Roberto Buonaurio

Sharing of quorum-sensing signals and role of interspecies communities in a bacterial plant disease

Pathogenic bacteria interact not only with the host organism but most probably also with the resident microbial flora. In the knot disease of the olive tree (Olea europaea), the causative agent is the bacterium Pseudomonas savastanoi pv. savastanoi (Psv). Two bacterial species, namely Pantoea agglomerans and Erwinia toletana, which are not pathogenic and are olive plant epiphytes and endophytes, have been found very often to be associated with the olive knot. We identified the chemical signals that are produced by strains of the three species isolated from olive knot and found that they belong to the N-acyl-homoserine lactone family of QS signals. The luxI/R family genes responsible for the production and response to these signals in all three bacterial species have been identified and characterized. Genomic knockout mutagenesis and in planta experiments showed that virulence of Psv critically depends on QS; however, the lack of signal production can be complemented by wild-type E. toletana or P. agglomerans. It is also apparent that the disease caused by Psv is aggravated by the presence of the two other bacterial species. In this paper we discuss the potential role of QS in establishing a stable consortia leading to a poly-bacterial disease.

Induction of systemic acquired resistance in pepper plants by acibenzolar-S-methyl against bacterial spot disease

The ability of acibenzolar-S-methyl to induce resistance in pepper plants against Xanthomonas campestris pv. vesicatoria was investigated in both growth chamber and open field conditions. Growth chamber experiments showed that acibenzolar-S-methyl (300μM) treatment protects pepper plants systemically and locally against X. campestris pv. vesicatoria. Evidence for this was a reduction in the number and diameter of bacterial spots and bacterial growth in planta. Systemic protection was also exerted by the acibenzolar-S-methyl acid derivative, CGA 210007, which may be produced by hydrolysis in the plant. The efficacy of acibenzolar-S-methyl was also found in open field conditions, where both leaves and fruit were protected from the disease. The highest efficacy (about 67%) was obtained by spraying the plants 6–7 times every 8–12 days with a mixture of acibenzolar-S-methyl and copper hydroxide (2.5 + 40ghl−1 active ingredient). Persistence and translocation data obtained from the growth chamber experiments revealed a persistence of acibenzolar-S-methyl lasting five days after treatment with rapid translocation and negligible levels of acid derivative formation. Since the protection exerted by acibenzolar-S-methyl against bacterial spot disease was observed when the inducer was completely degraded, it would appear to be due to SAR activation.

Soluble superoxide dismutase (SOD) in susceptible and resistant host-parasite complexes of Phaseolus vulgaris and Uromyces phaseoli☆

Abstract The level of soluble superoxide dismutase (E.G.1.15.1.1) was determined in Phaseolus vulgaris leaves susceptible (cv. Pinto 111) and hypersensitive resistant (cvs K.W. 765 and K.W. 814) to Uromyces phaseoli. In all cultivars tested increased levels of extracted Superoxide dismutase (SOD) appeared to be associated with the appearance of flecks and the differentiation of the uredosori, in the susceptible cultivar, and of microscopic or macroscopic necrotic lesions in the hypersensitive cultivars. Different SOD enzymes (Mn or Cu, Zn-SOD) were selectively stimulated by compatible and incompatible host-pathogen interactions. There was an increase in activity of the Mn-enzyme in the susceptible leaves during stroma differentiation in the mesophyll. By contrast there was an increase in activity of the Cu,Zn-enzyme in the hypersensitive response of resistant leaves. The above mentioned increases were due to a host response to infection since the contribution of fungal SOD was negligible. SOD and peroxidase increased simultaneously. The increase of peroxidasr greatly exceeded that of SOD only in the hypersensitive cultivars. The infection did not change the electrophoretic behaviour of SOD enzymes.

Sphingomonas melonis sp. nov., a novel pathogen that causes brown spots on yellow Spanish melon fruits

A polyphasic taxonomic study was performed on the phytopathogenic bacterial strains DAPP-PG 224(T) and DAPP-PG 228, which cause brown spot on yellow Spanish melon (Cucumis melo var. inodorus) fruits. Based on the presence of glucuronosyl ceramide (SGL-1) in cellular lipids, the results of fatty acid analysis and 16S rDNA sequence comparison, the strains had been identified as belonging to the genus Sphingomonas and as phylogenetically related to Sphingomonas mali, Sphingomonas pruni and Sphingomonas asaccharolytica. The levels of 16S rDNA sequence similarity of these three species to strain DAPP-PG 224(T) were respectively 98.0, 98.0 and 97.4%. DNA-DNA hybridization experiments between strains pathogenic on melon fruit and S. mali, S. pruni and S. asaccharolytica revealed < or = 16% relatedness. Based on these results, the two isolates studied are regarded as independent from the type strains of the three species mentioned above. Sphingomonas strains from melon fruits are recognized as forming a genetically and phenotypically discrete species and to be differentiated by phenotypic characteristics from all 29 named species of the genus. Thus, the name Sphingomonas melonis sp. nov. is proposed for the isolates from diseased melon fruits. The type strain is DAPP-PG 224(T) (= LMG 19484(T) = DSM 14444(T)). The G+C content of DNA of the type strain is 65.0 mol%.

The Arabidopsis thaliana cysteine-rich receptor-like kinase CRK20 modulates host responses to Pseudomonas syringae pv. tomato DC3000 infection

In plants, the cysteine-rich repeat kinases (CRKs) are a sub-family of receptor-like protein kinases that contain the DUF26 motif in their extracellular domains. It has been shown that in Arabidopsis thaliana, CRK20 is transcriptionally induced by pathogens, salicylic acid and ozone (O(3)). However, its role in responses to biotic and abiotic stress remains to be elucidated. To determine the function of CRK20 in such responses, two CRK20 loss-of-function mutants, crk20-1 and crk20-2, were isolated from public collections of Arabidopsis T-DNA tagged lines and examined for responses to O(3) and Pseudomonas syringae pv. tomato (Pst) DC3000. crk20-1 and crk20-2 showed similar O(3) sensitivities and no differences in the expression of defense genes when compared with the wild-type. However, pathogen growth was significantly reduced, while there were no differences in the induction of salicylic acid related defense genes or salicylic acid accumulation. Furthermore, correlation analysis of CRK20 gene expression suggests that it has a role in the control of H(2)O and/or nutrient transport. We therefore propose that CRK20 promotes conditions that are favorable for Pst DC3000 growth in Arabidopsis, possibly through the regulation of apoplastic homeostasis, and consequently, of the environment of this biotrophic pathogen.

Contributions of the effector gene hopQ1-1 to differences in host range between Pseudomonas syringae pv. phaseolicola and P. syringae pv. tabaci

To study the role of type III-secreted effectors in the host adaptation of the tobacco (Nicotiana sp.) pathogen Pseudomonas syringae pv. tabaci, a selection of seven strains was first characterized by multilocus sequence typing (MLST) to determine their phylogenetic affinity. MLST revealed that all strains represented a tight phylogenetic group and that the most closely related strain with a completely sequenced genome was the bean (Phaseolus vulgaris) pathogen P. syringae pv. phaseolicola 1448A. Using primers designed to 21 P. syringae pv. phaseolicola 1448A effector genes, it was determined that P. syringae pv. phaseolicola 1448A shared at least 10 effectors with all tested P. syringae pv. tabaci strains. Six of the 11 effectors that failed to amplify from P. syringae pv. tabaci strains were individually expressed in one P. syringae pv. tabaci strain. Although five effectors had no effect on phenotype, growth in planta and disease severity of the transgenic P. syringae pv. tabaci expressing hopQ1-1(Pph1448A) were significantly increased in bean, but reduced in tobacco. We conclude that hopQ1-1 has been retained in P. syringae pv. phaseolicola 1448A, as this effector suppresses immunity in bean, whereas hopQ1-1 is missing from P. syringae pv. tabaci strains because it triggers defences in Nicotiana spp. This provides evidence that fine-tuning effector repertoires during host adaptation lead to a concomitant reduction in virulence in non-host species.

Erwinia oleae sp. nov., isolated from olive knots caused by Pseudomonas savastanoi pv. savastanoi

Three endophytic bacterial isolates were obtained in Italy from olive knots caused by Pseudomonas savastanoi pv. savastanoi. Phenotypic tests in combination with 16S rRNA gene sequence analysis indicated a phylogenetic position for these isolates in the genera Erwinia or Pantoea, and revealed two other strains with highly similar 16S rRNA gene sequences (>99 %), CECT 5262 and CECT 5264, obtained in Spain from olive knots. Rep-PCR DNA fingerprinting of the five strains from olive knots with BOX, ERIC and REP primers revealed three groups of profiles that were highly similar to each other. Multilocus sequence analysis (MLSA) based on concatenated partial atpD, gyrB, infB and rpoB gene sequences indicated that the strains constituted a single novel species in the genus Erwinia. The strains showed general phenotypic characteristics typical of the genus Erwinia and whole genome DNA-DNA hybridization data confirmed that they represented a single novel species of the genus Erwinia. The strains showed DNA G+C contents ranging from 54.7 to 54.9 mol%. They could be discriminated from phylogenetically related species of the genus Erwinia by their ability to utilize potassium gluconate, l-rhamnose and d-arabitol, but not glycerol, inositol or d-sorbitol. The name Erwinia oleae sp. nov. (type strain DAPP-PG 531(T)= LMG 25322(T) = DSM 23398(T)) is proposed for this novel taxon.

Bacterial multispecies studies and microbiome analysis of a plant disease.

Although the great majority of bacteria found in nature live in multispecies communities, microbiological studies have focused historically on single species or competition and antagonism experiments between different species. Future directions need to focus much more on microbial communities in order to better understand what is happening in the wild. We are using olive knot disease as a model to study the role and interaction of multispecies bacterial communities in disease establishment/development. In the olive knot, non-pathogenic bacterial species (e.g. Erwinia toletana) co-exist with the pathogen (Pseudomonas savastanoi pv. savastanoi); we have demonstrated cooperation among these two species via quorum sensing (QS) signal sharing. The outcome of this interaction is a more aggressive disease when co-inoculations are made compared with single inoculations. In planta experiments show that these two species co-localize in the olive knot, and this close proximity most probably facilitates exchange of QS signals and metabolites. In silico recreation of their metabolic pathways showed that they could have complementing pathways also implicating sharing of metabolites. Our microbiome studies of nine olive knot samples have shown that the olive knot community possesses great bacterial diversity; however. the presence of five genera (i.e. Pseudomonas, Pantoea, Curtobacterium, Pectobacterium and Erwinia) can be found in almost all samples.

The olive knot disease as a model to study the role of interspecies bacterial communities in plant disease.

There is an increasing interest in studying interspecies bacterial interactions in diseases of animals and plants as it is believed that the great majority of bacteria found in nature live in complex communities. Plant pathologists have thus far mainly focused on studies involving single species or on their interactions with antagonistic competitors. A bacterial disease used as model to study multispecies interactions is the olive knot disease, caused by Pseudomonas savastanoi pv. savastanoi (Psv). Knots caused by Psv in branches and other aerial parts of the olive trees are an ideal niche not only for the pathogen but also for many other plant-associated bacterial species, mainly belonging to the genera Pantoea, Pectobacterium, Erwinia, and Curtobacterium. The non-pathogenic bacterial species Erwinia toletana, Pantoea agglomerans, and Erwinia oleae, which are frequently isolated inside the olive knots, cooperate with Psv in modulating the disease severity. Co-inoculations of these species with Psv result in bigger knots and better bacterial colonization when compared to single inoculations. Moreover, harmless bacteria co-localize with the pathogen inside the knots, indicating the formation of stable bacterial consortia that may facilitate the exchange of quorum sensing signals and metabolites. Here we discuss the possible role of bacterial communities in the establishment and development of olive knot disease, which we believe could be taking place in many other bacterial plant diseases.

Effect of tobacco mosaic virus infection of levels of soluble superoxide dismutase (SOD) in nicotiana tabacum and nicotiana glutinosa leaves

Abstract The activity of superoxide dismutase (SOD: E.C. 1.15.1.1) was evaluated on Nicotiana tabacum and Nicotiana glutinosa leaf tissue after Tobacco Mosaic Virus (TMV) infection. Significant increase in extracted SOD appeared to be directly related to the appearance of necrotic and systemic symptoms in hypersensitive ( N. glutinosa and N. tabacum cv. Havana 425) and susceptible ( N. tabacum cv. Bright BC 60) leaves, respectively. SOD activity did not change significantly during the replication of TMV in the inoculated susceptible leaves up to 4 days after inoculation. Both cyanide-insensitive (2 days after inoculation) and sensitive (3–4 days after inoculation) enzymes increased during the expression of the hypersensitivity. Only cyanide-sensitive enzyme increased in systematically infected leaves. SOD and peroxidase increased simultaneously and the enhancement of peroxidase was higher than that of SOD. The values of peroxidase greatly exceeded that of SOD only in the hypersensitive leaves during local lesion differentiation. In N. tabacum leaves 4 clear SOD bands were separated by polyacrylamide gel electrophoresis: 3 cyanide-sensitive (Cu,Zn enzyme) and 1 cyanide-insensitive, while N. glutinosa had 3 bands: 2 cyanide-sensitive and 1 cyanide-insensitive. The cyanide-insensitive band, both in N. tabacum and N. glutinosa , was sensitive to H 2 O 2 and insensitive to chloroform-ethanol treatment and thus supposed to be Fe enzyme. The infection did not induce change in the electrophoretic pattern of SOD enzymes. In summary, our results indicate that the pathogenic alteration caused by TMV infection both in the compatible and in the incompatible combinations are characterized by an induction of SOD activity, particularly cyanide-sensitive Cu,Zn-SOD. The connection between the induction of SOD and a possible activation of O 2 − production in the hypersensitive tissue following TMV infection is discussed.