Delineation of Pseudomonas savastanoi pv. savastanoi strains isolated in Tunisia by random-amplified polymorphic DNA analysis.

AIMS: To investigate the genetic diversity of Pseudomonas savastanoi pv. savastanoi strains and to look whether these strains were distributed to geographical location. METHODS AND RESULTS: Random amplification of polymorphic DNA (RAPD) was used to discriminate between 58 Tunisian strains and 21 strains from various other countries of P. savastanoi pv. savastanoi, the causal agent of olive knot disease. Isolates were separated into three groups by cluster analysis and principal coordinate analysis of RAPD fingerprint data obtained with three primers (OPR-12, OPX-7 and OPX-14). Group 1 contained isolates from the southeast of Tunisia and European strains. Group 2 comprised strains isolated from the north of Tunisia exclusively while group 3 encompassed the majority of isolates obtained from five orchards located in the centre of Tunisia. CONCLUSIONS: The results indicated that isolates of P. savastanoi pv. savastanoi were genetically distinct according to geographic regions. RAPD grouped isolates derived from the same orchard as identical. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first application of RAPD in the delineation of P. savastanoi pv. savastanoi strains.

Factors Affecting Pseudomonas savastanoi pv. savastanoi Plant Inoculations and Their Use for Evaluation of Olive Cultivar Susceptibility.

ABSTRACT Pseudomonas savastanoi pv. savastanoi causes olive knot disease, which is present in most countries where olive trees are grown. Although the use of cultivars with low susceptibility may be one of the most appropriate methods of disease control, little information is available from inoculation assays, and cultivar susceptibility assessments have been limited to few cultivars. We have evaluated the effects of pathogen virulence, plant age, the dose/response relationship, and the induction of secondary tumors in olive inoculation assays. Most P. savastanoi pv. savastanoi strains evaluated were highly virulent to olive plants, but interactions between cultivars and strains were found. The severity of the disease in a given cultivar was strongly dependent of the pathogen dose applied at the wound sites. Secondary tumors developed in noninoculated wounds following inoculation at another position on the stem, suggesting the migration of the pathogen within olive plants. Proportion and weight of primary knots and the presence of secondary knots were evaluated in 29 olive cultivars inoculated with two pathogen strains at two inoculum doses, allowing us to rate most of the cultivars as having either high, medium, or low susceptibility to olive knot disease. None of the cultivars were immune to the disease.

Tumorigenic Agrobacterium sp. Isolated from Weeping Fig in Spain.

Agrobacterium-like colonies were recovered onto Roy-Sasser's medium from a young tumor (4 cm in diameter) on the stem of weeping fig (Ficus benjamina L.), 10 cm from the crown. The galled plant was collected in 1999 from a garden center in Valencia, Spain. After colony purification and tomato and weeping fig plant inoculations, one nonpathogenic and five Agrobacterium isolates that were tumorigenic in both plant species were characterized. On the basis of biovar classification tests, the nonpathogenic isolate was identified as belonging to biovar 1 of Agrobacterium (now called A. tumefaciens), whereas the tumorigenic isolates could not be assigned to any of the known Agrobacterium biovars. The isolates were positive for oxidase, growth in 2% NaCl, production of alkali from l-tartaric acid, and production of acid from mannitol-CaCO3 and negative for 3-ketolactose production, growth and pigmentation in ferric ammonium citrate, growth at 35°C, citrate utilization, acid production from sucrose and melezitose, and alkali production from malonic acid. Nopaline was the unique opine found in galls induced in weeping fig plants inoculated with the pathogenic isolates. Moreover, all isolates utilize the opine nopaline, but not octopine, manopine, agropine, chrysopine, cucumopine, or mikimopine. They were susceptible to agrocin 84 produced by strain K84. Heat-treated bacterial suspensions of these isolates yielded the expected amplification product using polymerase chain reaction (PCR) with the FGPtmr530/FGPtmr701' primers pair from the tmr gene (3). Aerial gall disease was first reported on F. benjamina in Florida (1), and the isolated agrobacteria belongs to a new species named A. larrymoorei (2). Later, tumorigenic agrobacteria from weeping fig galls were isolated in Italy and the Netherlands (4). Our data suggest that the tumorigenic strains isolated in Spain differ greatly from those first described in the United States (1) on the basis of alkali production from l-tartaric acid, chrysopine detection on tumors, susceptibility to agrocin 84, and tmr amplification, but they might be similar to some of the Italian isolates (4). To our knowledge, this is the first report of isolation of tumorigenic Agrobacterium sp. from F. benjamina L. in Spain. References: (1) H. Bouzar et al. Appl. Environ. Microbiol. 61:65, 1995. (2) H. Bouzar and J. B. Jones. Int. J. Syst. Evol. Microbiol. 51:1023. 2001. (3) X. Nesme et al. Pages 47-50 in: Endocytobiology IV. P. Nardon et al. eds. INRA, France, 1989. (4) A. Zoina et al. Plant Pathol. 50:620, 2001.

Iron-binding compounds from Agrobacterium spp.: biological control strain Agrobacterium rhizogenes K84 produces a hydroxamate siderophore.

Iron-binding compounds were produced in various amounts in response to iron starvation by a collection of Agrobacterium strains belonging to the species A. tumefaciens, A. rhizogenes, and A. vitis. The crown gall biocontrol agent A. rhizogenes strain K84 produced a hydroxamate iron chelator in large amounts. Production of this compound, and also of a previously described antibiotic-like substance called ALS84, occurred only in cultures of strain K84 grown in iron-deficient medium. Similarly, sensitivity to ALS84 was expressed only when susceptible cells were tested in low-iron media. Five independent Tn5-induced mutants of strain K84 affected in the production of the hydroxamate iron chelator showed a similar reduction in the production of ALS84. One of these mutants, M8-10, was completely deficient in the production of both agents and grew poorly compared to the wild type under iron-limiting conditions. Thus, the hydroxamate compound has siderophore activity. A 9.1-kb fragment of chromosomal DNA containing the Tn5 insertion from this mutant was cloned and marker exchanged into wild-type strain K84. The homogenote lost the ability to produce the hydroxamate siderophore and also ALS84. A cosmid clone was isolated from a genomic library of strain K84 that restored to strain M8-10 the ability to produce of the siderophore and ALS84, as well as growth in iron-deficient medium. This cosmid clone contained the region in which Tn5 was located in the mutant. Sequence analysis showed that the Tn5 insert in this mutant was located in an open reading frame coding for a protein that has similarity to those of the gramicidin S synthetase repeat superfamily. Some such proteins are required for synthesis of hydroxamate siderophores by other bacteria. Southern analysis revealed that the biosynthetic gene from strain K84 is present only in isolates of A. rhizogenes that produce hydroxamate-type compounds under low-iron conditions. Based on physiological and genetic analyses showing a correlation between production of a hydroxamate siderophore and ALS84 by strain K84, we conclude that the two activities share a biosynthetic route and may be the same compound.

Detection of Pseudomonas savastanoi pv. savastanoi in olive plants by enrichment and PCR.

The sequence of the gene iaaL of Pseudomonas savastanoi EW2009 was used to design primers for PCR amplification. The iaaL-derived primers directed the amplification of a 454-bp fragment from genomic DNA isolated from 70 strains of P. savastanoi, whereas genomic DNA from 93 non-P. savastanoi isolates did not yield this amplified product. A previous bacterial enrichment in the semiselective liquid medium PVF-1 improved the PCR sensitivity level, allowing detection of 10 to 100 CFU/ml of plant extract. P. savastanoi was detected by the developed enrichment-PCR method in knots from different varieties of inoculated and naturally infected olive trees. Moreover, P. savastanoi was detected in symptomless stem tissues from naturally infected olive plants. This enrichment-PCR method is more sensitive and less cumbersome than the conventional isolation methods for detection of P. savastanoi.

Cocolonization of the rhizosphere by pathogenic agrobacterium strains and nonpathogenic strains K84 and K1026, used for crown gall biocontrol

The crown gall biocontrol agent strain K84 and three mutants derived from it, K1026 (Tra- deletion mutant of pAgK84), K84 Agr- (lacking pAgK84), and K1143 (lacking pAgK84 and pNoc), significantly reduced gall formation caused by two pathogenic strains resistant to agrocin 84 in peach x almond seedlings planted in infested soil. Cocolonization of roots by pathogenic and nonpathogenic strains was observed in these biocontrol experiments under field conditions. In spite of the efficient biocontrol observed, average populations consisting of 10(2) and 10(6) pathogenic agrobacteria per g of root were found 8 months after planting. The total numbers of pathogenic bacteria on roots were similar for plants treated with the biocontrol strains and for the untreated plants. Strain K84 and the genetically engineered organism K1026 survived at a level of 10(6) agrocin 84-producing bacteria per g of root. The population size of genetically engineered strain K1026 was not significantly different than the population size of wild-type strain K84 8 months after root inoculation. Strains K84 and K1026 controlled two pathogens resistant to agrocin 84 without reducing the total number of pathogenic bacteria in the root system. In addition, this study shows that some biological control activity of strain K84 against agrocin 84-resistant pathogens is independent of plasmids pAgK84 and pNoc.