The N-acetyltransferase gene-implicated iron acquisition contributes to host specificity of Pseudomonas cichorii strain SPC9018 and its virulence

A pathogenicity island within the genome of a multi-host plant bacterium, Pseudomonas cichorii strain SPC9018, comprises the hrp genes encoding a type III secretion system and the pat gene encoding an N-acetyltransferase proposed to play a role in virulence. However, the function of the N-acetyltransferase remains poorly characterized. Interestingly, limiting the iron condition using a phytosiderophore, mugineic acid, resulted in reduced virulence of strain SPC9018 on respective host plants, including eggplant, similar to the reduced virulence observed with a pat gene-deletion mutant. Spectroscopic analyses showed that the pat deletion reduced the concentration of pyoverdine, which is the main siderophore produced by strain SPC9018, leading to a reduction in pyoverdine-mediated iron acquisition. Furthermore, the pat gene deletion mutant showed enhanced expression of the fecA, pvdL, and pvdR genes, whose expression is induced under deficient siderophore-mediated iron uptake. The pat-deletion mutant showed a hyper-swarming phenotype, and the addition of iron decreased this swarming motility. The pat deletion also reduced the adhesion ability of the bacteria, similar to the effect of iron-limited conditions. Furthermore, deletion of the pat gene enhanced expression of the hrp genes. These findings suggested that the pat gene encoding the N-acetyltransferase may be implicated in iron acquisition, contributing to host specificity of P. cichorii strain SPC9018 and its virulence.     

Induction of reactive oxygen generation and functional changes in mitochondria and their involvement in the development of bacterial rot in lettuce caused by Pseudomonas cichorii

Pseudomonas cichorii is the major causal agent of bacterial rot disease in lettuce, and apoptosis-like programmed cell death is closely associated with disease development. Depletion of cellular ATP and expression of the alternative oxidase gene was observed in lettuce leaves inoculated with P. cichorii suggesting mitochondrial dysfunction. Cytochemical observation showed production of reactive oxygen species in mitochondria of P. cichorii-inoculated lettuce leaf cells. Release of cytochrome c into the cytosol was also induced by inoculation with the bacterium. Superoxide production was observed in the mitochondrial fraction isolated from P. cichorii-infected leaves much more intense than water-treated leaves. Loss of swelling ability was also observed in the mitochondrial fractions following inoculation with P. cichorii. Intriguingly, inhibitors of the mitochondrial respiratory complex III prevented loss of swelling ability, whereas superoxide generation was scarcely affected by inhibitors of mitochondrial permeability transition in the mitochondrial fractions. Inhibitors of the mitochondrial respiratory complex III and mitochondrial permeability transition delayed not only P. cichorii-induced cell death, but also disease development. In contrast, P. cichorii-induced DNA fragmentation was not inhibited in the presence of either type of inhibitor. The findings suggest that mitochondria may play a crucial role in DNA fragmentation-independent cell death pathway(s).     

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.     

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.     

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.     

Characterization of Erwinia sp. causing black rot of papaya (Carica papaya) first recorded in Okinawa Main Island,Japan

Since 2002, papaya black rot has been spreading over several islands of Okinawa Prefecture, Japan. To devise a prevention strategy for the disease, microbiological research on the pathogen was conducted. Twelve strains were isolated from papaya infected with black rot showing symptoms such as water-soaked lesions on stems and petioles, black spots on fruits, and rotted leaves turning yellow with necrotic spots. Through Koch's postulates, we confirmed that the isolated strains caused papaya black rot. Bacteriological assays showed that the strains have characteristics different from the type strains of Erwinia mallotivora, E. papayae, and E. psidii. Moreover, 16S rDNA sequence similarity searches showed that the isolated strains had less than 98.6% similarity with type strains. Additionally, phylogenetic analysis of 16S rDNA sequences suggested that the isolated strains were possibly a novel species belonging to the genus Erwinia, as the strains formed an independent cluster and had low sequence similarity with the type strains. Earlier studies indicated that papaya black rot is caused by E. cypripedii. Therefore, we propose to add the Erwinia sp. isolated in this study to the list of papaya black rot pathogens.     

A strawberry disease caused by Acremonium strictum

We report Acremonium strictum as the causal agent of a new disease in strawberry plants (Fragaria x ananassa Duch.) in the Northwest of Argentina. Both the structure of conidiophores and the sequence spanning the internal transcribed spacers 1 and 2 (ITS1 and ITS2) of the nuclear ribosomal DNA (rDNA) allowed confirming the affiliation of the isolate, corresponding to A. strictum. An analysis of symptoms and lesions caused by the strain of A. strictum in susceptible cultivars showed that the typical symptoms are as follows: in an early stage, small necrotic light-brown spots in leaves and petioles increase in number and size as the disease progresses; in a more advanced stage, dark necrotic areas expand over petioles and leaves causing strangulation of petioles and the plant wilt. Crown rot was not observed even at a very advanced stage of the disease.     

First report of leaf spot on lettuce caused by <Emphasis Type="Italic">Curvularia aeria</Emphasis>

In 2017, leaf spots were found on lettuce growing in fields in Songkhla Province, southern Thailand. The fungus isolated from the spot lesions on the leaves was identified as Curvularia aeria (Bat., J.A.Lima and C.T.Vasconc.) Tsuda based on morphological characteristics and DNA sequences of the ITS region of the rRNA gene. After a conidial suspension of the isolate was sprayed on lettuce seedlings, the leaf spots developed on lettuce seedlings, and the fungus was reisolated; leaves of plants inoculated with water did not develop spots. This is the first report of C. aeria causing leaf spot on lettuce.     

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.     

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).     

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.     

Regulatory network of hrp gene expression in Xanthomonas oryzae pv. oryzae

Like other plant-pathogenic bacteria, Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, has hrp genes that are indispensable for its virulence. The hrp genes are involved in the construction of the type III secretion (T3S) apparatus, through which dozens of virulence-related proteins, called effectors, are directly secreted into plant cells to suppress and disturb plant immune systems and/or induce plant susceptibility genes. The expression of hrp genes is strictly regulated and induced only in plants and in certain nutrient-poor media. Two proteins, HrpG and HrpX, are known as key regulators for hrp gene expression. Great efforts by many researchers have revealed unexpectedly that, besides HrpG and HrpX, many regulators are involved in this regulation, some of which also regulate the expression of virulence-related genes other than hrp. Moreover, it has been found that HrpG and HrpX regulate not only hrp genes and effector genes but also genes unrelated to the T3S system. These findings suggest that the expression of the hrp gene is orchestrally regulated with other virulence-related genes by a complicated, sophisticated regulatory network in X. oryzae pv. oryzae.     

Phenotypic,molecular and pathogenic characterization of Phlyctema vagabunda,causal agent of olive leprosy

Olive leprosy, caused by the fungus Phlyctema vagabunda, is a classic fruit rot disease widespread in the Mediterranean basin. From 2009 to 2013, new disease symptoms consisting of small circular necrotic leaf lesions, coin branch canker and shoot dieback were observed in Spanish and Portuguese olive orchards showing intense defoliation. Phlyctema‐like anamorphs were consistently isolated from leaves and shoots with symptoms. Representative isolates from affected leaves, shoots and fruits were characterized based on morphology of colonies and conidia, optimum growth temperature and comparison of DNA sequence data from four regions: ITS, tub2, MIT and rpb2. In addition, pathogenicity tests were performed on apple and olive fruits, and on branches and leaves of olive trees. Maximum mycelial growth rate ranged between 0.54 and 0.73 mm per day. Conidia produced on inoculated apple fruits showed slight differences in morphology among the representative fungal isolates evaluated. Phylogenetic analysis clustered all of the Phlyctema‐like isolates in the same clade, identifying them as Phlyctema vagabunda. On fruits, influence of wounding, ripening and cultivar resistance was studied, with cv. Blanqueta being the most susceptible cultivar. On branches, a mycelial‐plug inoculation method reproduced olive leprosy symptoms and caused shoot dieback. On leaves, Koch's postulates were fulfilled and the pathogen caused characteristic necrotic spots and plant defoliation. This is the first time that the pathogenicity of P. vagabunda in olive leaves has been demonstrated.     

First report on the whole genome sequence of Pseudomonas cichorii strain JBC1 and comparison with other Pseudomonas species

Pseudomonas cichorii, a plant pathogen that infects a wide range of host plants worldwide, causes several diseases in economically important vegetable crops. Availability of the genome sequences of pathogens can greatly enhance research necessary for the advancement of disease management programmes. Despite the significance of P. cichorii, its whole genome sequence has not been reported previously. The genome sequence of P. cichorii JBC1, described for the first time in this study, is 5 986 012 bp with an average GC content of 58·1% and has 5174 coding sequences (CDS). The genes related to virulence, transport mechanisms, phytotoxic compounds, and secondary metabolite products were analysed and the genome was compared to eight other Pseudomonas species to understand the diversity at species level. Despite the high similarity (up to 80·85%), significant diversity was found among the different Pseudomonas species at the genome level. A comparison of JBC1 pathogenicity island (PAI) regions indicated that the P. viridiflava UASWS0038 PAI has more similarity than the P. syringae PAI region, and the analysis revealed significant divergence at PAI regions among the Pseudomonas species, providing an insight into the differences in host specificity and degree of virulence. In addition, JBC1 encodes antibiotic resistance and tolerance to heavy metals, and two different prophage segments were inserted at three different regions. The genome sequence of JBC1, which was deposited into the NCBI GenBank (accession no. CP007039 ), will be a reference sequence for other P. cichorii strains and a useful resource for further research.     

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.     

Erwinia amylovora hrpN mutants,blocked in harpin synthesis,express a reduced virulence on host plants and elicit variable hypersensitive reactions on tobacco

Erwinia amylovora is the bacterium responsible for fire blight, a necrotic disease affecting many rosaceous plants and especially pear tree and apple tree. A protein named harpin, secreted through the Hrp secretion pathway and able to elicit an hypersensitive reaction (HR) on tobacco has recently been isolated. Mutants inhrpN, the gene encoding harpin were described as non pathogenic on immature pear fruit and unable to elicit an HR on tobacco [Weiet al., 1992; Wei and Beer, 1993]. In this paper, the phenotype on plant ofhrpN mutants was carefully determined.hrpN mutants expressed a weak but significant virulence on host plants. Furthermore, when infiltrated into tobacco leaf mesophyll, thehrpN mutants elicited varied responses that fluctuated from null reaction to full necrosis of the infiltrated area. These results show that harpin is not absolutely required neither for pathogenicity on host plant nor for elicitation of an hypersensitive reaction on tobacco. Furthermore, in all the tests performed, mutant blocked in harpin secretion remained non pathogenic and unable to elicit an HR on tobacco. This suggests that factor(s), different from harpin, involved both in pathogenicity and HR eliciting ability is (are) secreted through the Hrp secretion pathway.Abbreviations HR hypersensitive reaction - NSI necrosis severity index - CFU colonie forming units     

Phylogenetic analyses of plant-growth-promoting rhizobacteria isolated from tomato,lettuce, and Japanese pepper plants in Hyogo Prefecture,Japan

Approximately 30,000 fluorescent bacterial strains isolated from tomato, lettuce, eggplant, Chinese cabbage, and Japanese pepper plants at seven different locations in Hyogo Prefecture, were screened for plant-growth-promoting (PGP) activity to induce disease resistance against bacterial wilt in tomato. The 37 strains that had higher PGP activity were subjected to molecular phylogenetic analyses using the sequences of the 16S rRNA, gyrB and rpoD genes. Most of the strains were identified as Pseudomonas fluorescens or its close relative, P. putida, while a few strains were grouped with more distantly related bacterial species such as Enterobacter and Stenotrophomonas. The phylogenetic relationships among tomato and lettuce isolates mostly coincided with the source locality and host plants, with a few exceptions. In contrast, isolates from Japanese pepper plants did not form their own cluster but represented several different bacterial species.     

Wild Lactuca species,their genetic diversity,resistance to diseases and pests,and exploitation in lettuce breeding

Current knowledge of wild Lactuca L. species, their taxonomy, biogeography, gene-pools, germplasm collection quality and quantity, and accession availability is reviewed in this paper. Genetic diversity of Lactuca spp. is characterized at the level of phenotypic and phenological variation, variation in karyology and DNA content, biochemical traits, and protein and molecular polymorphism. The reported variation in reaction to pathogens and pests of wild Lactuca spp. is summarized, including the viral pathogens (Lettuce mosaic virus-LMV, Mirafiori lettuce virus/Lettuce big vein virus-LBV, Beet western yellows virus-BWYV, Tomato spotted wilt virus-TSWV, Cucumber mosaic virus-CMV, Lettuce necrotic stunt virus-LNSV), bacterial pathogens (corky root-Rhizomonas suberifaciens, bacterial leaf spot-Xanthomonas campestris pv. vitians), fungal pathogens (downy mildew-Bremia lactucae, powdery mildew-Golovinomyces cichoracearum, anthracnose-Microdochium panattoniana, stemphylium leaf spot-Stemphylium spp., sclerotinia drop-Sclerotinia spp., verticillium wilt-Verticillium dahliae, fusarium wilt-Fusarium spp., pythium wilt-Pythium tracheiphylum, P. uncinulatum), nematodes (potato cyst nematode-Globodera rostochiensis, root-knot nematode-Meloidogyne spp., incognita, hapla, javanica, enterolobii), insects and mites (the green lettuce aphid-Nasonovia ribisnigri, the green peach aphid-Myzus persicae, the potato aphid-Macrosiphum euphorbiae, leafminer-Liriomyza spp., L. langei). The approaches used to exploit wild Lactuca spp. in lettuce breeding (interspecific hybridization, cell and tissue culture, transformation) are dicussed, and known examples of lettuce cultivars with traits derived from wild Lactuca spp. are described.     

Differential necrotic lesion formation in soybean cultivars in response to soybean mosaic virus

In this study, we examined the necrosis phenotype on leaves of two cultivars of soybean (ZheA8901 and Nannong1138-2) that show varying level of resistance to soybean mosaic virus (SMV). The necrotic symptoms seen on inoculated and systemic leaves of soybean cultivar ZheA8901 were reminiscent of programmed cell death (PCD). The cell death phenotypes were evaluated using the TUNEL method, quantification of hydrogen peroxide (H₂O₂) and salicylic acid, callose production, as well as by monitoring expression of defence genes GmPR-1 and GmNPR1. Our results show that SMV inoculation induced PCD on ZheA8901 is associated with rapid increase in H₂O₂, increased SA and callose accumulation and higher defence gene expression.