Search

Bacteria were isolated from necrotic apple and pear tree tissue and from dead wood in Germany and Austria as well as from pear tree exudate in China. They were selected for growth at 37 °C, screened for levan production and then characterized as Gram-negative, facultatively anaerobic rods. Nucleotide sequences from 16S rRNA genes, the housekeeping genes dnaJ, gyrB, recA and rpoB alignments, BLAST searches and phenotypic data confirmed by MALDI-TOF analysis showed that these bacteria belong to the genus Gibbsiella and resembled strains isolated from diseased oaks in Britain and Spain. Gibbsiella-specific PCR primers were designed from the proline isomerase and the levansucrase genes. Acid secretion was investigated by screening for halo formation on calcium carbonate agar and the compound identified by NMR as acetic acid. Its production by Gibbsiella spp. strains was also determined in culture supernatants by GC/MS analysis after derivatization with pentafluorobenzyl bromide. Some strains were differentiated by the PFGE patterns of SpeI digests and by sequence analyses of the lsc and the ppiD genes, and the Chinese Gibbsiella strain was most divergent. The newly investigated bacteria as well as Gibbsiella querinecans, Gibbsiella dentisursi and Gibbsiella papilionis, isolated in Britain, Spain, Korea and Japan, are taxonomically related Enterobacteriaceae, tolerate and secrete acetic acid. We therefore propose to unify them in the species Gibbsiella acetica sp. nov.

Flower infections caused by Erwinia amylovora can be prevented only during the short period of epiphytic colonization. Bacterial antagonists like Erwinia tasmaniensis and Bacillus spp. are able to repress E. amylovora growth at this initial stage and prevent further intrusion of the pathogen. They can be applied as biological control agents if a suitable formulation can be produced. To investigate inhibitory effects of different antagonists and dried formulations, we used several test systems in the laboratory, including co-cultivation experiments, agar diffusion assay and detached-flower assays. A luminescent reporter strain of E. amylovora was used to distinguish between pathogen and antagonist growth. In laboratory studies, application of dry formulations was as efficient as with antagonists derived from batch culture. Samples from test-formulations were also applied in field trials according to EPPO guideline 1/166.

We identified a compound in culture supernatants of Erwinia species, such as Erwinia amylovora, E. pyrifoliae, E. billingiae, E. tasmaniensis, E. persicina and E. rhapontici absorbing at 340 nm, which was associated before with the yellow pigment produced by E. amylovora on media containing copper ions. The compound was purified from E. tasmaniensis strain Et1/99 supernatants by chromatography on Dowex-1 and Dowex-50 columns and identified by HPLC/MS and NMR analysis as 6-thioguanine (6TG). Its signal at 167 Da matched with the expected molecular mass. By random mutagenesis with miniTn5, we obtained mutants defective in the genes for pyrimidine and purine metabolism. A specific gene cluster with ycf genes described by us before, absent in the corresponding region of Escherichia coli, was identified in the genome sequence of three Erwinia species and named tgs region for thioguanine synthesis. Clones of the tgs gene cluster promoted 6TG synthesis and secretion in E. coli, when the bacteria were grown in minimal medium supplemented with amino acids. 6TG was bacteriostatic for E. coli and Salmonella typhimurium strains, with cell growth resumed after prolonged incubation. Similar results were obtained with P. agglomerans strains. Bacteria from the genus Pectobacterium were barely and Rahnella or Gibbsiella species were not inhibited by 6TG. Adenine and guanine relieved the toxic effect of 6TG on E. coli. Non-producing strains were fully virulent on host plants. 6TG synthesis may help erwinias to interfere with growth of some microorganisms in the environment.

Aims: To detect the apple and pear pathogens Erwinia amylovora and Erwinia pyrifoliae as well as the related epiphytes Erwinia tasmaniensis and Erwinia billingiae, we created novel PCR primers and also applied them to a series of other plant-associated bacteria as control. To facilitate fast diagnosis, we used matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI–TOF MS). Methods and Results: The PCR primers were deduced from the pstS–glmS regions, which can include the gene for levansucrase, and also from regions encoding capsular polysaccharide synthesis. All primer combinations were specific for their associated Erwinia species to detect them with conventional PCR, also in mixed cultures from necrotic plant tissue. Other primers designed for quantitative PCR with SYBR Green or together with TaqMan probes were applied for real-time detection to determine growth of Erw. amylovora, Erw. billingiae, Erw. pyrifoliae and Erw. tasmaniensis in apple blossoms. From whole-cell protein extracts, profiles were generated using a Bruker microflex machine and Erwinia strains classified according to a score scheme. Conclusions: The designed PCR primers identified the Erwinia species unambiguously and can be applied to qualitative and quantitative tests. MALDI–TOF MS data were in agreement with the PCR assays. Significance and Impact of the Study: The applied diagnosis methods allow fast and precise monitoring of two pathogenic and two epiphytic Erwinia species. They are valuable for population studies with apple and pear flowers and with diseased plant material.

Erwinia amylovora produces a compound with an absorbance maximum at 340 nm that forms a yellow colored complex with copper. This compound was identified as 6-thioguanine, a guanine analogue that is used in chemotherapy and the treatment of inflammatory bowel disease. Synthesis of 6-thioguanine could be linked to five genes common in Erwinia genomes, but missing in related genera. Expression of Erwinia tasmaniensis genes tgsA-D in Escherichia coli was sufficient for heterologous production of 6-thioguanine. Transfer of the four biosynthetic E. tasmaniensis genes did not enhance resistance of E. coli against 6-thioguanine. Bacterial and synthetic 6-thioguanine have a strong growth inhibitory effect on many bacteria, such as E. coli or a number of Pantoea agglomerans isolates. While this inhibition of competing species might provide an advantage to Erwinia, further biological functions of 6-thioguanine cannot be excluded. A direct link between 6-thioguanine synthesis of E. amylovora and its pathogenicity was not observed, as two 6-thioguanine negative mutants produced as severe symptoms on apple and pear shoots as did producing strains.

The harlequin ladybird Harmonia axyridis (Coleoptera: Coccinellidae) is a polyphagous predatory beetle native to Central and Eastern Asia. Since 2007 it has established all over Central Europe. In order to elucidate which defense strategy is responsible for its high resistance to diseases, we tested hemolymph as well as eleven main components of the headspace of H. axyridis for antimicrobial activity against Gram-positive (Bacillus subtilis, B. thuringiensis ssp. tenebrionis, Micrococcus luteus) and Gram-negative bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). While three of the volatile compounds weakly reduced the growth of microorganisms, hemolymph of adults and larvae of H. axyridis strongly inhibited the growth of Gram-positive and Gram-negative bacteria as well as yeast. Furthermore, we compared the antimicrobial activity in the hemolymph of H. axyridis and Coccinella septempunctata. Antimicrobial activity in H. axyridis was about a thousand times higher compared to hemolymph from C. septempunctata. In contrast to C. septempunctata, the antimicrobial activity in H. axyridis was present without prior challenge. Minimal inhibitory concentration (MIC) of the hemolymph of H. axyridis was lowest against E. coli and yeast followed by B. subtilis, and was highest against entomopathogenic B. thuringiensis ssp. tenebrionidae. Furthermore, MIC values of the hemolymph obtained from live beetles were significantly lower than from frozen insects. This suggests that the active antimicrobial compound is affected by freezing and subsequent thawing of the beetles. Potential implications of our findings for the competitive advantages of H. axyridis over C. septempunctata are discussed.

For fast and cost-efficient identification of bacteria we tested the Biotyper system (Bruker Daltonics) to detect Erwinia amylovora and other plant-associated bacteria by protein profiles from whole cells. Strains could also be identified by direct transfer of cells to the sample target, and the signals were compared to preceding protein extraction with formic acid. The identification scores obtained for extracted proteins were higher and more reliable than those of directly applied agar cultured cells. For verification of MALDI-TOF identification and for detection of several Erwinia species in mixed cultures we applied PCR primer sets designed from specific chromosomal sequence regions such as the gene cluster for biosynthesis of capsular EPS or the nucleotide sequences flanked by genes pstS and glmS. The novel primers readily detected E. amylovora fruit tree and raspberry strains, E. pyrifoliae, E. billingiae and E. tasmaniensis. The identification obtained by PCR agreed with the MALDI-TOF MS results. While PCR analysis is also applicable for detection of bacteria in mixed cultures obtained from plant samples, MALDI-TOF analysis identifies essentially cells from single colonies applied from agar plates or cultured in liquid medium. Both methods were combined for identification of bacteria in field samples and from artificially inoculated plant material.

Pantoea stewartii subsp. stewartii is the causative agent of Stewart's wilt, a bacterial disease transmitted by the corn flea beetle mainly to sweet corn ( Zea mays ). In many countries, it is classified as a quarantine organism and must be differentiated from other yellow enteric bacteria frequently occurring with corn. We have created novel primers from the pstS-glmS region of P. stewartii for use in conventional PCR (cPCR) and quantitative PCR (qPCR). To facilitate rapid diagnosis, we applied matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Using whole-cell protein extracts, profiles were generated with a Bruker microflex machine, and the bacteria classified. P. stewartii strains were clearly distinguished from strains of Pantoea agglomerans , Pantoea dispersa , and Pantoea ananatis . Dendrogram analysis of the protein profiles confirmed the score values and showed the formation of separate clades for each species. The identification achieved by MALDI-TOF MS analysis agrees with the diagnosis by specific PCR primers. The combination of both methods allows a rapid and simple identification of the corn pathogen. P. stewartii subsp. stewartii and P. stewartii subsp. indologenes are highly related and can be distinguished not only by virulence assays and indole tests but also by a characteristic pattern in the nucleotide sequence of recA .