Virulence factors analysis of native isolates of Xanthomonas albilineans and Xanthomonas sacchari from Tucumán,Argentina, reveals differences in pathogenic strategies

Xanthomonas albilineans (Xa) and X. sacchari (Xs) are both sugarcane pathogens. Xa is the causal agent of leaf scald disease, but there is limited information about the pathogenicity of Xs. The aim of this work was to study virulence factors of native strains of Xa (Xa32, Xa33, and XaM6) and Xs (Xs14 and Xs15) previously isolated from sugarcane with leaf scald symptoms, to gain insight into the biology of each microorganism. We analysed epiphytic survival, sensitivity to oxidative stress, extracellular degradative enzymes, cell motilities, exopolysaccharide (EPS) characteristics, cell adhesion, biofilm development, and control of stomatal regulation of the five strains. We observed that each species presented similar phenotypes for every factor analysed. Xa strains appeared to be more sensitive to oxidative stress and presented lower epiphytic survival than Xs. All strains presented endoglucanase activity; however, we could only detect protease and amylase activities in Xs strains. Swimming and sliding were higher in Xs, but twitching was variable among species. We also observed that only Xs strains produced a xanthan-like EPS, presented a strong cell adhesion, and structured biofilm. We detected some intraspecific variations showing that higher amounts of EPS produced by Xs14 correlated with its higher sliding motility and its homogenous and more adhesive biofilm. In addition, EPSs of Xs14 and Xs15 presented differences in strand height and acetyl percentage. Finally, we found that strains of both species were able to interfere with stomatal aperture mechanism. All these differences could influence the colonization strategies and/or disease progression in each species.     

Pathogenicity of Meloidogyne incognita and M. javanica on recombinant inbred lines from a crossing of Cucurbita pepo subsp. pepo × C. pepo subsp. ovifera

The response of recombinant inbred lines (RILs) from a cross of zucchini × scallop (Cucurbita pepo subsp. pepo ‘Murcia MU-CU-16’ × C. pepo subsp. ovifera ‘Scallop UPV-196’) to Meloidogyne incognita and M. javanica was determined after completion of a nematode reproduction cycle in experiments carried out in a growth chamber. The nematode differentiated the C. pepo genotypes at the subspecies level due to lower egg mass production on subspecies pepo than ovifera, and thus subspecies pepo was a poorer host than ovifera. In addition, Murcia MU-CU-16 discriminated M. incognita from M. javanica in terms of egg masses (EM), eggs per gram of root and reproduction factor (Rf), whereas Scallop UPV-196 did so in eggs per gram of root and Rf. The RILs differed in gall formation and EM production depending on the nematode × line combination. Comparisons between nematode isolates resulted in four significant combinations for pathogenic potential (galls/initial population (Pi) × 100), seven for parasitic success (egg masses/Pi × 100), and nine for host efficiency (egg masses/galls per root system × 100) which included all the lines tested against both isolates. Lines that restricted nematode development by at least 90% were considered as having intermediate resistance to M. incognita based on the definition of the International Seed Federation. They included lines 28-1, 35A, 107A, 110-3 and 153-2. All the RILs were susceptible hosts for M. javanica. The information presented here will be helpful for nematode management and also for plant breeders working on pathogen resistance on C. pepo.     

Molecular cloning and expression of a novel eukaryotes elongation factor1A gene (ZjeEF-1α) from Chinese jujube in response to phytoplasma infection

Eukaryotic translation elongation factor 1A (eEF1A) in plant is a multifunctional protein. In our previous study, we identified a 651-bp EST sequence of eEF-1α from a suppression subtractive hybridization (SSH) library of Chinese jujube (Ziziphus jujuba Mill.), which was established under jujube witches' broom (JWB) phytoplasma stress. Here we isolated the eEF1A gene (ZjeEF-1α, JF488063) from Chinese jujube by single oligonucleotide nested (SON)-PCR and rapid amplification of cDNA ends (RACE). ZjeEF-1α contains an open reading frame (ORF) of 1344 bp, encoding a polypeptide of 447 amino acids with an estimated molecular mass of 49.29 kDa and a theoretical pI of 9.10. ZjeEF-1α shared over 92% similarity with eEF-1α proteins from other 24 eukaryotes including plants and animals. Search Pfam predicted that ZjeEF-1A contains three typical eEF1A functional domains. Moreover, the corresponding genomic sequence of ZjeEF-1α was also sequenced, in which a 115-bp intervening sequence with splicing site GT-AG was found. Real-time quantitative PCR (RT-qPCR) and western blotting assays revealed that the expression of ZjeEF-1α was enhanced in resistant cultivar ‘Xingguang’ and decreased in sensitive cultivar ‘Zanhuangdazao’ at early stages of phytoplasma infection. It is proved that ZjeEF-1A expression is early response to phytoplasma infection and its possible functions in resistant process to JWB phytoplasma are discussed.     

Quantification of viable cells of Clavibacter michiganensis subsp. sepedonicus in digester material after heat treatment by TaqMan® BIO-PCR

Journal of Plant Diseases and Protection - With bacterial ring rot affected potato lots must be decontaminated under official control without causing a risk for the dissemination of the disease....     

MALDI-Qq-TOF-MS and transient gene expression analysis indicated co-enhancement of β-1,3-glucanase and endochitinase by tMEK2 and the involvement of divergent pathways

A mitogen-activated protein kinase (MAPK) pathway has been demonstrated as a key pathway in plant defense against pathogen attacks. With proteomics approaches, we specifically studied activation events downstream of a MAPK kinase, tMEK2, in tomato. Overexpression of a constitutively activated tomato MAPK kinase gene (tMEK2^MUT) enhanced resistance of transgenic tomato lines to the virulent bacterial pathogen Pseudomonas syringae pv. tomato. Pathogenesis-related genes, PR1b1, β-1,3-glucanase, and endochitinase were up-regulated by tMEK2^MUT. Two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation-time-of-flight-mass spectrometry analysis of total soluble leaf proteins indicated that β-1,3-glucanase and endochitinase are among the up-regulated proteins in these transgenic plants. Co-expression studies using a transient gene expression system have indicated that β-1,3-glucanase and endochitinase genes up-regulated by tMEK2^MUT were down-regulated by different specific phosphatases through dephosphorylation of certain downstream signaling molecules. Our observations indicate that increased products of β-1,3-glucanase and endochitinase genes downstream of tMEK2 may play an important role in achieving disease resistance.     

Spatial pattern analysis of citrus canker-infected plantings in são paulo, Brazil, and augmentation of infection elicited by the asian leafminer

ABSTRACT Eradication of Asiatic citrus canker (ACC) has become increasingly difficult over the last decade, following the introduction of the Asian leafminer into Brazil and Florida, which has led to changes in the eradication protocols. The present study, undertaken in Brazil, was aimed at characterizing the spatial patterns of ACC in commercial citrus plantings to gain better understanding of the dynamics of the disease subsequent to introduction of the leafminer. The spatial patterns of ACC were mapped in 326 commercial citrus plantings and statistically assessed at various spatial dimensions. The presence of "within-group" aggregation in each plot was examined via beta-binomial analysis for groups of trees parsed into three-by-three-tree quadrats. The relative intensity of aggregation was expressed as a binomial index of dispersion (D) and heterogeneity among plots expressed as the intracluster correlation coefficient, rho. The population of data sets was found to fall into three D categories, D < 1.3, 1.3 3.5. These categories then were related to other spatial characteristics. The binary form of Taylor's power law was used to assess the overdispersion of disease across plots and was highly significant. When the overall population of plots was parsed into D categories, the Taylor's R (2) improved in all cases. Although these methods assessed aggregation well, they do not give information on the number of foci or aggregations within each plot. Therefore, the number of foci per 1,000 trees was quantified and found to relate directly to the D categories. The lowest D category could be explained by a linear relationship of number of foci versus disease incidence, whereas the higher two categories were most easily explained by a generalized beta function for the same relationship. Spatial autocorrelation then was used to examine the spatial relationships "among groups" composed of three-by-three-tree quadrats and determine common distances between these groups of ACC-infected trees. Aggregation was found in >84% of cases at this spatial level and there was a direct relationship between increasing D category and increasing core cluster size, and aggregation at the among-group spatial hierarchy was generally stronger for the within-row than for the across-row orientation. Clusters of disease were estimated to average between 18 and 33 tree spaces apart, and the presence of multiple foci of infection was commonplace. The effectiveness of the eradication protocol of removing all "exposed" trees within 30 m surrounding each "ACC-infected tree" was examined, and the distance of subsequent infected trees beyond this 30-m zone from the original focal infected tree was measured for each plot. A frequency distribution was compiled over all plots to describe the distance that would have been needed to circumscribe all of these outliers as a theoretical alternative protocol to the 30-m eradication protocol. The frequency distribution was well described by a monomolecular model (R(2) = 0.98) and used to determine that 90, 95, and 99% of all newly infected trees occurred within 296, 396, and 623 m of prior-infected trees in commercial citrus plantings, respectively. These distances are very similar to previously reported distances determined for ACC in residential settings in Florida.     

Functional analysis of the G-protein α subunits FGA1 and FGA3 in the banana pathogen Fusarium oxysporum f. sp. cubense

The asexual fungus Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of fusarium wilt in bananas (Musa spp.). This fungus poses a threat to banana production throughout the world. Here, two Foc genes, fga1 and fga3, were functionally characterized. These genes encode proteins homologous to the G-protein α subunits GPA1 from Saccharomyces cerevisiae and MAGC from Magnaporthe grisea, respectively. The deletion of fga1 leads to a phenotypic defect in colony morphology and reductions in vegetative growth, conidiation and pathogenicity against the banana plant (Musa spp. cv. Brazil), which was not observed for the Δfga3 deletion mutant. Intriguingly, both Δfga1 and Δfga3 deletion mutants showed declines in intracellular cyclic AMP levels and increases in heat resistance, suggesting that FGA1 regulates growth, development, pathogenicity, and heat resistance, whereas FGA3 modulates heat resistance, potentially through the cAMP-dependent protein kinase A pathway. These findings offer insights into the roles of the G-protein α subunits in the development and pathogenicity of the fungus Foc.     

Cloning, developmental and tissue-specific expression of γ-aminobutyric acid (GABA) receptor alpha2 subunit gene in Spodoptera exigua (Hübner)

The full-length cDNA sequence of γ-aminobutyric acid (GABA) receptor alpha2 subunit gene was cloned from Spodoptera exigua, an economically important pest species in China, through rapid amplification of cDNA ends (RACE) and polymerase chain reaction using primers based on the 5′ and 3′-ends of the mRNA. The gene (named as SeGABARα2) was 1620 bp long, with an open reading frame of 1500 bp encoding a predicted GABA receptor protein of 499 amino acids with a molecular weight of 55 kDa. The predicted GABA receptor protein shared 96.19%, 82.16%, 74.30%, 73.95%, 73.42%, 71.40%, 67.11% and 65.13% identity with other GABA receptors proteins isolated from Heliothis virescens, Plutella xylostella, Aedes aegypti, Laodelphax striatellus, Lucilia cuprina, Musca domestica, Drosophila simulans and Drosophila melanogaster, at amino acid level, respectively. The developmental changes and tissue-specificity of the relative mRNA expression levels of SeGABARα2 gene were investigated in S. exigua. The highest expression level was observed in adult and lowest was in the first instar, there was a stable expression level during the developmental period from the second instar to the adult. Expression levels were 1.41-, 1.57-, 1.45-, 1.80-, 1.82- and 1.93-fold higher in the second instar, third instar, fourth instar, fifth instar, pupa and adult than in the first instar larva, respectively. The relative mRNA expression levels of SeGABARα2 gene were 3.51-fold higher in head and 1.77-fold higher in thorax than in abdomen     

Need for flagella for complete virulence of Pseudomonas syringae pv. tabaci: genetic analysis with flagella-defective mutants ?fliC and ?fliD in host tobacco plants

The flagellins purified from Pseudomonas syringae pv. tabaci induce a hypersensitive reaction in nonhost tomato cells. To investigate the role of flagella and flagellin in the compatible interaction, we generated two types of flagella-defective mutant. The fliC mutant lost the fliC gene that encodes flagellin protein, whereas the fliD mutant lost the fliD gene that encodes HAP2-capping protein. The two mutants had markedly reduced ability to cause disease symptoms in tobacco leaves. Furthermore, propagation of the mutants in tobacco leaves was less than that in wild-type pv. tabaci. Compared to the inoculation with wild-type pv. tabaci, inoculation with the two mutants did not markedly induce the expression of typical defense response-related genes such as PAL and hsr203J. Complementation of each fliC and fliD gene to the corresponding deficient mutant restored motility and virulence. These results indicate that flagella of P. syringae pv. tabaci are indispensable organelles for complete virulence on host tobacco plants.     

Scientific critique of the paper “Climatic distribution of citrus black spot caused by <Emphasis Type="Italic">Phyllosticta citricarpa</Emphasis>. A historical analysis of disease spread in South Africa” by Martínez-Minaya et al. (2015)

The global distribution of citrus black spot (CBS) disease, caused by Phyllosticta citricarpa, is climatically constrained, which is evident from its occurrence in citrus growing areas with warm, summer rainfall and its absence from areas with cooler, Mediterranean-type winter rainfall. Various epidemiological and modelling studies have supported this observation, predominantly estimating unsuitability for P. citricarpa in Mediterranean type climates, with no more than marginal suitability estimated at a few localities within some regions with Mediterranean type climates. The study by Martínez-Minaya et al. (European Journal of Plant Pathology, 143, 69–83, 2015), describes an historic sequence of recorded CBS occurrence in parts of South Africa, conducts an autocorrelation analysis and a correlative analysis with Köppen-Geiger climate zones and makes observations about the occurrence of certain Köppen-Geiger climate zones in the European Union. The study suggests that significant portions of the European Union and the broader Mediterranean basin are climatically similar to warm, summer rainfall areas in South Africa where P. citricarpa persists and causes CBS disease and concludes that the potential distribution of P. citricarpa is less constrained by climatic factors than spatial contagion. However, in this critique we expose methodological shortcomings in the Martínez-Minaya et al. (European Journal of Plant Pathology, 143, 69–83, 2015) study and conclude that the study grossly overestimated the extent of the geographical area that could support P. citricarpa, thereby rendering the findings scientifically unreliable.     

Response to the letter on “Climatic distribution of citrus black spot caused by <Emphasis Type="Italic">Phyllosticta citricarpa.</Emphasis> A historical analysis of disease spread in South Africa” by Fourie et al. (2017)

In a previous study, Martínez-Minaya et al. (European Journal of Plant Pathology 143, 69–83, 2015) performed an analysis of climate-based distribution of citrus black spot (CBS) in South Africa. It was found that CBS was initially confined to humid areas with summer rainfall, but later spread to arid steppe and even desert climates. A strong spatial autocorrelation of CBS distribution was found. Fourie et al. (European Journal of Plant Pathology, 2017) take a critical view of our study, but without presenting any analysis of results to refute our findings. Furthermore, Fourie et al. (European Journal of Plant Pathology, 2017) appear to have misunderstood our work, since many of their criticisms relate to the potential distribution of CBS in Europe, which is beyond the scope of our original study. Fourie et al. (European Journal of Plant Pathology, 2017) highlight the limitations of climate classifications in species distribution modelling. However, this was made explicit in our study, indicating that it was a preparatory work and further advanced modelling studies, including spatial effects, will be needed. Fourie et al. (European Journal of Plant Pathology, 2017) incorrectly assume that we used all of South Africa as the background in the spatial autocorrelation analysis. However, only citrus areas were used and a strong spatial autocorrelation was detected at all distances evaluated. Contrary to what Fourie et al. (European Journal of Plant Pathology, 2017) suggest, similar climate distributions of CBS were obtained at 5′ and 30′ resolution, and also with the national land-cover map of South Africa. The figure comparison presented by Fourie et al. (European Journal of Plant Pathology, 2017) appears to ignore the fact that the maps we used were grid cells of 10 × 10 km and not the line polygons they suggest. Therefore, we consider the conclusions from the Martínez-Minaya et al. (European Journal of Plant Pathology 143, 69–83, 2015) remain entirely valid.