance phenomenon.     

Involvement of a vitronectin-like protein in attachment of Agrobacterium tumefaciens to carrot suspension culture cells.

Infections of dicotyledonous plants by Agrobacterium tumefaciens result in the formation of crown gall tumors. Attachment of the bacteria to plant host cells is required for tumor formation. Human vitronectin and antivitronectin antibodies both inhibited the binding of A. tumefaciens to carrot cells. Wild-type bacteria are able to bind radioactive vitronectin; nonattaching mutants showed a reduction in the ability to bind vitronectin. The binding of biotype 1 A. tumefaciens to carrot cells or to radioactive vitronectin was not affected by high ionic strength. Detergent extraction of carrot cells removed the receptor to which the bacteria bind. The extract was found to contain a vitronectin-like protein. These results suggest that A. tumefaciens utilizes a vitronectin-like protein on the plant cell surface as the receptor for its initial attachment to host cells.