Phytotoxin produced by the netted scab pathogen, <Emphasis Type="Italic">Streptomyces turgidiscabies</Emphasis> strain 65, isolated in Sweden

Streptomyces spp. are a highly diverse group of bacteria most of which are soil-inhabiting saprophytes. A few are plant pathogens that produce a family of phytotoxins called thaxtomins and cause significant economic losses, e.g., by reducing the marketability of potato tubers (Solanum tuberosum). In northern Europe, S. scabies, S. turgidiscabies and S. europaeiscabiei are the most common plant pathogenic species. In this study, a Streptomyces strain isolated from a netted scab lesion on a tuber of potato cv. Bintje in northern Sweden was identified as S. turgidiscabies but was found to differ in the genomic region carrying genes required for thaxtomin biosynthesis. Our results showed that the strain did not produce thaxtomin but rather phytotoxin fridamycin E, which is an anthraquinone novel to plant pathogenic Streptomyces spp. Fridamycin E was shown to reduce or inhibit sprouting of potato microtubers in vitro. While fridamycin E is known to have antibiotic activity against Gram-positive bacteria, the inhibitory activity of fridamycin E on plant growth is a novel finding.     

Association of ‘Candidatus Liberibacter asiaticus’ root infection,but not phloem plugging with root loss on huanglongbing‐affected trees prior to appearance of foliar symptoms

Huanglongbing (HLB) is a systemic disease of citrus caused by phloem‐limited bacteria ‘Candidatus Liberibacter’ spp. with ‘Ca. Liberibacter asiaticus’ (Las) the most widespread. Phloem‐limited bacteria such as liberibacters and phytoplasmas are emerging as major pathogens of woody and herbaceous plants. Little is known about their systemic movement within a plant and the disease process in these tissues. Las movement after initial infection was monitored in leaves and roots of greenhouse trees. Root density, storage starch content, and vascular system anatomy in relation to Las presence in field and greenhouse trees, both with and without symptoms, showed the importance of root infection in disease development. Las preferentially colonized roots before leaves, where it multiplied and quickly invaded leaves when new foliar flush became a sink tissue for phloem flow. This led to the discovery that roots were damaged by root infection prior to development of visible foliar symptoms and was not associated with carbohydrate starvation caused by phloem‐plugging as previously hypothesized. The role of root infection in systemic insect‐vectored bacterial pathogens has been underestimated. These findings demonstrate the significance of early root infection to tree health and suggest a model for phloem‐limited bacterial movement from the initial insect feeding site to the roots where it replicates, damages the host root system, and then spreads to the rest of the canopy during subsequent leaf flushes. This model provides a framework for testing movement of phloem‐limited bacteria to gain greater understanding of how these pathogens cause disease and spread.     

Expression-based genotyping of the rice blast resistance genes in the elite maintainer line Yixiang1B

Plants employ extracellular immune receptors to perceive conserved pathogen-associated molecular patterns (PAMPs), triggering the first layer of defense known as pattern-triggered immunity (PTI). The understanding of PTI is mainly based on studies focusing on leaves. Plants are vulnerable to attack by various root pathogens including plant-parasitic nematodes. Evidence is accumulating that phytonematodes utilize their secreted effectors to suppress PTI to enable infection. PTI assays used for characterizing nematode effectors are often conducted in a non-host plant or tissue, such as leaves, because of lacking of root assays. Thus, there is a need for PTI assays in roots of host plants. Here, we tested two bacterial PAMPs (flg22 and flgII-28) and two nonpathogenic bacteria (Pseudomonas fluorescens and P. syringae strain DC3000 ΔhrcQ-U) for their ability to induce PTI responses, including the induction of defense gene expression and callose deposition, in roots of tomato and potato. We found that flg22 and the two nonpathogenic bacteria are potent in inducing defense gene expression and callose deposition in tested roots, demonstrating for the first time induction of PTI in roots of solanaceous plants. Effectors GrCEP12 and Hs10A06 were previously indicated to be involved in PTI suppression. Consistently, upon elicitor treatment, roots of transgenic plants overexpressing GrCEP12 and Hs10A06, respectively, showed a reduced level of defense gene expression or no induction of callose deposition compared to control roots. Taken together, our established root PTI assays represent a valuable tool that will facilitate the study of phytonematodes and potentially other root pathogens in their manipulation of plant immunity.     

Seeing the forest for the trees: Use of phages to treat bacterial tree diseases

Trees and woody plants can be attacked by many pests and pathogens either individually or as polymicrobial infections. In particular, infections caused by tree-specific bacterial pathogens have become more common during the last decade, causing serious concern for important tree and woody plant species in horticulture, urban environments, and forests. For example, Xylella and Pseudomonas bacteria are causing significant economic and ecological devastation throughout Europe in olive, cherry, and other stone fruits, mainly because of lack of efficient control methods and the emergence of bacterial resistance to traditional antimicrobial compounds such as copper and antibiotics. Hence, there is an urgent need for innovative approaches to tackle bacterial plant diseases. One way to achieve this could be through the application of biological control, which offers a more environmentally friendly and targeted approach for pathogen management. This review will explore recent advances in use of pathogen-specific viruses, bacteriophages (or phages), for the biocontrol of bacterial tree diseases. Phages are an important component of plant microbiomes and are increasingly studied in plant pathogen control due to their highly specific host ranges and ability to selectively kill only the target pathogenic bacteria. However, their use still poses several challenges and limitations, especially in terms of managing the bacterial diseases of long-lived trees. A particular insight will be given into phage research focusing on controlling Pseudomonas syringae pathovars, Erwinia amylovora, Xanthomonas species, Ralstonia solanacearum, and Agrobacterium tumefaciens. Recent milestones, current challenges, and future avenues for phage therapy in the management of tree diseases are discussed.     

A semiaxenic phototrophic system to study interactions between arbuscular mycorrhizal and pathogenic fungi in woody plants

Among other benefits, arbuscular mycorrhizal (AM) fungi may increase plant tolerance to root diseases. The research on the underlying mechanisms requires growth conditions that are both controlled and realistic. To study these interactions, a semiaxenic phototrophic system was developed in which the roots grow in a controlled environment and can be inoculated with both pathogenic and symbiotic fungi. Micropropagated fig plantlets were grown in containers having shoots in the outside and roots in a growth medium without sugar, inoculated or not with the AM fungus Rhizophagus irregularis and the pathogenic fungus Armillaria mellea. Dual inoculated plants developed the mycorrhizal association and pathogen infection symptoms. Mycorrhizal inoculation lowered disease index and increased plant growth. Colonization of A. mellea in fig roots was quantified by real-time PCR, showing that R. irregularis did not significantly lower the quantity of Armillaria, suggesting that other mechanisms were involved in increased tolerance to the pathogen. The results show that the system proposed is suitable to study the triple interaction involving plant, AM and root pathogenic fungi.     

Microbial community responses associated with the development of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">cucumerinum</Emphasis> after 24-epibrassinolide applications to shoots and roots in cucumber

Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum (FO), is one of the major diseases in cucumber (Cucumis sativus) production. Root and foliar applications of 24-epibrassinolide (EBL), an immobile phytohormone with antistress activity, were evaluated for their effects on the incidence of Fusarium wilt and changes in the microbial population and community in roots of cucumber plants. EBL pre-treatment to either roots or shoots significantly reduced disease severity followed by an improved plant growth regardless of the treatment methods applied. EBL applications decreased the Fusarium population on root surfaces and in nutrient solution, but increased the population of fungi and actinobacteria on root surfaces. PCR-DGGE analysis showed that FO-inoculation had significant effects on the bacterial community on root surfaces as expressed by a decreased diversity index and evenness index, but EBL applications alleviated these changes. Moreover, several kinds of decomposing bacteria and growth-promoting bacteria were identified from root surfaces of FO-inoculated plants and EBL-pre-treated plants, respectively. Overall, these results show that the microbial community on root surfaces was affected by a complex interaction between phytohormone-induced resistance and plant pathogens.     

Plant responses to plant growth-promoting rhizobacteria

Non-pathogenic soilborne microorganisms can promote plant growth, as well as suppress diseases. Plant growth promotion is taken to result from improved nutrient acquisition or hormonal stimulation. Disease suppression can occur through microbial antagonism or induction of resistance in the plant. Several rhizobacterial strains have been shown to act as plant growth-promoting bacteria through both stimulation of growth and induced systemic resistance (ISR), but it is not clear in how far both mechanisms are connected. Induced resistance is manifested as a reduction of the number of diseased plants or in disease severity upon subsequent infection by a pathogen. Such reduced disease susceptibility can be local or systemic, result from developmental or environmental factors and depend on multiple mechanisms. The spectrum of diseases to which PGPR-elicited ISR confers enhanced resistance overlaps partly with that of pathogen-induced systemic acquired resistance (SAR). Both ISR and SAR represent a state of enhanced basal resistance of the plant that depends on the signalling compounds jasmonic acid and salicylic acid, respectively, and pathogens are differentially sensitive to the resistances activated by each of these signalling pathways. Root-colonizing Pseudomonas bacteria have been shown to alter plant gene expression in roots and leaves to different extents, indicative of recognition of one or more bacterial determinants by specific plant receptors. Conversely, plants can alter root exudation and secrete compounds that interfere with quorum sensing (QS) regulation in the bacteria. Such two-way signalling resembles the interaction of root-nodulating Rhizobia with legumes and between mycorrhizal fungi and roots of the majority of plant species. Although ISR-eliciting rhizobacteria can induce typical early defence-related responses in cell suspensions, in plants they do not necessarily activate defence-related gene expression. Instead, they appear to act through priming of effective resistance mechanisms, as reflected by earlier and stronger defence reactions once infection occurs.     

Bacterial diseases of potatoes: from classical phytobacteriology to molecular pathogenicity

Various bacterial pathogens attack potato plants and tubers. These pathogens includeErwinia, Corynebacterium (Clavibacter), Psuedomonas andStreptomyces. Over the past few years there have been significant advances in the molecular biological analysis of several of these pathogens and this is now helping us to understand the major aspects of virulence mechanisms. However, to date, such information is not sufficiently useful to allow us to intervene rationally in these potato diseases, except by the standard practises of good husbandry.     

Ultrastructural and cell wall modifications during infection of <Emphasis Type="Italic">Eucalyptus viminalis</Emphasis> roots by a pathogenic <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strain

Fusarium species are soil-borne fungal pathogens that produce a variety of disease symptoms when attacking crop plants. The mode of root colonization of Eucalyptus viminalis seedlings by a pathogenic F. oxyporum strain (Foeu1) at the ultrastructural level and changes in cell wall pectin during host pathogen interactions are described. Root systems of E. viminalis plants were inoculated with F. oxysporum in an in vitro model system. Hyphae of F. oxysporum adhered to the outer epidermal cell walls through fibrillar material, and after penetration they spread into the internal tissues. They developed intercellularly and intracellularly in the root cortex and invaded vascular tissues. Papillae were induced, and the host plasma membrane ruptured in colonized cells, causing rapid host tissue and cell damage. Changes in distribution and occurrence of nonesterified and methyl-esterified pectins were evaluated after root colonization by F. oxysporum using two monoclonal antibodies, JIM 5 and JIM 7, respectively. Nonesterified pectin in control roots was mainly localized in the epidermal cell walls and middle lamellae in parenchymal cortex, whereas methyl-esterified pectin accumulated more in primary cell walls of the cortex and phloem. Decreases in immunodetected nonesterified and methyl-esterified pectins were associated with extensive plant tissue degradation after root colonization by the pathogenic fungus.     

Soft rot of root chicory in Hokkaido and its causal bacteria

In August 2010, bacterial soft rot was found on root chicory (Cichorium intybus var. sativum) in Hokkaido, Japan. Severely infected plants in fields were discolored, had wilted foliage, and black necrosis of petioles near the crown. Wilted leaves subsequently collapsed and died, forming a dry, brown or black rosette. The root and crown became partially or wholly soft-rotted. Slimy masses on infected areas of roots, turned dark brown or black. Gram-negative, rod-shaped, peritrichously flagellated, facultatively anaerobic bacteria were exclusively isolated from rotted roots, and typical symptoms were reproduced after inoculation with the strains. The bacteria were identified as Dickeya dianthicola, Pectobacterium carotovorum subsp. carotovorum, and Pectobacterium carotovorum subsp. odoriferum based on further bacteriological characterization and the sequence analysis of the malate dehydrogenase gene and 16S rRNA gene. These bacteria should be included with the previously reported Dickeya (=Erwinia) chrysanthemi in Saitama Prefecture, Japan, as causal pathogens of bacterial wilt of chicory.     

Mechanisms of symptom production by foliar bacterial pathogens

Most foliar bacterial pathogens are pathovars ofPseudomonas syringae andXanthomonas campestris. Many of them live during the greater part of their life-cycle on the upper parts of the plant. In order to survive and develop in this environment, the pathogens have elaborated an array of mechanisms which enable them to penetrate the plant foliage, produce a substantial endophytic population, and use the host tissue as a nutrient source. These activities result in the formation of different categories of symptoms,e.g. necrotic and chlorotic lesions, wilting, gall formation, leaf abscission and inhibition of plant growth. The operating mechanisms of foliar phytopathogenic bacteria can be divided into four major groups: (i) production of toxins (mostly non-specific), mainly byP. syringae pathovars; (ii) excessive production of plant growth hormones; (iii) enzymatic hydrolyzing activity; and (iv) inhibition of seedling growth by unknown mechanism(s). More than one pathogenic mechanism may operate in a given leaf disease or by a single pathogen. Despite the large amount of literature describing visual, biochemical and biophysical aspects of any leaf disease, basic knowledge is fragmental and comprehensive understanding of the mode of action of any disease is lacking. It is proposed that in order to close up these gaps in our knowledge other approaches, such as molecular biology technology and more intensive use of both bacterial and plant mutants, should be incorporated in pathological studies of foliar bacterial diseases.     

New insights into radiata pine seedling root infection by Fusarium circinatum

Pine root infection by Fusarium circinatum has been reported in the literature, but the underlying pathogenic interaction is poorly understood. A green fluorescent protein (GFP)‐tagged F. circinatum isolate, together with confocal microscopy, was used in order to monitor the events associated with root infection of Pinus radiata seedlings. It was found that in order to reach and successfully infect pine roots, F. circinatum employed features that are similar to those previously described for other root‐infecting pathogens, such as mycelial strands, single runner hyphae and simple hyphopodia as well as other features that are reminiscent of those that are known to be involved in biotrophic invasion, such as bulbous invasive hyphae and filamentous invasive hyphae. Abundant sporulation was observed at the root surface as well as inside tracheids both in roots and in the root collar region. The fungus can spread from the roots to the aerial parts of the plant, and once there, colonization appears to be similar to the process that occurs when the pathogen is inoculated in the stem. Wilting symptoms and plant demise may be the result of a reduction in water uptake by roots and of the blockage of the vascular system by fungal hyphae and resin.     

Detection of bacterial aggregation in tobacco cell suspensions treated with pathogenic bacteria

Previous studies of this model system involving plant cell suspensions inoculated with bacteria, have documented that interactions with incompatible pathogens, which cause a hypersensitive response on whole plants, will cause a transient increase in oxygen uptake 2–4 h after inoculation. The initial objective of this study was to determine whether this oxygen uptake burst was a result of increased bacterial multiplication, possibly due to nutrient leakage from plant cells. The adaptation of flow cytometry and the use of fluorescent nucleic acid stains provided the precision needed to monitor bacterial concentrations in tobacco suspension cells inoculated with pathogenic and non-pathogenic Pseudomonas species. Surprisingly, there was a transient decrease in the planktonic, or free-living, bacteria in cell suspensions inoculated with isolate Pseudomonas syringae pv. syringae WT (HR+), an incompatible pathogen of tobacco. This decrease in planktonic numbers was followed by an apparent increase in bacterial multiplication. Examination of the samples with fluorescent microscopy revealed the formation of bacterial aggregates in the extracellular fluid of the Pss WT (HR+) inoculated plant cells. The size of the aggregates increased at the onset of the oxygen uptake response, and contained increasing numbers of bacterial cells. These aggregated bacterial cells appear to be removed along with plant cells, as a result of filtration during sample preparation, causing the apparent decrease in planktonic bacteria detected by flow cytometry. This bacterial aggregation was also observed with the compatible Pseudomonas tabaci pathogen, which does not induce a noticeable oxygen uptake burst. No aggregation was observed with suspension inoculated with Pseudomonas fluorescens, a saprophyte, or Pss B7 (HR−), a Tn5 mutant of P. s. syringae. This aggregation response was rapid, once initiated, and appeared similar to reports of adhesion involving Hrp pili.     

Microscopic analysis of plant–bacterium interactions using auto fluorescent proteins

Plant growth promoting rhizobacteria (PGPR) include bacteria that fix nitrogen (e.g., Rhizobiaceae, Herbaspirillum, Azoarcus), produce phytohormones (e.g., Azospirillum) and provide protection against fungal and/or bacterial pathogens (e.g., Pseudomonas, Bacillus, Streptomyces). Interactions between PGPR and plants can be divided into different steps which include initial attraction, attachment, proliferation and colonization e.g., of roots, stem, leaves and flowers. At the genetic level the expression of many bacterial genes are altered during these processes. In addition to the interaction with the plant, PGPR interact and compete with the endogenous microflora, consisting of other bacteria, fungi and/or mycorrhizal fungi. In the case of biocontrol bacterial strains, a direct interaction with the pathogen is often required to suppress the disease. Microscopic analyses of plant growth promoting rhizobacteria (PGPR) in their natural environment and in specific during their interaction(s) with the host plant(s) and/or their target organism(s) is essential for the elucidation of their functioning and the successful application of commercial inoculants. With the discovery and development of auto fluorescent proteins (AFPs) as markers and the development of highly sophisticated fluorescence microscopes such as confocal laser scanning microscopes, a new dimension has been created for studying PGPR in their natural environment. This paper will give a short overview on available tools, the application of AFPs in PGPR research and some future perspectives. Several recent reviews will give the reader an option for further reading (Bloemberg and Lugtenberg 2004; Chalfie and Kain 2005; Larrainzar et al. 2005; Rediers et al. 2005; Bloemberg and Camacho 2006).     

Dynamics of the root/soil pathogens and antagonists in organic and integrated production of potato

Microbial communities in the root, rhizoplane, and rhizosphere and non-rhizosphere soil in potato, in organic and integrated production systems, were compared at the emergence and flowering phases of plant development. Microorganisms were identified on the basis of their morphology. The dominant groups included Clonostachys + Gliocladium + Trichoderma, Fusarium + Gibberella + Haematonectria + Neonectria, Paecilomyces, Penicillium and Phoma. Microbial density at the flowering phase was often significantly greater in roots and non-rhizosphere soil than in the rhizoplane and rhizosphere. Diversity of the communities often remained stable or was greater at the emergence phase. The density of bacteria changed with time. The density of Pseudomonas often decreased while Streptomyces significantly increased with time. Changes in densities of pathogens and antagonists decreased the suppressiveness of the habitat towards soil-borne potato pathogens at the flowering phase. The study contributes information that will help to: (a) understand the epidemiology of some potato diseases, (b) make decisions on the economic and ecological aspects of chemical control in potato, (c) develop strategies for manipulation of the soil microbial environment as a viable crop management technique, and (d) develop prognosis models for potato diseases in central Europe.     

Coffee bacterial diseases: a plethora of scientific opportunities

Coffee is a very important crop for several tropical countries across different continents. The diseases bacterial halo blight (BHB), bacterial leaf spot (BLS), bacterial leaf blight (BLB) and coffee leaf scorch (CLS), caused by the bacterial pathogens Pseudomonas syringae pv. garcae (Psgc), P. syringae pv. tabaci (Psta), Pseudomonas cichorii (Pch) and Xylella fastidiosa subsp. pauca (Xfp), respectively, cause significant reductions in coffee production, although other minor bacterial diseases have also been reported in some countries. Little research progress has been made on aspects that are relevant for control and management of these diseases. In all cases, there is an urgent need to develop rapid and more reliable methods for early detection of the pathogens in order to minimize their negative impact on coffee production. Because of the high rate of intra- and intersubspecific recombination occurring in X. fastidiosa, a permanent revision of the detection methods is necessary. Greater efforts should be made to understand the genetic and virulence diversity of Psgc, Psta and Pch populations. Early studies reported the identification of potential sources of resistance against Psgc and Psta, but, to date, no resistance gene has been isolated. Little effort has been made to understand the biology and molecular mechanisms underlying the interaction between Coffea spp. and these pathogenic bacteria. This review discusses the recent progress on the molecular mechanisms used by these bacteria to cause diseases on other plant species, in order to provide a guideline for the establishment of future research programmes.     

Evaluation of Gram-positive rhizosphere and endophytic bacteria for biological control of fungal rice (<Emphasis Type="Italic">Oryzia sativa</Emphasis> L.) pathogens

Gram-positive bacteria isolated from the rhizosphere and inside the roots of rice were characterized for plant growth promoting (PGP) traits and antifungal activity against some rice plant pathogenic fungi of rice. The results showed the endophytic and rhizosphere isolates had different PGP traits and antifungal activity. Only one rhizosphere isolate and one endophytic isolate showed highly inhibitory effects against the mycelial growth of all fungal rice pathogens tested in this study. The best bacterial isolates, based on multiple PGP traits and inhibitory effects against the mycelial growth of all fungal rice pathogens, were identified. Based on biochemical tests and by comparison of 16S rDNA sequences, the endophytic isolate REN₃ and the rhizosphere isolate REN₄ were closely related to Bacillus cereus and Bacillus mojavensis respectively. The broad-spectrum antifungal strains, the REN₃ and REN₄ isolates analyzed here, exert multiple PGP and antagonistic activity and represent an excellent option to be used as either potent bio-promoting or bio-control agents in rice under in vitro conditions. This application may help to minimize dependence on pesticides, which have adverse effects on the environment, finally leading to have sustainable environments. In conclusion, the results of antifungal activity showed rice harbors bacteria with a good potential in biocontrol of rice fungal pathogens.     

Bacterial wilt disease: Host resistance and pathogen virulence mechanisms

Bacterial wilt (BW), caused by Ralstonia solanacearum, is one of the most destructive bacterial diseases of Solanaceous species worldwide. The species infects plants in more than 200 species and 50 families and was ranked second in a list of the top 10 most scientifically and economically important bacterial plant pathogens [1]. The molecular mechanisms underlying resistance and the functions of R. solanacearum effectors are beginning to be uncovered, and much remains to be discovered. In this mini-review, we provide a summary of host resistance and R. solanacearum virulence mechanisms, with a focus on tomato.     

Thaxtomin A: evidence for a plant cell wall target

Thaxtomins are unique 4-nitroindol-3-yl containing dioxopiperazines that cause dramatic plant cell hypertrophy and seedling stunting. This family of phytotoxins is produced by Streptomyces species that cause diseases of root and tuber crops; its members are essential for pathogenicity. The symptoms produced by thaxtomin A suggest several potential plant cell targets including the plasma membrane, various components of the cytoskeleton and the cell wall. Dramatic increases in cell volume in onion seedling hypocotyls, radish seedling hypocotyls and tobacco suspension cultures, in response to 0.05–1.0 μM thaxtomin A, suggested that this phytotoxin is interacting with one or more conserved plant cell targets. Onion root tip cells treated with thaxtomin A concentrations at or below that which inhibited onion root growth were binucleate or had abnormal cell plates. Thaxtomin A (1.0–3.0 μM) inhibited normal cell elongation of tobacco protoplasts in a manner that suggested an effect on primary cell wall development. In summary, these data suggest that thaxtomin A alters, either directly or indirectly, the deposition or composition of monocot and dicot plant cell walls in ways that affect the wall integrity and the ability of the cell to progress normally through cytokinesis.