Biocontrol of the Potato Blackleg and Soft Rot Diseases Caused by Dickeya dianthicola

Development of protection tools targeting Dickeya species is an important issue in the potato production. Here, we present the identification and the characterization of novel biocontrol agents. Successive screenings of 10,000 bacterial isolates led us to retain 58 strains that exhibited growth inhibition properties against several Dickeya sp. and/or Pectobacterium sp. pathogens. Most of them belonged to the Pseudomonas and Bacillus genera. In vitro assays revealed a fitness decrease of the tested Dickeya sp. and Pectobacterium sp. pathogens in the presence of the biocontrol agents. In addition, four independent greenhouse assays performed to evaluate the biocontrol bacteria effect on potato plants artificially contaminated with Dickeya dianthicola revealed that a mix of three biocontrol agents, namely, Pseudomonas putida PA14H7 and Pseudomonas fluorescens PA3G8 and PA4C2, repeatedly decreased the severity of blackleg symptoms as well as the transmission of D. dianthicola to the tuber progeny. This work highlights the use of a combination of biocontrol strains as a potential strategy to limit the soft rot and blackleg diseases caused by D. dianthicola on potato plants and tubers.     

Interplay of classic Exp and specific Vfm quorum sensing systems on the phenotypic features of Dickeya solani strains exhibiting different virulence levels

Bacteria from the genus Dickeya cause severe symptoms on numerous economically important plants. Dickeya solani is the Dickeya species most frequently found on infected potato plants in Europe. D. solani strains from different countries show high genetic homogeneity, but significant differences in their virulence level. Dickeya species possess two quorum sensing (QS) mechanisms: the Exp system based on classic N‐acyl‐homoserine lactone (AHL) signals and a specific system depending on the production and perception of a molecule of unknown structure, Virulence Factor Modulating (VFM). To study the interplay between these two QS systems, five D. solani strains exhibiting different virulence levels were selected. Mutants were constructed by inactivating genes coding for each QS system. Double mutants were obtained by simultaneous inactivation of genes coding for both QS systems. Most of the D. solani mutants showed an attenuation of chicory maceration and a decreased production of plant cell wall‐degrading enzymes (PCWDEs) and motility, but to different degrees depending on the strain. The VFM‐QS system seems to regulate virulence in both D. solani and Dickeya dadantii, but the AHL‐QS system has greater effects in D. solani than in D. dadantii. The inactivation of both QS systems in D. solani did not reveal any additive effect on the tested features. The inactivation of vfm genes generally has a more dominant effect relative to that of exp genes. Thus, VFM‐ and AHL‐QS systems do not work in synergy to modulate the production of diverse virulence factors and the ability to macerate plant tissue.     

Differentiation of Pectobacterium and Dickeya spp. phytopathogens using infrared spectroscopy and machine learning analysis

Pectobacterium and Dickeya spp. are soft rot Pectobacteriaceae that cause aggressive diseases on agricultural crops leading to substantial economic losses. The accurate, rapid and low‐cost detection of these pathogenic bacteria are very important for controlling their spread, reducing the consequent financial loss and for producing uninfected potato seed tubers for future generations. Currently used methods for the identification of these bacterial pathogens at the strain level are based mainly on molecular techniques, which are expensive. We used an alternative method, infrared spectroscopy, to measure 24 strains of five species of Pectobacterium and Dickeya. Measurements were then analyzed using machine learning methods to differentiate among them at the genus, species and strain levels. Our results show that it is possible to differentiate among different bacterial pathogens with a success rate of ~99% at the genus and species levels and with a success rate of over 94% at the strain level.     

A new clade of Dickeya spp. plays a major role in potato blackleg outbreaks in North Finland

Until recently Dickeya was regarded as a pathogen not established in Finland. As a result the blackleg symptom observed on potato was often associated with Pectobacterium atrosepticum. The occurrence of Dickeya spp. on potato in Finland was first reported in 2004. Since then the prevalence of Dickeya has been monitored through surveys and routine test of seed lots produced in the country. The results of monitoring of Dickeya spp. in seed lots produced in Finland between the years 2004 and 2008 indicated a steady increase in the incidence of Dickeya spp. The highest incidence was observed in samples from the 2006 growing season where about 37% were positive for Dickeya spp. The summer in 2006 was one of the warmest summers recorded in 100 years in Finland. The majority of infected lots were imported varieties. Since recently heavy blackleg outbreaks have occurred in production fields in the High Grade (HG) zone. A detailed study of these incidents of blackleg outbreaks in North Finland during the years 2008 and 2010 indicated that Dickeya spp. was the major component in the observed blackleg complex. It was detected and isolated from almost all symptomatic plants investigated. Repetitive sequences PCR (REP‐PCR) and Pulsed Field Gel Electrophoresis (PFGE) analysis of strains isolated in Finland showed identical pattern with those isolated recently in other European countries with a proposed name ‘Dickeya solani’. Moreover, the dnaX gene sequence of the representative strains isolated in Finland indicated 100% similarity to the dnaX sequences of ‘D. solani’. The study presents the first report of a detailed analysis of bacteria involved in potato blackleg complex from natural field outbreaks in North Finland HG zone and characterisation of the ‘D. solani’ strains playing the major role in the disease complex.     

The complete genome sequence of Dickeya zeae EC1 reveals substantial divergence from other Dickeya strains and species

Background

Dickeya zeae is a bacterial species that infects monocotyledons and dicotyledons. Two antibiotic-like phytotoxins named zeamine and zeamine II were reported to play an important role in rice seed germination, and two genes associated with zeamines production, i.e., zmsA and zmsK, have been thoroughly characterized. However, other virulence factors and its molecular mechanisms of host specificity and pathogenesis are hardly known.

Results

The complete genome of D. zeae strain EC1 isolated from diseased rice plants was sequenced, annotated, and compared with the genomes of other Dickeya spp.. The pathogen contains a chromosome of 4,532,364 bp with 4,154 predicted protein-coding genes. Comparative genomics analysis indicates that D. zeae EC1 is most co-linear with D. chrysanthemi Ech1591, most conserved with D. zeae Ech586 and least similar to D. paradisiaca Ech703. Substantial genomic rearrangement was revealed by comparing EC1 with Ech586 and Ech703. Most virulence genes were well-conserved in Dickeya strains except Ech703. Significantly, the zms gene cluster involved in biosynthesis of zeamines, which were shown previously as key virulence determinants, is present in D. zeae strains isolated from rice, and some D. solani strains, but absent in other Dickeya species and the D. zeae strains isolated from other plants or sources. In addition, a DNA fragment containing 9 genes associated with fatty acid biosynthesis was found inserted in the fli gene cluster encoding flagellar biosynthesis of strain EC1 and other two rice isolates but not in other strains. This gene cluster shares a high protein similarity to the fatty acid genes from Pantoea ananatis.

Conlusion

Our findings delineate the genetic background of D. zeae EC1, which infects both dicotyledons and monocotyledons, and suggest that D. zeae strains isolated from rice could be grouped into a distinct pathovar, i.e., D. zeae subsp. oryzae. In addition, the results of this study also unveiled that the zms gene cluster presented in the genomes of D. zeae rice isolates and D. solani strains, and the fatty acid genes inserted in the fli gene cluster of strain EC1 were likely derived from horizontal gene transfer during later stage of bacterial evolution.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1545-x) contains supplementary material, which is available to authorized users.     

Pseudomonas chlororaphis L5 and Enterobacter asburiae L95 biocontrol Dickeya soft rot diseases by quenching virulence factor modulating quorum sensing signal

Virulence factor modulating (VFM) is a quorum sensing (QS) signal shared by and specific to Dickeya bacteria, regulating the production of plant cell wall degrading enzymes (PCWDEs) and virulence of Dickeya. High polarity and trace of VFM signal increase the difficulty of signal separation and structure identification, and thus limit the development of quorum quenching strategy to biocontrol bacterial soft rot diseases caused by Dickeya. In order to high-throughput screen VFM quenching bacteria, a vfmE-gfp biosensor VR2 (VFM Reporter) sensitive to VFM signal was first constructed. Subsequently, two bacterial strains with high quenching efficiency were screened out by fluorescence intensity measurement and identified as Pseudomonas chlororaphis L5 and Enterobacter asburiae L95 using multilocus sequence analysis (MLSA). L5 and L95 supernatants reduced the expression of vfm genes, and both strains also decreased the production of PCWDEs of D. zeae MS2 and significantly reduced the virulence of D. oryzae EC1 on rice seedlings, D. zeae MS2 on banana seedlings, D. dadantii 3937 on potato and D. fangzhongdai CL3 on taro. Findings in this study provide a method to high-throughput screen VFM quenching bacteria and characterize novel functions of P. chlororaphis and E. asburiae in biocontrolling plant diseases through quenching VFM QS signal.     

Specificity and genetic polymorphism in the Vfm quorum sensing system of plant pathogenic bacteria of the genus Dickeya

The Vfm quorum sensing (QS) system is preponderant for the virulence of different species of the bacterial genus Dickeya. The vfm gene cluster encodes 26 genes involved in the production, sensing or transduction of the QS signal. To date, the Vfm QS signal has escaped detection by analytical chemistry methods. However, we report here a strain-specific polymorphism in the biosynthesis genes vfmO and vfmP, which is predicted to be related to the production of different analogues of the QS signal. Consequently, the Vfm communication could be impossible between strains possessing different variants of the genes vfmO/P. We constructed three Vfm QS biosensor strains possessing different vfmO/P variants and compared these biosensors for their responses to samples prepared from 34 Dickeya strains possessing different vfmO/P variants. A pattern of specificity was demonstrated, providing evidence that the polymorphism in the genes vfmO/P determines the biosynthesis of different analogues of the QS signal. Unexpectedly, this vfmO/P-dependent pattern of specificity is linked to a polymorphism in the ABC transporter gene vfmG, suggesting an adaptation of the putative permease VfmG to specifically bind different analogues of the QS signal. Accordingly, we discuss the possible involvement of VfmG as co-sensor of the Vfm two-component regulatory system.     

Jasmonate‐dependent modifications of the pectin matrix during potato development function as a defense mechanism targeted by Dickeya dadantii virulence factors

The plant cell wall constitutes an essential protection barrier against pathogen attack. In addition, cell‐wall disruption leads to accumulation of jasmonates (JAs), which are key signaling molecules for activation of plant inducible defense responses. However, whether JAs in return modulate the cell‐wall composition to reinforce this defensive barrier remains unknown. The enzyme 13–allene oxide synthase (13–AOS) catalyzes the first committed step towards biosynthesis of JAs. In potato (Solanum tuberosum), there are two putative St13–AOS genes, which we show here to be differentially induced upon wounding. We also determine that both genes complement an Arabidopsis aos null mutant, indicating that they encode functional 13–AOS enzymes. Indeed, transgenic potato plants lacking both St13–AOS genes (CoAOS1/2 lines) exhibited a significant reduction of JAs, a concomitant decrease in wound‐responsive gene activation, and an increased severity of soft rot disease symptoms caused by Dickeya dadantii. Intriguingly, a hypovirulent D. dadantii pel strain lacking the five major pectate lyases, which causes limited tissue maceration on wild‐type plants, regained infectivity in CoAOS1/2 plants. In line with this, we found differences in pectin methyl esterase activity and cell‐wall pectin composition between wild‐type and CoAOS1/2 plants. Importantly, wild‐type plants had pectins with a lower degree of methyl esterification, which are the substrates of the pectate lyases mutated in the pel strain. These results suggest that, during development of potato plants, JAs mediate modification of the pectin matrix to form a defensive barrier that is counteracted by pectinolytic virulence factors from D. dadantii.     

The effects of different light–dark cycles on the metabolism of the diazotrophic,unicellular cyanobacteria Cyanothece sp. ATCC 51142, and Cyanothecesp. PCC 7822

The diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142 demonstrates circadian patterns in nitrogenase activity, H₂ production and glycogen storage when grown under nitrogen‐fixing, 12:12 light:dark (L:D) conditions. In this study, we grew Cyanothece sp. ATCC 51142, and another strain in this genus, Cyanothece sp. PCC 7822, under long‐day (16:8 L:D) and short‐day (8:16 L:D) nitrogen‐fixing conditions to determine if they continued to display circadian rhythms. Both strains demonstrated similar circadian patterns for all three metabolic parameters when grown under long‐day conditions. However, the strains responded differently to short‐day growth conditions. Cyanothece sp. ATCC 51142 retained reasonable circadian patterns under 8:16 L:D conditions, whereas Cyanothece sp. PCC 7822 had quite damped patterns without a clear circadian pattern. In particular, glycogen storage changed very little throughout the day and we ascribe this to the difference in the type of glycogen granules in Cyanothece sp. PCC 7822 which has small β‐granules, compared to the large, starch‐like granules in Cyanothece sp. ATCC 51142. The results suggested that both mechanistic and regulatory processes play a role in establishing the basis for these metabolic oscillations.     

An unusual overrepresentation of genetic factors related to iron homeostasis in the genome of the fluorescent Pseudomonas sp. ABC1

Members of the genus Pseudomonas inhabit diverse environments, such as soil, water, plants and humans. The variability of habitats is reflected in the diversity of the structure and composition of their genomes. This cosmopolitan bacterial genus includes species of biotechnological, medical and environmental importance. In this study, we report on the most relevant genomic characteristics of Pseudomonas sp. strain ABC1, a siderophore-producing fluorescent strain recently isolated from soil. Phylogenomic analyses revealed that this strain corresponds to a novel species forming a sister clade of the recently proposed Pseudomonas kirkiae. The genomic information reveals an overrepresented repertoire of mechanisms to hoard iron when compared to related strains, including a high representation of fecI-fecR family genes related to iron regulation and acquisition. The genome of the Pseudomonas sp. ABC1 contains the genes for non-ribosomal peptide synthetases (NRPSs) of a novel putative Azotobacter-related pyoverdine-type siderophore, a yersiniabactin-type siderophore and an antimicrobial betalactone; the last two are found only in a limited number of Pseudomonas genomes. Strain ABC1 can produce siderophores in a low-cost medium, and the supernatants from cultures of this strain promote plant growth, highlighting their biotechnological potential as a sustainable industrial microorganism.     

珠江口沉积物来源链霉菌SCSIO 40020中bafilomycins的鉴定及其生物合成基因簇的异源表达

【目的】从珠江口沉积物来源的菌株SCSIO40020中分离bafilomycins,并对其生物合成基因簇进行克隆和异源表达研究。【方法】通过分析菌株SCSIO 40020的16S rRNA基因序列并构建系统发育树以鉴定菌种,以柱层析法和制备色谱法对次级代谢产物进行分离纯化,借助波谱学手段完成单体化合物的结构鉴定,采用生物信息学分析定位bafilomycins的生物合成基因簇,通过筛选菌株SCSIO 40020基因组的细菌人工染色体文库和接合转移将bafilomycins生物合成基因簇导入3种链霉菌进行异源表达,利用高效液相色谱检测异源表达菌株的发酵产物。【结果】菌株SCSIO 40020被鉴定为链霉菌属菌株,从其发酵产物中分离鉴定了2个单体化合物bafilomycinsA1和D。克隆了链霉菌SCSIO40020中bafilomycins的生物合成基因簇并推导了其生物合成途径,在3种链霉菌中表达产生了bafilomycins。【结论】从珠江口环境中获得了一株产生bafilomycins的链霉菌SCSIO 40020,成功建立了该菌株次级代谢产物生物合成基因簇的异源表达体系,并首次在链霉菌...     

Culture Independent Detection of Sphingomonas sp. EPA 505 Related Strains in Soils Contaminated with Polycyclic Aromatic Hydrocarbons (PAHs)

The Sphingomonas genus hosts many interesting pollutant-degrading strains. Sphingomonas sp. EPA505 is the best studied polycyclic aromatic hydrocarbon (PAH)-degrading Sphingomonas strain. Based on 16S rRNA gene sequence analysis, Sphingomonas sp. strain EPA505 forms a separate branch in the Sphingomonas phylogenetic tree grouping exclusively PAH-degrading isolates. For specific PCR detection and monitoring of Sphingomonas sp. EPA505 and related strains in PAH-contaminated soils, a new 16S rRNA gene-based primer set was designed. The new primer set was shown to be highly selective for Sphingomonas sp. strain EPA505 as it only amplified DNA from strain EPA505 and not from other tested Sphingomonas strains or soil bacteria not belonging to the Sphingomonas genus. Using DNA extracts of a variety of inoculated PAH-contaminated soils, the primer pair was able to detect EPA505 in concentrations as low as 10² cells per gram of soil. Applying the new primer set, 16S rRNA gene fragments which were 99–100% similar to the corresponding gene of strain EPA505 were amplified from four of five PAH-contaminated soils. On the other hand, no PCR products were obtained from any of five tested uncontaminated soils. The preferential presence of EPA505 related Sphingomonas strains in PAH-contaminated soils with very different contamination profiles and different origin suggests an important role of this type of Sphingomonas in the natural Sphingomonas community colonizing PAH-contaminated sites.     

Molecular phylogeny and evolution of subsection Magnicellulatae (Erysiphaceae: Podosphaera) with special reference to host plants

The subsection Magnicellulatae of the genus Podosphaera section Sphaerotheca belongs to the tribe Cystotheceae of the Erysiphaceae, which has the characteristic of producing catenate conidia with distinct fibrosin bodies. In this study, we newly determined the nucleotide sequences of the D1/D2 domains of the 28S rDNA region and the sequences of the rDNA internal transcribed spacer (ITS) region to investigate the relationships between the phylogeny of this fungal group and their host plants. The results indicated that the 28S rDNA region is too conservative for phylogenetic analysis of this fungal group. The phylogenetic analysis using 95 ITS sequences demonstrated that two or more Magnicellulatae taxa often infect the same plant genus or species. Although there is a close relationship between Magnicellulatae and asteraceous hosts, this association seems to be not as strict as that between Golovinomyces and the Asteraceae. The difference between the two fungal groups may be explained by their different evolutionary timing.     

Tilletiopsis derxii, Tilletiopsis oryzicola and Tilletiopsis penniseti, three new species of the ustilagionomycetous anamorphic genus Tilletiopsis isolated from leaves in Thailand

Four strains of ballistoconidium-forming yeast-like fungi (K-95, K-125, K-132 and K139), isolated from plants collected in Bangkok, Thailand, were assigned to the genus Tilletiopsis based on morphological and chemotaxonomical characteristics. On the basis of sequence data of 18S rDNA and the D1/D2 region of 26S rDNA, strains K-95, K-125 and K-132 were close to T. flava and T. fulvescens, and strain K-139 each formed related to T. minor. DNA-DNA reassociation experiments with related species revealed that strains K-125, K-132 and K-139 each formed a new and distinct species whereas strain K-95 was identified as T. flava. Tilletiopsis derxii Takashima et Nakase sp. nov. (JCM 10217^T; K-125), Tilletiopsis oryzicola Takashima et Nakase sp. nov. (JCM 10218^T; K-132), and Tilletiopsis penniseti Takashima et Nakase sp. nov. (JCM 10216^T; K-139) are the names proposed for the new taxa.     

Salicylic and jasmonic acid pathways are necessary for defence against Dickeya solani as revealed by a novel method for Blackleg disease screening of in vitro grown potato

Potato is major crop ensuring food security in Europe, and blackleg disease is increasingly causing losses in yield and during storage. Recently, one blackleg pathogen, Dickeya solani has been shown to be spreading in Northern Europe that causes aggressive disease development. Currently, identification of tolerant commercial potato varieties has been unsuccessful; this is confounded by the complicated etiology of the disease and a strong environmental influence on disease development. There is currently a lack of efficient testing systems. Here, we describe a system for quantification of blackleg symptoms on shoots of sterile in vitro potato plants, which saves time and space compared to greenhouse and existing field assays. We found no evidence for differences in infection between the described in vitro‐based screening method and existing greenhouse assays. This system facilitates efficient screening of blackleg disease response of potato plants independent of other microorganisms and variable environmental conditions. We therefore used the in vitro screening method to increase understanding of plant mechanisms involved in blackleg disease development by analysing disease response of hormone‐ related (salicylic and jasmonic acid) transgenic potato plants. We show that both jasmonic (JA) and salicylic (SA) acid pathways regulate tolerance to blackleg disease in potato, a result unlike previous findings in Arabidopsis defence response to necrotrophic bacteria. We confirm this by showing induction of a SA marker, pathogenesis‐related protein 1 (StPR1), and a JA marker, lipoxygenase (StLOX), in Dickeya solani infected in vitro potato plants. We also observed that tubers of transgenic potato plants were more susceptible to soft rot compared to wild type, suggesting a role for SA and JA pathways in general tolerance to Dickeya.     

Ciliate Protozoa in the Rumen of Brazilian Water Buffalo,Bubalus bubalis Linnaeus*

SYNOPSIS Total numbers and distribution of genera, subgenera and species were determined for the ciliate protozoa in rumen contents of 4 Brazilian water buffalo Bubalus bubalis Linnaeus. The fauna of one animal, housed in close proximity to European and zebu-type cattle, differed considerably from that of the remaining animals, which were somewhat isolated on a large ranch. Several of the protozoan species observed in the semi-isolated animals were first described in rumen contents from humped Indian cattle, and their subsequent occurrence in other hosts and geographic locations has been limited or absent. In all, 49 different species of protozoa were found, 8 of which have not been previously described. Three of the new species belong to the genus Entodinium: E. ciculum sp. n., E. spinonucleatum sp. n. and E. triangulum sp. n.: 4 to Diplodinium (Ostracodinium): D. (O.) brazili sp. n., D. (O.) esalqum sp. n., D. (O.) nucleolobum sp. n., and D. (O.) tiete sp. n.; and one to Diplodinium (Eudiplodinium): D. (E.) bubalus sp. n.     

Two new species of Duplicibothrium Williams & Campbell, 1978 (Tetraphyllidea: Serendipidae) from the Pacific cownose ray Rhinoptera steindachneri

Two new tetraphyllidean species, Duplicibothrium cairae n. sp. and D. paulum n. sp., are described from Pacific cownose rays Rhinoptera steindachneri collected from the Gulf of California. D. cairae n. sp. differs from the only other known species in this genus, D. minutum, in its possession of two posteriorly bifurcating longitudinal septa on each bothridium and having a greater number of loculi per bothridium, wider bothridia and a greater number of segments per worm. D. paulum n. sp. differs from the above two species in being shorter, having two continuous longitudinal septa on each bothridium, rather than posteriorly bifurcating or absent longitudinal septa, having a greater number of loculi per bothridium and having fewer segments per worm. The generic diagnosis of Duplicibothrium is amended to reflect the inclusion of the two new species. Species of Duplicibothriumappear to be restricted to host species of the genus Rhinoptera. Systematic information gained from the study of these two new species supports the monophyly of the tetraphyllidean family Serendipidae and suggests that Duplicibothrium shares a phylogenetic heritage with species in the genera Glyphobothrium and Serendip, taxa that are also parasitic in cownose rays. Evidence for a phylogenetic relationship between serendipid species and species of Dioecotaenia is discussed, as are potential molluscan hosts for cestode species in Duplicibothrium and Dioecotaenia.