Phylogenetic,toxigenic and virulence profiles of <Emphasis Type="Italic">Alternaria</Emphasis> species causing leaf blight of tomato in Egypt

Species of Alternaria are serious plant pathogens, causing major losses on a wide range of crops. Leaf blight symptoms were observed on tomato leaves, and samples were collected from various regions. Isolation was done from symptomatic tomato leaves, and 15 representatives were selected from a collection of 65 isolates of Alternaria species. The virulence of Alternaria isolates was investigated on detached leaves (DL) and whole plants of tomato cv. Super strain B. A phylogenetic analysis was performed based on three partial gene regions, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the RNA polymerase second largest subunit (RPB2) and the Alternaria major allergen gene (Alt a 1). The potentiality of Alternaria isolates to produce toxins was also investigated on the basis of thin-layer chromatography (TLC). Our investigations revealed that Alternaria isolates showed different levels of virulence either on tomato plants or DL. Based on the phylogeny of three genes, Alternaria isolates encompassed two species of small-spored morphospecies: A. alternata (14 isolates) and A. arborescens (single isolate). The produced toxins varied among Alternaria isolates with tenuazonic acid (TeA) being the most abundant mycotoxin produced by most isolates. This study highlighted on other Alternaria species in Egypt that might represent a serious concern for tomato producers as causal agents of leaf blight over other species, i.e. A. solani.     

Down-regulation of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">DND1</Emphasis> orthologs in potato and tomato leads to broad-spectrum resistance to late blight and powdery mildew

Multiple susceptibility genes (S), identified in Arabidopsis, have been shown to be functionally conserved in crop plants. Mutations in these S genes result in resistance to different pathogens, opening a new way to achieve plant disease resistance. The aim of this study was to investigate the role of Defense No Death 1 (DND1) in susceptibility of tomato and potato to late blight (Phytophthora infestans). In Arabidopsis, the dnd1 mutant has broad-spectrum resistance against several fungal, bacterial, and viral pathogens. However this mutation is also associated with a dwarfed phenotype. Using an RNAi approach, we silenced AtDND1 orthologs in potato and tomato. Our results showed that silencing of the DND1 ortholog in both crops resulted in resistance to the pathogenic oomycete P. infestans and to two powdery mildew species, Oidium neolycopersici and Golovinomyces orontii. The resistance to P. infestans in potato was effective to four different isolates although the level of resistance (complete or partial) was dependent on the aggressiveness of the isolate. In tomato, DND1-silenced plants showed a severe dwarf phenotype and autonecrosis, whereas DND1-silenced potato plants were not dwarfed and showed a less pronounced autonecrosis. Our results indicate that S gene function of DND1 is conserved in tomato and potato. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of DND1 orthologs, as well as additional S gene orthologs from Arabidopsis, to breed for resistance to pathogens in crop plants.     

Identification and Pathogenicity of Fungal Pathogens Causing Black Point in Wheat on the North China Plain

Fungi associated with black point were isolated from three highly susceptible wheat genotypes in the North China Plain. The 21 isolates represented 11 fungal genera. The most prevalent genera were Alternaria (isolation frequency of 56.7%), Bipolaris (16.1%), and Fusarium (6.0%). The other eight genera were Curvularia, Aspergillus, Cladosporium, Exserohilum, Epicoccum, Nigrospora, Penicillium, and Ulocladium; their isolation frequencies ranged from 0.8 to 4.8%. The pathogenicity of the isolates was individually assessed in the greenhouse by inoculating wheat plants with spore suspensions. Ten of the 21 isolates caused significantly higher incidences of black point than that the controls. These isolates belonged to eight fungal species (A. alternata, B. sorokiniana, B. crotonis, B. cynodontis, C. spicifera, F. equiseti, E. rostratum, and E. sorghinum) based on morphological traits and phylogenetic analysis. The average incidences of black point in the eight fungal species were 32.4, 54.3, 43.0, 41.9, 37.2, 38.8, 50.1, and 34.1%, respectively. B. sorokiniana and A. alternata were determined to be the most important pathogens in the North China Plain based on fungal prevalence and symptom severity. This study is the first to identify E. rostratum as a major pathogen causing black point in wheat.     

Molecular characterization and expression analysis of the <Emphasis Type="Italic">SPL</Emphasis> gene family with <Emphasis Type="Italic">BpSPL9</Emphasis> transgenic lines found to confer tolerance to abiotic stress in <Emphasis Type="Italic">Betula platyphylla</Emphasis> Suk.

Christolea crassifolia HARDY: gene (CcHRD) belongs to the AP2/ERF-like tanscritpion factor family, and overexpression of HRD gene has been proved to result in improved water use efficiency and enhanced drought resistance in multiple plant species. In the present study, we cloned the CcHRD gene from Christolea crassifolia, which shares 99.1% sequence similarity with the HRD gene from Arabidopsis thaliana. We generated transgenic tomato plants expressing CcHRD gene by agrobacterium-mediated genetic transformation. Our results revealed that the transgenic tomato plants showed a more developed root system and higher fruit yield than the wild-type plants. Furthermore, the leaf relative water content, chlorophyll content and Fv/Fm value in transgenic plants were significantly higher than the wild type, while the relative conductivity and MDA content of transgenic plant leaves were markedly lower than those of wild type under drought stress. We also observed that the major agronomic traits of transgenic tomato plants were improved under natural drought stress compared with those of the wild type. In summary, results in this transgenic study showed that the CcHRD gene could enhance the drought resistance in tomato, and also provided important information for the application of drought-responsive genes in improving crop plant resistance to abiotic stresses.     

SlAGO4A,a core factor of RNA-directed DNA methylation (RdDM) pathway,plays an important role under salt and drought stress in tomato

In Arabidopsis, it has been clarified that AGO4 protein is implicated in a phenomenon termed RNA-directed DNA methylation (RdDM). Previously, four orthologs of AtAGO4 were cloned in tomato, designated as SlAGO4A–SlAGO4D. Here, we studied the role of the SlAGO4A gene in regulating salt and drought tolerance in tomato. SlAGO4A-down-regulating (AS) transgenic tomato plants showed enhanced tolerance to salt and drought stress compared to wild-type (WT) and SlAGO4A-overexpressing (OE) transgenic plants, as assessed by physiological parameters such as seed germination rate, primary root length, chlorophyll/proline/MDA/soluble sugar/RWC content, and survival rate. Moreover, several genes involved in ROS scavenging and plant defense, including CAT, SOD, GST, POD, APX, LOX, and PR1, were up- or down-regulated consistently under salt and drought stress. Notably, expression levels of some DNA methyltransferase genes and RNAi pathway genes were significantly lower in AS plants than in WT. Taken together, our results suggest that SlAGO4A gene plays a negative role under salt and drought stress in tomato probably through the modulation of DNA methylation as well as the classical RNAi pathway. Hence, it may serve as a useful biotechnological tool for the genetic improvement of stress tolerance in crops.     

Characterization of fungi transferred by dust storms in Kuwait and their plant pathogenicity

Dust storms carry large amounts of plant detritus and microorganisms that may cause diseases in humans, animals or plants. These storms are frequent in Kuwait throughout the year. This research was conducted to identify the fungal species carried by the dust storms in Kuwait, originating from the northwesterly direction, with emphasis on plant pathogens. Fungi were isolated from settled dust samples and identified using established microbiological and molecular approaches. Fungal isolates identified as Fusarium oxysporum from settled dust were examined for pathogenicity using a number of crop plants. In total, 17 genera of fungi were identified in the dust samples. These fungi included plant pathogens or facultative plant parasites that were transported in the dust storms as viable propagules. The most common dust-carried fungi belonged to the genera Fusarium, Alternaria, Ulocladium, Phoma, Aspergillus, Acremonium and Penicillium. The F. oxysporum isolates that had been characterized by partial 18S rRNA gene sequencing were pathogenic, causing root and stem rot in tomato, bean and cucumber, but not squash.     

Overexpression of Tomato Homolog of Glycolate/Glycerate Transporter Gene <Emphasis Type="Italic">PLGG1/AtLrgB</Emphasis> Leads to Reduced Chlorophyll Biosynthesis

LrgA and LrgB genes have been identified as new components in regulation of programmed cell death (PCD) in bacteria. While in Arabidopsis, it has been documented that AtLrgB plays a crucial role in chloroplast development and photorespiration by acting as a glycolate/glycerate translocator (PLGG1) in the chloroplast inner membrane. However, little is known about LrgB homologs in other plant species, especially those with fleshy fruits. In this study, a homologous gene of AtLrgB, here designated SlLrgB, was identified in tomato. Similar to AtLrgB, structure analysis suggests that the LrgA and LrgB genes have evolved into two domains of the SlLrgB protein. Expression pattern analysis showed that SlLrgB accumulated mainly in green tissues and could be regulated by light, hormone, and abiotic stress treatments. Compared to wild-type plants, parts of SlLrgB overexpression plants displayed etiolated leaves and a growth retardation phenotype, with significantly reduced chlorophyll content both in leaves and fruits. The qPCR results revealed that the SGR gene, which was associated with chlorophyll degradation, was severely repressed. Two key genes in the chlorophyll biosynthesis pathway, CAO and POR, were also suppressed in the SlLrgB overexpression plants. Taken together, we suggest that SlLrgB may play important roles in the regulation of chlorophyll metabolism pathways in tomato.     

Contribution of the non-effector members of the HrpL regulon, <Emphasis Type="Italic">iaaL</Emphasis> and <Emphasis Type="Italic">matE</Emphasis>, to the virulence of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. tomato DC3000 in tomato plants

Background

The phytohormone indole-3-acetic acid (IAA) is widely distributed among plant-associated bacteria. Certain strains of the Pseudomonas syringae complex can further metabolize IAA into a less biologically active amino acid conjugate, 3-indole-acetyl-ε-L-lysine, through the action of the iaaL gene. In P. syringae and Pseudomonas savastanoi strains, the iaaL gene is found in synteny with an upstream gene, here called matE, encoding a putative MATE family transporter. In P. syringae pv. tomato (Pto) DC3000, a pathogen of tomato and Arabidopsis plants, the HrpL sigma factor controls the expression of a suite of virulence-associated genes via binding to hrp box promoters, including that of the iaaL gene. However, the significance of HrpL activation of the iaaL gene in the virulence of Pto DC3000 is still unclear.

Results

A conserved hrp box motif is found upstream of the iaaL gene in the genomes of P. syringae strains. However, although the promoter region of matE is only conserved in genomospecies 3 of this bacterial group, we showed that this gene also belongs to the Pto DC3000 HrpL regulon. We also demonstrated that the iaaL gene is transcribed both independently and as part of an operon with matE in this pathogen. Deletion of either the iaaL or the matE gene resulted in reduced fitness and virulence of Pto DC3000 in tomato plants. In addition, we used multicolor fluorescence imaging to visualize the responses of tomato plants to wild-type Pto DC3000 and to its ΔmatE and ΔiaaL mutants. Activation of secondary metabolism prior to the development of visual symptoms was observed in tomato leaves after bacterial challenges with all strains. However, the observed changes were strongest in plants challenged by the wild-type strain, indicating lower activation of secondary metabolism in plants infected with the ΔmatE or ΔiaaL mutants.

Conclusions

Our results provide new evidence for the roles of non-type III effector genes belonging to the Pto DC3000 HrpL regulon in virulence.

     

Fungi from leaves of lotus (<Emphasis Type="Italic">Nelumbo nucifera</Emphasis>)

In spite of the self-cleaning property of its leaves called the lotus effect, leaves of lotus (Nelumbo nucifera) provide a habitat for an unknown fungal diversity. The aim of this study was to detect and identify fungi from leaves of N. nucifera, including ectophytic, parasitic and endophytic fungi, in Taiwan using different collection strategies, as well as morphological and diverse molecular markers established in the different systematic groups of fungi. Among ectophytic and parasitic fungi, a new species of Dissoconium and of Pseudocercospora are described, respectively. Phyllosticta nelumbonis Sawada is transferred to Diaporthe. Among plant parasitic fungi, Erysiphe takamatsui and Ps. nymphaeacea are recorded in Taiwan for the first time. Euryale is recorded as a new host genus for Ps. nymphaeacea. The basidiomycetous yeast Fereydounia khargensis is recorded for the first time from living plants and in East Asia. Endophytic fungi from lotus were studied for the first time. From 1002 plant segments, 476 endophytic isolates were produced in culture, comprising 33 typical terrestrial species mainly belonging to the genera Colletotrichum (mainly C. siamense), Diaporthe (D. tulliensis and D. ueckerae) and Fusarium (F. solani species 6, hitherto known from clinical samples), as well as to Xylariaceae, but no Ingoldian fungi. Most isolates were from leaf laminas (71%) compared to those from petioles (29%). From this observation, we conclude that the fungi of the aquatic lotus plant appear to have terrestrial origin and, after dispersal by wind and in spite of the lotus effect, may enter the plant from the lamina. Only three species isolated as endophytes were also found as ectophytic or parasitic fungi.     

Molecular analyses of tomato GS,GOGAT and GDH gene families and their response to abiotic stresses

Glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) are closely related enzymes in plant nitrogen metabolism and potential targets for improving nitrogen use efficiency. However, little research has focused on the enzyme-encoding genes in tomato. Here, a comprehensive study of these genes was conducted. Six GS genes, two GOGAT genes and five GDH genes were identified in tomato. Bioinformatics and gene expression analyses suggested that these genes evolved species-specific regulatory properties and biological functions in tomato. SlNADH-GOGAT, SlGS1.1 and SlNAD-GDHB1 were abundantly expressed in roots, SlGS1.1 can be induced by nitrogen deprivation, and SlGS1.2, SlGS1.3, SlNADH-GOGAT and SlNAD-GDHB1 can be induced by the re-supply of nitrogen after 5 days of deprivation, they may play key roles in primary nitrogen assimilation. SlFd-GOGAT, SlGS1.1 and SlNAD-GDHA1-A2 were also highly expressed in fruits, indicating their important roles in fruit development and ripening. Tomato GS, GOGAT and GDH may be involved in stress responsiveness, since most of these genes modified their expression levels under drought, cold or heat stress treatment. We believe these findings will assist in the exploration of the genes’ biological functions and regulatory mechanisms, as well as the studies to improve nitrogen use efficiency, stress resistance and fruit quality in tomato.     

Cloning,identification, and expression analysis of a Dicer-Like gene family from Solanum lycopersicum

Dicer proteins belong to the RNase III family of proteins, which are key components in small RNA biogenesis. In Solanum lycopersicum, seven Dicer-like (DCL) genes have been identified and have been named SlDCL. In this study, we cloned the full-length sequence of the SlDCL genes including untranslated regions using RNA ligase-mediated rapid amplification of cDNA ends. Our analysis indicates that 7 SlDCLs were located on 5 tomato chromosomes (6, 7, 8, 10, and 11). The gene structure of the SlDCLs covered long genomic regions and contained more than 20 exons. Phylogenetic analysis divided the seven SlDCL members into four subgroups. In general, all seven SlDCLs were expressed in all organs but more in flowers and fruits than in the other parts. Moreover, the expressions of some genes changed slightly after treatment with ethylene or 1-methylcyclopropene suggesting their likely roles in plant responses to ethylene. Our findings provide essential information on SlDCL genes in tomato and will aid in the functional classification of DCL families in plants.     

The tomato cytosolic fructokinase FRK1 is important for phloem fiber development

Tomato (Solanum lycopersicum) plants have four fructokinase genes, SlFRK1-4. The SlFRK4 is expressed only in pollen whereas the other three are expressed in all plant parts. While SlFRK2 and SlFRK3 are involved in vascular tissue development and affects the shape, size, and cell-wall width of xylem vessels and xylem fibers, the role of SlFRK1 has not been studied previously. The current work investigates the expression of SlFRK1 using transgenic tomato plants expressing the β-glucuronidase reporter gene under the SlFRK1 promoter, as well as the role of SlFRK1 using transgenic plants with antisense suppression of SlFRK1. The SlFRK1 promoter is expressed primarily in vascular tissues and specific suppression of SlFRK1 reduces water transport in stems, but has no other anatomical or phenotypic effects. Combined suppression of SlFRK1 and SlFRK2 severely inhibited plant growth and an anatomical analysis revealed a reduction in secondary xylem area and distorted phloem fibers characterized by thin cell walls and reduced lignification. The results suggest that SlFRK1 is involved in vascular tissue development and hydraulic conductivity in tomato plants and that SlFRK1 is important for normal phloem fiber development, together with SlFRK2.