Mechanism of disease development caused by a multihost plant bacterium, Pseudomonas cichorii, and its virulence diversity

Pseudomonas cichorii causes rot on lettuce leaves, distinct from the necrotic spots of infected eggplant leaves. On lettuce leaves, P. cichorii invades intercellular spaces through stomata and grows vigorously, causing rot on the leaves. Surprisingly, P. cichorii does not produce pectate lyase, the most important enzyme for degrading plant cell walls. Alternatively, infection with P. cichorii causes heterochromatin condensation and DNA laddering in lettuce cells, followed by induced cell death, which results in disease symptoms. Thus, apoptotic programmed cell death (PCD) is associated with the symptoms on lettuce leaves. Although PCD in P. cichorii-infected eggplant leaves is also associated with necrotic spots caused by the bacteria, there are differences in the responses of the two hosts. P. cichorii harbors the N-acetyltransferase family gene (pat) and the hrp genes (hrp) encoding a type III secretion system, adjacent to an aldehyde dehydrogenase gene (aldH). The virulence of P. cichorii is hrp-dependent in eggplant, but not in lettuce. Furthermore, hrp, pat and aldH are implicated in the diversity of P. cichorii virulence on susceptible Asteraceae species. The involvement of hrp, aldH and pat in bacterial virulence on the respective species has no relationship with the phylogeny of the plants species. When these results are considered together, P. cichorii has multiple virulence determinants. The involvement of not only hrp but also aldH and pat in P. cichorii virulence arose after species diversification of host plants. Host responses implicated in symptom development have been responsible for the development of virulence determinants of P. cichorii.     

A new genomovar of Pseudomonas cichorii, a causal agent of tomato pith necrosis

Recent taxonomic advances, based on biochemical and genotypic processes demonstrate that the plant pathogenic species Pseudomonas cichorii consists of a cluster of closely related genomic groups. Prior to this study, three morphotype groups had been described (C1-C3), all sharing various phenotypic and biochemical characters but partially differing in their DNA content. All entities of the complex could cause disease among a variety of hosts, including lettuce, celery, chrysanthemum and others. In this study, we present the biochemical and molecular characterization of P. cichorii isolates as the causal agent of pith necrosis of tomato plants. A detailed characterization of the genetic variability among strains belonging to P. cichorii was achieved using BOX-PCR and Multi Locus Sequence Analysis utilizing three housekeeping genes (gyrB, rpoD, rpoB). In addition, a number of biochemical and physiological tests were also used for the identification of the tomato P. cichorii isolates. To our knowledge, this is the first complete biochemical, molecular and phylogenetic study of P. cichorii strains isolated from tomato plants affected by pith necrosis disease. Our findings demonstrate the emergence of a new genomovar of P. cichorii, yet another indication for the genetic heterogeneity of the species.     

Biology of Pseudomonas cichorii in chrysanthemum1

Since 1982, outbreaks of stem blight caused by Pseudomonas cichorii are sometimes found in greenhouse-grown chrysanthemums in The Netherlands. In this study, 13 Dutch chrysanthemum isolates and, for comparison, six from other origins were biochemically, serologically and pathologically characterized. All isolates were biochemically homogeneous in most of some 40 tests. Different serotypes were observed, which were not host or virulence-correlated. After inoculation into cut and potted chrysanthemum, endive and chicory, P. cichorii isolates were found to be non-host-specific. Isolates showed differences in virulence after inoculation into potted chrysanthemum. For five isolates, disease development at high humidity (85–95% RH, 18°C) was more than 100% greater than at low humidity (55–70% RH, 18°C). Using a selective medium and immunosorbent dilution plating (ISDP), P. cichorii could be isolated from symptomless leaves and soil from nurseries where the disease was observed and from soil of a disease-free nursery. ISDP did not significantly increase the recovery of the pathogen. It was concluded that: (a) P. cichorii is an opportunistic pathogen; (b) epidemic infections of stem blight are due to a combination of high humidity, higher temperatures, high plant density and use of very susceptible cultivars; (c) control measures should concentrate on preventing this combination.     

Development of a real‐time PCR assay for Pseudomonas cichorii,the causal agent of midrib rot in greenhouse‐grown lettuce,and its detection in irrigating water

Midrib rot is an emerging disease in greenhouse production of lettuce caused by Pseudomonas cichorii, and probably introduced through contaminated irrigation water. Concentrations of 100 CFU mL^?1 are enough to induce the typical midrib rot symptoms. A sensitive real‐time PCR assay was developed, based on a 90‐bp amplicon from the pathogenicity gene cluster hrcRST and a Taqman Minor Groove Binding probe. Specificity of the assay was tested with 39 P. cichorii strains, including the type strain, and 89 strains from 83 other Pseudomonas species. The relationship between detection signals and P. cichorii DNA concentrations was linear over 6‐logs. Detection threshold with excellent reproducibility was 500 fg of DNA or about 70 genome copies. Sample preparation and DNA isolation were optimized to allow detection in 1 L water samples. The assay was first evaluated with greenhouse irrigation water spiked with serial dilutions of P. cichorii. The calculated cell numbers obtained with real‐time PCR were 10‐fold lower than plate counts of actual spiked cells. However, the assay consistently detected 100 CFU per reaction, corresponding to the detection of 1 CFU mL^?1 of irrigation water, which is well below the concentration needed for midrib rot infection. Finally, the assay proved to be valuable for detecting infective P. cichorii concentrations in the irrigation water of a commercial lettuce production greenhouse.     

First report of bacterial leaf blight on cosmos (Cosmos bipinnatus Cav.) caused by Pseudomonas cichorii in Japan

Since 2006, a commercial grower in Japan has noted a leaf blight symptom on potted cosmos plants grown in a field. In August 2012, a Pseudomonas-like bacterium was isolated from the symptomatic leaves and found to cause the same symptom on cosmos seedlings after inoculation. On the basis of bacteriological and phylogenetic analyses, the causative bacterium was identified as Pseudomonas cichorii. This is the first report of bacterial leaf blight on cosmos caused by P. cichorii in Japan.     

Seedling rot of garland chrysanthemum caused by Gibellulopsis chrysanthemi and ecological characters of the causal fungus

A new disease found on garland chrysanthemum in Osaka, Japan in 2009 and its causal agent were identified and characterized. Light brown spots first appeared on lower leaves of seedlings, and the leaves blighted or rotted. A fungus isolated from diseased plants, described recently as Gibellulopsis chrysanthemi, was demonstrated to reproduce the natural symptoms in inoculation tests. Sufficiently long, moist periods after inoculation promoted the infection even after lengthy dry periods. The pathogen also caused the disease on chrysanthemum and lettuce, but not on seven other vegetables. The fungus caused the disease after 6 months of dry storage. The disease was termed seedling rot (“nae-fuhai-byo” in Japanese).     

Hypersensitive cell death and post-haustorial defence responses arrest the orange rust (Hemileia vastatrix) growth in resistant coffee leaves

The growth of a coffee orange rust fungus (Hemileia vastatrix Berk and Br.) isolate (race II) and the sequence of responses it induced in leaves of resistant Coffea arabica L. and C. congensis Froehner as well as on a susceptible C. arabica were investigated cytologically and biochemically. The percentages of germinated urediospores and of appressoria formed over stomata as well as the fungal growth inside leaf tissues were similar in resistant and susceptible leaves until the 3rd day after the inoculation. In the susceptible leaves, at the majority of the infection sites (70%) the fungus pursued its growth without apparent inhibition while in the resistant leaves the fungus ceased its growth with higher frequency (34% in C. arabica and 54% in C. congensis) after the formation of at least one haustorium. The first signs of incompatibility, detected 2 days after the inoculation, were cytologically expressed by hypersensitive host cell death (HR), host cell wall autofluorescence and haustoria encasement with callose and β-1,4-glucans. Biochemically, two peaks of phenylalanine ammonia-lyase (PAL) activity were detected by 2 and 5 days after the inoculation. The 1st peak coincided with the early accumulation of phenolic compounds and with the beginning of cell death. The 2nd peak could be related to later accumulation of phenols and the lignification of the host cell walls. About 5–7 days after the inoculation, ultrastructural observations revealed the accumulation of a material partially crystallized in the intercellular spaces around the senescent hyphae, next to dead host cells and in close association with the middle lamella that initially labelled for pectins. It also contained polysaccharides and phenolic-like compounds. Cellulose, hemicellulose, extensins, hydroxyproline-rich glycoproteins and proteins were not detected. The hypertrophy of the host cells in the infection area were also observed around 12 days after the inoculation corresponding macroscopically to the reaction flt.In susceptible plants, cell death was also observed 3 days after the inoculation but only in a reduced percentage of infection sites in which the fungus aborted at an early stage. A late haustorium encasement and stimulation of PAL activity were also observed but these delayed host responses did not prevent fungal growth and sporulation.The intercellular material, only observed in the resistant plants, is here reported for the first time and although its role is unknown it might be the result of plant cell death.     

The application of lettuce in the qualitative and quantitative detection of <Emphasis Type="Italic">Rhizoctonia Solani</Emphasis> AG- 1 IA toxins

The toxins produced by Rhizoctonia solani are important causal agents of rice sheath blight. Effective detection of such toxins could improve the determination of the virulence of this agronomically important fungal pathogen. As such, the objective of the current study was to investigate the use of a variety of plant species [annual sowthistle (Sonchus oleraceus L.), Chinese cabbage (Brassica chinensis), spinach (Spinacia oleracea L.), lettuce (Lactuca sativa L. var. sativa), long leaf lettuce (Lactuca sativa var. ramosa Hort) and tobacco (Nicotiana tabacum)] for qualitative detection of R. solani crude toxins (RHCT) to replace the current rice leaf sheath based assay. This is constrained as rice plant takes long time to grow before the leaf sheath can be harvested From the initial screen, it was found that detached lettuce leaves provided the best alternative to rice material. Quantitative determination of RHCT activity by the phosphorus extravasation method was then performed on both rice (Oryza sativa L.) and lettuce. The results demonstrated that the detached lettuce leaves had the advantages of fast onset of symptoms, high sensitivity and non-perishability after inoculation. The quantity of phosphorus exosmosis observed in both lettuce leaves and rice leaf sheaths were significantly positively correlated. These data indicate that lettuce leaves can be used as a substitute material for rice leaf sheaths, with which to study the RHCT both qualitatively and quantitatively. The current study provides a new way to qualitatively and quantitatively detect RHCT.     

Pathogenicity of <Emphasis Type="Italic">Plectosphaerella</Emphasis> species on lettuce and susceptibility of lettuce cultivars

Plectosphaerella rot affects hydroponically grown lettuce, especially those grown using deep flow technique. Plectosphaerella species such as P. pauciseptata and P. cucumerina are reported as causal agents of this disease. However, the relation between fungal lineage and pathogenicity on lettuce has been unclear. From inoculation tests, we discovered that various lineages of Plectosphaerella can infect lettuce tissues. Even strains isolated from non-lettuce plants were pathogenic on lettuce. Furthermore, various lettuce cultivars were equally susceptible to a particular strain. These results indicate that strains from a wide lineage of Plectosphaerella can be pathogenic on various lettuce cultivars.     

Interaction of perfluidone with mitochondrial,thylakoid, and liposome membranes

Perfluidone (1,1,1-trifluoro-N-[2-methyl-4-(phenylsulfonyl)phenyl]methanesulfonamide) was shown to interfere with phosphorylation and electron transport in isolated mung bean (Phaseolus aureus Roxb.) mitochondria. At low molar concentrations (<100 μM), perfluidone acted as an uncoupler of oxidative phosphorylation as evidenced by stimulation of state 4 respiration, induction of ATPase activity, and circumvention of oligomycin-inhibited state 3 respiration. At higher molar concentrations (>100 μM), perfluidone inhibited electron transport by acting on complexes I and II, and on the alternate (cyanide-insensitive) oxidase. In isolated spinach thylakoids (Spinacia oleracea L.), perfluidone also acted as an uncoupler, at low concentrations, as evidenced by stimulation of photoinduced electron transport with water as the reductant and methyl viologen and ferricyanide as oxidants, and from reduced dichlorophenolindophenol to methyl viologen. In addition, perfluidone inhibited the rate and magnitude of valinomycin-induced mitochondrial swelling in isotonic potassium chloride and potassium thiocyanate, and with thylakoids suspended in potassium thiocyanate at concentrations that inhibited ATP generation (<100 μM). Passive swelling in mitochondria was induced at higher concentrations. The permeability of lecithin liposomes to protons was also increased by perfluidone in a manner characteristic of uncouplers. The results obtained suggested that the partitioning of perfluidone perturbs the inner mitochondrial and thylakoid membranes. The perturbations increase the permeability of the membranes to protons and cations (at least potassium) and decrease membrane “fluidity.” As a consequence of the perturbations, the ATP-generating pathway in both mitochondria and chloroplasts is uncoupled and the structural organization of the electron transport components in mitochondria is disrupted, resulting in multisite inhibition of respiration. No evidence was obtained for a direct interaction between perfluidone and redox components of the electron transport pathways.     

In vitro sensitivity of wheat and oat mitochondria to the selective herbicide,diclofop-methyl

Compared to diclofop-methyl (methyl 2-[4-(2′,4′-dichlorophenoxy)phenoxy]propanoate), diclofop (the demethylated derivative) was a more potent inhibitor of polarographically monitored state 3 respiration of mitochondrial preparations isolated from shoots of dark-grown wheat (Triticum aestivum L. cv. Neepawa) and oat (Avena sativa L. cv. Terra) seedlings. Wheat and oat mitochondria demonstrated essentially similar concentration-response patterns for the uncoupler-like stimulation of state 4 respiration and the inhibition of state 3 respiration by diclofop, thereby intimating that differential mitochondrial sensitivity was not a selectivity factor between these species. Diclofop suppression of unconstrained oxygen utilization elicited by the respiratory uncoupler FCCP indicated that inhibition of state 3 respiration involved interference with some site(s) on the mitochondrial electron transport chain and not with energy transfer directly. Cytochrome c oxidase activity was not affected by diclofop, but succinate- and malate-PMS oxidoreductase activities were inhibited by diclofop. Enhanced rates of passive mitochondrial swelling in isotonic KCl medium in the presence of diclofop pointed to a direct influence on the permeability properties of the inner mitochondrial membrane and indicated that membrane disruption could have been a factor in the effects elicited by diclofop on mitochondrial respiration. However, it does not appear that specific interference with mitochondrial functionality is the primary mechanism of phytotoxicity in susceptible plants.     

Environmental conditions influencing Sclerotinia sclerotiorum infection and disease development in lettuce

The environmental factors that influence infection of lettuce by ascospores of Sclerotinia sclerotiorum , and subsequent disease development, were investigated in controlled environment and field conditions. When lettuce plants were inoculated with a suspension of ascospores in water or with dry ascospores and exposed to a range of wetness durations or relative humidities at different temperatures, all plants developed disease but there was no relationship between leaf wetness duration or humidity and percentage of diseased plants. Ascospores started to germinate on lettuce leaves after 2–4 h of continuous leaf wetness at optimum temperatures of 15–25°C. The rate of development of sclerotinia disease and the final percentage of plants affected after 50 days were greatest at 16–27°C, with disease symptoms first observed 7–9 days after inoculation, and maximum final disease levels of 96%. At lower temperatures, 8–11°C, disease was first observed 20–26 days after inoculation, with maximum final disease levels of 10%. Disease symptoms were always observed first at the stem base. In field-grown lettuce in Norfolk, 2000 and 2001, inoculated with ascospore suspensions, disease occurred only in lettuce planted in May and June, with a range of 20–49% of plants with disease by 8 weeks after inoculation. In naturally infected field-grown lettuce in Cheshire, 2000, disease occurred mainly in lettuce planted throughout May, with a maximum of 31% lettuce diseased within one planting, but subsequent plantings had little (≤ 4%) or no disease. Lack of disease in the later plantings in both Norfolk and Cheshire could not be attributed to differences in weather factors.     

Evidence for Lettuce big‐vein associated virus as the causal agent of a syndrome of necrotic rings and spots in lettuce

Lettuce big‐vein associated virus (LBVaV, genus Varicosavirus) was shown to be responsible for characteristic necrotic symptoms observed in combination with big‐vein symptoms in lettuce breeding lines when tested for their susceptibility to lettuce big‐vein disease (BVD) using viruliferous Olpidium virulentus spores in a nutrient film technique (NFT) system. Lettuce plants showing BVD are generally infected by two viruses: Mirafiori lettuce big‐vein virus (MiLBVV, genus Ophiovirus) and LBVaV. New mechanical inoculation methods were developed to separate the two viruses from each other and to transfer both viruses to indicator plants and lettuce. After mechanical inoculation onto lettuce plants MiLBVV induced vein‐band chlorosis, which is the characteristic symptom of BVD. LBVaV caused a syndrome of necrotic spots and rings which was also observed earlier in lettuce plants inoculated in the NFT system, resembling symptoms described for lettuce ring necrosis disease (RND). This observation is in contrast with the idea that LBVaV only causes latent infections in lettuce. De novo next‐generation sequencing demonstrated that LBVaV was the only pathogen present in a mechanically inoculated lettuce plant with symptoms, providing evidence that LBVaV was the causal agent of the observed necrotic syndrome and thus fulfilling Koch’s postulates for this virus. The necrotic syndrome caused by LBVaV in lettuce is referred to as LBVaV‐associated necrosis (LAN).     

Ultrastructural study on the interaction between Physcomitrella patens and Botrytis cinerea

Little is known about the interactions of bryophytes with their pathogens compared with those of flowering plants. This study used the moss Physcomitrella patens and fungal pathogen Botrytis cinerea to investigate their interaction at the ultrastructural level. Infection behaviour of B. cinerea in moss tissues was observed for 1–7 days after inoculation. Some appressoria directly penetrated P. patens cytoplasm through leaf cell walls, and others entered the intercellular spaces of leaves and infected cytoplasm. Infection hyphae were observed in sclerenchyma cells of P. patens stems for the first time. The results demonstrated that the ratio of hyphae‐inoculated leaf cells to leaf numbers could be used to quantify the disease development process at 24 h after inoculation. The ultrastructural studies revealed two responses of P. patens to B. cinerea inoculation: reinforcement of cell walls, including papillae formation; and cell death. This was the first observation of papillae for P. patens–B. cinerea interaction at the ultrastructural level. Papillae in P. patens had a similar ultrastructure to those of higher plants, and may be involved in the defence response to B. cinerea infection. Thus papillae formation is probably an evolutionarily conserved defence mechanism from the early land colonization by plants. Cell death during the P. patens–B. cinerea interaction had some features of programmed cell death, with hydrogen peroxide produced in cytoplasm membranes, suggesting something like a hypersensitive response. These ultrastructural studies suggest that P. patens could be a useful system for studying phytopathogens and could provide theoretical bases for defence mechanisms in evolutionary development.     

Effects of β-pinene,a nonsubstituted monoterpene,on rat liver mitochondria

β-Pinene uncouples oxidative phosphorylation and inhibits respiration in isolated rat liver mitochondria. The uncoupling effects are observed at lower concentrations (100 to 200 μM) than the inhibition of respiration (400 μM). At low concentrations, the effects observed could be explained by an increase of the passive permeability of the mitochondrial membrane produced by the terpene. Higher concentrations seemed to inhibit respiration through an effect on the electron transport chain. At the highest concentrations tested (600 to 1200 μM), β-pinene seemed to produce a partial resealing of the mitochondrial membrane. All effects can be explained by the interaction of β-pinene with the mitochondrial membrane. Other hydrophobic molecules tested do not show the effects of β-pinene or limonene on mitochondria.     

Histopathology of durable adult plant resistance to leaf rust in the Brazilian wheat variety Toropi

Leaf rust, caused by the fungus Puccinia triticina is a major disease of wheat (Triticum aestivum) worldwide. This disease is prevalent in southern South America where the environmental conditions and high genetic variability of P. triticina favour epidemics. The primary means of controlling pathogenic P. triticina races has been through using wheat varieties containing race-specific resistance genes. The defence mechanisms involved in durable race non-specific resistance to P. triticina are probably distinct from those involved in non-durable race-specific resistance. We investigated the histological components of resistance to P. triticina present in three wheat genotypes: the race non-specific resistant Brazilian variety Toropi; the race-specific resistant line RL6010 Lr9; and the susceptible Brazilian variety BRS 194. Plants of these three genotypes were inoculated with P. triticina race MFP and tissue samples excised from flag leaves at various times after inoculation to assess the number of infective structures, frequency of cell death and the accumulation of autofluorescent cells and hydrogen peroxide. The genotypes showed different resistance mechanisms active at different times during the infection process. Our results for Toropi indicate that there was a reduction in the extent of formation of stomatal appressoria and all subsequent structures. During attempted penetration we also observed the production of autofluorescent compounds and late cell death, but not peroxide formation. This non-specific resistance to P. triticina involves both pre-haustorial and post-haustorial mechanisms which may be responsible for maintaining the low disease severity observed in this variety even under high inoculum pressure.     

Bacterial leaf blight of sweet pepper (Capsicum annuum) caused by Pseudomonas cichorii in Japan

Sweet peppers (cv. Tosajishi beauty) with leaf blight symptoms were observed in Kochi Prefecture, 2003. Initially, small spots formed on leaves, and later enlarged to brown to dark brown spots, and eventually blighted leaves fell. Several bacteria that caused the same symptoms were isolated and subsequently reisolated. These bacteria were identified as Pseudomonas cichorii on the basis of bacterial characteristics and the 16S rRNA gene sequence. This is the first report of bacterial leaf blight in the sweet pepper in the world.     

Histological and histochemical changes in groundnut leaves inoculated withCercospora Arachidicola andCercosporidium Personatum

Histological and histochemical characteristics of leaves from resistant and susceptible groundnut genotypes (Arachis hypogaea L.) were observed after inoculation withCercospora arachidicola Hori andCercosporidium personatum (Berk. and Curt.) Deighton. Both the pathogens induced almost similar anatomical responses in the inoculated leaves. The epidermal and mesophyll cells were shrunken or collapsed; damage to protoplasts was more obvious than damage to cell walls. The histochemical localizations revealed a gradual depletion of polysaccharides, proteins, ascorbic acid, and nucleic acids from the diseased host tissue at the site of contact with the pathogen and their subsequent accumulation in the pathogen in the later stages of disease development. Differential staining for these metabolites was not observed in healthy tissues of these genotypes.