The apocarotenoid ß-ionone regulates the transcriptome of Arabidopsis thaliana and increases its resistance against Botrytis cinerea.

Carotenoids are isoprenoid pigments indispensable for photosynthesis. Moreover, they are the precursor of apocarotenoids, which include the phytohormones abscisic acid (ABA) and strigolactones (SLs) as well as retrograde signaling molecules and growth regulators, such as ß-cyclocitral and zaxinone. Here, we show that the application of the volatile apocarotenoid ß-ionone (ß-I) to Arabidopsis plants at micromolar concentrations caused a global reprogramming of gene expression, affecting thousands of transcripts involved in stress tolerance, growth, hormone metabolism, pathogen defense, and photosynthesis. This transcriptional reprogramming changes, along with induced changes in the level of the phytohormones ABA, jasmonic acid, and salicylic acid, led to enhanced Arabidopsis resistance to the widespread necrotrophic fungus Botrytis cinerea (B.c.) that causes the gray mold disease in many crop species and spoilage of harvested fruits. Pre-treatment of tobacco and tomato plants with ß-I followed by inoculation with B.c. confirmed the effect of ß-I in increasing the resistance to this pathogen in crop plants. Moreover, we observed reduced susceptibility to B.c. in fruits of transgenic tomato plants overexpressing LYCOPENE ß-CYCLASE, which contains elevated levels of endogenous ß-I, providing a further evidence for its effect on B.c. infestation. Our work unraveled ß-I as a further carotenoid-derived regulatory metabolite and indicates the possibility of establishing this natural volatile as an environmentally friendly bio-fungicide to control B.c.

Molecular Characterization and Disease Control of Stem Canker on Royal Poinciana (Delonix regia) Caused by Neoscytalidium dimidiatum in the United Arab Emirates.

In the United Arab Emirates (UAE), royal poinciana (Delonix regia) trees suffer from stem canker disease. Symptoms of stem canker can be characterized by branch and leaf dryness, bark lesions, discoloration of xylem tissues, longitudinal wood necrosis and extensive gumming. General dieback signs were also observed leading to complete defoliation of leaves and ultimately death of trees in advanced stages. The fungus, Neoscytalidium dimidiatum DSM 109897, was consistently recovered from diseased royal poinciana tissues; this was confirmed by the molecular, structural and morphological studies. Phylogenetic analyses of the translation elongation factor 1-a (TEF1-α) of N. dimidiatum from the UAE with reference specimens of Botryosphaeriaceae family validated the identity of the pathogen. To manage the disease, the chemical fungicides, Protifert®, Cidely® Top and Amistrar® Top, significantly inhibited mycelial growth and reduced conidial numbers of N. dimidiatum in laboratory and greenhouse experiments. The described "apple bioassay" is an innovative approach that can be useful when performing fungicide treatment studies. Under field conditions, Cidely® Top proved to be the most effective fungicide against N. dimidiatum among all tested treatments. Our data suggest that the causal agent of stem canker disease on royal poinciana in the UAE is N. dimidiatum.

Molecular Identification and Disease Management of Date Palm Sudden Decline Syndrome in the United Arab Emirates.

Date palm orchards suffer from serious diseases, including sudden decline syndrome (SDS). External symptoms were characterized by whitening on one side of the rachis, progressing from the base to the apex of the leaf until the whole leaf dies; while the internal disease symptoms included reddish roots and highly colored vascular bundles causing wilting and death of the tree. Although three Fusarium spp. (F. oxysporum, F. proliferatum and F. solani) were isolated from diseased root samples, the fungal pathogen F. solani was associated with SDS on date palm in the United Arab Emirates (UAE). Fusarium spp. were identified based on their cultural and morphological characteristics. The internal transcribed spacer regions and large subunit of the ribosomal RNA (ITS/LSU rRNA) gene complex of the pathogens was further sequenced. Pathogenicity assays and disease severity indices confirm the main causal agent of SDS on date palm in the UAE is F. solani. Application of Cidely® Top (difenoconazole and cyflufenamid) significantly inhibited the fungal mycelial growth in vitro and reduced SDS development on date palm seedlings pre-inoculated with F. solani under greenhouse conditions. This is the first report confirming that the chemical fungicide Cidely® Top is strongly effective against SDS on date palm.

Detection and Management of Mango Dieback Disease in the United Arab Emirates.

Mango is affected by different decline disorders causing significant losses to mango growers. In the United Arab Emirates (UAE), the pathogen was isolated from all tissues sampled from diseased trees affected by Lasiodiplodia theobromae. Symptoms at early stages of the disease included general wilting appearance of mango trees, and dieback of twigs. In advanced stages, the disease symptoms were also characterized by the curling and drying of leaves, leading to complete defoliation of the tree and discolouration of vascular regions of the stems and branches. To substantially reduce the devastating impact of dieback disease on mango, the fungus was first identified based on its morphological and cultural characteristics. Target regions of 5.8S rRNA (ITS) and elongation factor 1-α (EF1-α) genes of the pathogen were amplified and sequenced. We also found that the systemic chemical fungicides, Score®, Cidely® Top, and Penthiopyrad®, significantly inhibited the mycelial growth of L. theobromae both in vitro and in the greenhouse. Cidely® Top proved to be a highly effective fungicide against L. theobromae dieback disease also under field conditions. Altogether, the morphology of the fruiting structures, molecular identification and pathogenicity tests confirm that the causal agent of the mango dieback disease in the UAE is L. theobromae.

Chemical Control of Black Scorch Disease on Date Palm Caused by the Fungal Pathogen Thielaviopsis punctulata in United Arab Emirates.

Date palm (Phoenix dactylifera L.) is one of the most important plants grown for its edible fruit. Palm diseases are among the major factors affecting its growth and productivity. In the United Arab Emirates (UAE), the causal agent of black scorch on date palm was found to be Thielaviopsis punctulata. The pathogen was isolated from all tissues of diseased trees affected by the virulent T. punctulata. Depending on the severity of the infection, symptoms included tissue necrosis, wilting, neck bending, death of terminal buds, and eventual plant mortality. This fungus, which was consistently isolated on potato dextrose agar from infected tissues, produced two types of conidia: the thick-walled aleuroconidia (chlamydospores) and phialoconidia (endoconidia). In addition, all target regions of 5.8S ribosomal RNA, 28S ribosomal DNA, ß-tubulin, and transcription elongation factor 1-α genes of the pathogen were amplified using polymerase chain reaction. We also found that the fungicide Score inhibited the mycelial growth of T. punctulata both in vitro and in vivo. Altogether, the morphology of the fruiting structures, pathogenicity tests, and molecular identification confirmed that the causal agent of symptomatic tissues is T. punctulata. This is the first report of the black scorch disease and the fungus T. punctulata on date palm in the UAE.

Co-expression Networks in Predicting Transcriptional Gene Regulation.

Recent progress in transcriptomics and co-expression networks have enabled us to predict the inference of the biological functions of genes with the associated environmental stress. Microarrays and RNA sequencing (RNA-seq) are the most commonly used high-throughput gene expression platforms for detecting differentially expressed genes between two (or more) phenotypes. Gene co-expression networks (GCNs) are a systems biology method for capturing transcriptional patterns and predicting gene interactions into functional and regulatory relationships. Here, we describe the procedures and tools used to construct and analyze GCN and investigate the integration of transcriptional data with GCN to provide reliable information about the underlying biological mechanism.

Metatranscriptomic Analysis of Multiple Environmental Stresses Identifies RAP2.4 Gene Associated with Arabidopsis Immunity to Botrytis cinerea.

In this study, we aimed to identify common genetic components during stress response responsible for crosstalk among stresses, and to determine the role of differentially expressed genes in Arabidopsis-Botrytis cinerea interaction. Of 1,554 B. cinerea up-regulated genes, 24%, 1.4% and 14% were induced by biotic, abiotic and hormonal treatments, respectively. About 18%, 2.5% and 22% of B. cinerea down-regulated genes were also repressed by the same stress groups. Our transcriptomic analysis indicates that plant responses to all tested stresses can be mediated by commonly regulated genes; and protein-protein interaction network confirms the cross-interaction between proteins regulated by these genes. Upon challenges to individual or multiple stress(es), accumulation of signaling molecules (e.g. hormones) plays a major role in the activation of downstream defense responses. In silico gene analyses enabled us to assess the involvement of RAP2.4 (related to AP2.4) in plant immunity. Arabidopsis RAP2.4 was repressed by B. cinerea, and its mutants enhanced resistance to the same pathogen. To the best of our knowledge, this is the first report demonstrating the role of RAP2.4 in plant defense against B. cinerea. This research can provide a basis for breeding programs to increase tolerance and improve yield performance in crops.

Growth Promotion of Salicornia bigelovii by Micromonospora chalcea UAE1, an Endophytic 1-Aminocyclopropane-1-Carboxylic Acid Deaminase-Producing Actinobacterial Isolate.

Salicornia bigelovii is a promising halophytic crop for saline soils in semi-arid regions. This study was designed to characterize isolates of endophytic actinobacteria from S. bigelovii roots and evaluate the effects associated with plant growth promotion. Twenty-eight endophytic isolates obtained from surface-sterilized roots of S. bigelovii were initially selected based on their production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase in vitro in a chemically defined medium. Application of Micromonospora chalcea UAE1, possessing the highest ACC deaminase activity, to S. bigelovii seedlings significantly enhanced the plant growth under gnotobiotic and greenhouse conditions. This was clear from the increases in the dry weight and length of both shoot and root, and seed yield compared to the non-ACC deaminase-producing isolate Streptomyces violaceorectus, or control treatment. The growth promotion was also supported by significant increases in the content of photosynthetic pigments and the levels of auxins, but significant decreases in the levels of ACC in planta. Under greenhouse conditions, M. chalcea recovered from inside the inoculated roots in all samplings (up to 12 weeks post inoculation), suggesting that the roots of healthy S. bigelovii are a suitable habitat for the endophytic actinobacterial isolates. Pure cultures of M. chalcea were not capable of producing auxins, gibberellic acid, cytokinins or polyamines in vitro. This indicates that the growth promotion is most likely to be due to the reduction of the endogenous levels of the stress hormone ethylene. Our findings suggest that growth and yields of S. bigelovii can be enhanced by the field application of the endophyte M. chalcea UAE1. This study is the first to report potential endophytic non-streptomycete actinobacteria to promote the growth of halophytic plants in semi-arid zones under greenhouse conditions.

Effect of phosphate nutrition on growth, physiology and phosphate transporter expression of cucumber seedlings.

Although abundantly present in soils, inorganic phosphate (Pi) acquisition by plants is highly dependent on the transmembrane phosphate transporter (PT) gene family. Cucumber (Cucumis sativus) requires a large amount of phosphorus (P). The purpose of this study was to isolate the CsPT2-1 from cucumber roots, and to determine the influence of Pi nutrition on cucumber growth, metabolism and transcript levels of CsPT2-1 in tissues. Full length CsPT2-1 was cloned and phylogenetically identified. In two greenhouse experiments, P-deficient seedlings provided with low or high P concentrations were sampled at 10 and 21 days post treatment, respectively. Addition of P dramatically reduced growth of roots but not shoots. Supplying plants with high P resulted in increased total protein in leaves. Acid phosphatase activity increased significantly in leaves at any rate higher than 4 mM P. Increasing P concentration had a notable decrease in glucose concentrations in leaves of plants supplied with >0.5 mM P. In roots, glucose and starch concentrations increased with increasing P supply. Steady-state transcript levels of CsPT2-1 were high in P-deprived roots, but declined when plants were provided >10 mM P. To our knowledge, this is the first report focusing on a PT and its expression levels in cucumber.

Streptomyces globosus UAE1, a Potential Effective Biocontrol Agent for Black Scorch Disease in Date Palm Plantations.

Many fungal diseases affect date palm causing considerable losses in date production worldwide. We found that the fungicide Cidely® Top inhibited the mycelial growth of the soil-borne pathogenic fungus Thielaviopsis punctulata, the causal agent of black scorch disease of date palm, both in vitro and in vivo. Because the use of biocontrol agents (BCAs) can minimize the impact of pathogen control on economic and environmental concerns related to chemical control, we aimed at testing local actinomycete strains isolated from the rhizosphere soil of healthy date palm cultivated in the United Arab Emirates (UAE) against T. punctulata. The selected isolate can thus be used as a potential agent for integrated disease management programs. In general, the BCA showed antagonism in vitro and in greenhouse experiments against this pathogen. The most promising actinomycete isolate screened showed the highest efficacy against the black scorch disease when applied before or at the same time of inoculation with T. punctulata, compared with BCA or fungicide application after inoculation. The nucleotide sequence and phylogenetic analyses using the 16S ribosomal RNA gene with other Streptomyces spp. in addition to morphological and cultural characteristics revealed that the isolated UAE strain belongs to Streptomyces globosus UAE1. The antagonistic activity of S. globosus against T. punctulata, was associated with the production by this strain of diffusible antifungal metabolites i.e., metabolites that can inhibit mycelial growth of the pathogen. This was evident in the responses of the vegetative growth of pure cultures of the pathogen when exposed to the culture filtrates of the BCA. Altogether, the pathogenicity tests, disease severity indices and mode of action tests confirmed that the BCA was not only capable of suppressing black scorch disease symptoms, but also could prevent the spread of the pathogen, as a potential practical method to improve disease management in the palm plantations. This is the first report of an actinomycete, naturally occurring in the UAE with the potential for use as a BCA in the management of the black scorch disease of date palms in the region.

Microarray analysis of Arabidopsis WRKY33 mutants in response to the necrotrophic fungus Botrytis cinerea.

The WRKY33 transcription factor was reported for resistance to the necrotrophic fungus Botrytis cinerea. Using microarray-based analysis, we compared Arabidopsis WRKY33 overexpressing lines and wrky33 mutant that showed altered susceptibility to B. cinerea with their corresponding wild-type plants. In the wild-type, about 1660 genes (7% of the transcriptome) were induced and 1054 genes (5% of the transcriptome) were repressed at least twofold at early stages of inoculation with B. cinerea, confirming previous data of the contribution of these genes in B. cinerea resistance. In Arabidopsis wild-type plant infected with B. cinerea, the expressions of the differentially expressed genes encoding for proteins and metabolites involved in pathogen defense and non-defense responses, seem to be dependent on a functional WRKY33 gene. The expression profile of 12-oxo-phytodienoic acid- and phytoprostane A1-treated Arabidopsis plants in response to B. cinerea revealed that cyclopentenones can also modulate WRKY33 regulation upon inoculation with B. cinerea. These results support the role of electrophilic oxylipins in mediating plant responses to B. cinerea infection through the TGA transcription factor. Future directions toward the identification of the molecular components in cyclopentenone signaling will elucidate the novel oxylipin signal transduction pathways in plant defense.

Identification of Arabidopsis candidate genes in response to biotic and abiotic stresses using comparative microarrays.

Plants have evolved with intricate mechanisms to cope with multiple environmental stresses. To adapt with biotic and abiotic stresses, plant responses involve changes at the cellular and molecular levels. The current study was designed to investigate the effects of combinations of different environmental stresses on the transcriptome level of Arabidopsis genome using public microarray databases. We investigated the role of cyclopentenones in mediating plant responses to environmental stress through TGA (TGACG motif-binding factor) transcription factor, independently from jasmonic acid. Candidate genes were identified by comparing plants inoculated with Botrytis cinerea or treated with heat, salt or osmotic stress with non-inoculated or non-treated tissues. About 2.5% heat-, 19% salinity- and 41% osmotic stress-induced genes were commonly upregulated by B. cinerea-treatment; and 7.6%, 19% and 48% of genes were commonly downregulated by B. cinerea-treatment, respectively. Our results indicate that plant responses to biotic and abiotic stresses are mediated by several common regulatory genes. Comparisons between transcriptome data from Arabidopsis stressed-plants support our hypothesis that some molecular and biological processes involved in biotic and abiotic stress response are conserved. Thirteen of the common regulated genes to abiotic and biotic stresses were studied in detail to determine their role in plant resistance to B. cinerea. Moreover, a T-DNA insertion mutant of the Responsive to Dehydration gene (rd20), encoding for a member of the caleosin (lipid surface protein) family, showed an enhanced sensitivity to B. cinerea infection and drought. Overall, the overlapping of plant responses to abiotic and biotic stresses, coupled with the sensitivity of the rd20 mutant, may provide new interesting programs for increased plant resistance to multiple environmental stresses, and ultimately increases its chances to survive. Future research directions towards a better dissection of the potential crosstalk between B. cinerea, abiotic stress, and oxylipin signaling are of our particular interest.

Transcriptome analysis reveals genes commonly induced by Botrytis cinerea infection, cold, drought and oxidative stresses in Arabidopsis.

Signaling pathways controlling biotic and abiotic stress responses may interact synergistically or antagonistically. To identify the similarities and differences among responses to diverse stresses, we analyzed previously published microarray data on the transcriptomic responses of Arabidopsis to infection with Botrytis cinerea (a biotic stress), and to cold, drought, and oxidative stresses (abiotic stresses). Our analyses showed that at early stages after B. cinerea inoculation, 1498 genes were up-regulated (B. cinerea up-regulated genes; BUGs) and 1138 genes were down-regulated (B. cinerea down-regulated genes; BDGs). We showed a unique program of gene expression was activated in response each biotic and abiotic stress, but that some genes were similarly induced or repressed by all of the tested stresses. Of the identified BUGs, 25%, 6% and 12% were also induced by cold, drought and oxidative stress, respectively; whereas 33%, 7% and 5.5% of the BDGs were also down-regulated by the same abiotic stresses. Coexpression and protein-protein interaction network analyses revealed a dynamic range in the expression levels of genes encoding regulatory proteins. Analysis of gene expression in response to electrophilic oxylipins suggested that these compounds are involved in mediating responses to B. cinerea infection and abiotic stress through TGA transcription factors. Our results suggest an overlap among genes involved in the responses to biotic and abiotic stresses in Arabidopsis. Changes in the transcript levels of genes encoding components of the cyclopentenone signaling pathway in response to biotic and abiotic stresses suggest that the oxylipin signal transduction pathway plays a role in plant defense. Identifying genes that are commonly expressed in response to environmental stresses, and further analyzing the functions of their encoded products, will increase our understanding of the plant stress response. This information could identify targets for genetic modification to improve plant resistance to multiple stresses.

A mutation in the expansin-like A2 gene enhances resistance to necrotrophic fungi and hypersensitivity to abiotic stress in Arabidopsis thaliana.

Expansins are cell wall loosening agents, known for their endogenous function in cell wall extensibility. The Arabidopsis expansin-like A2 (EXLA2) gene was identified by its down-regulation in response to infection by the necrotrophic pathogen Botrytis cinerea, and by the reduced susceptibility of an exla2 mutant to the same pathogen. The exla2 mutant was equally susceptible to Pseudomonas syringae pv. tomato, but was more resistant to the necrotrophic fungus Alternaria brassicicola, when compared with the wild-type or with transgenic, ectopic EXLA2-overexpressing lines. The exla2 mutants also enhanced tolerance to the phytoprostane-A1 . This suggests that the absence or down-regulation of EXLA2 leads to increased resistance to B. cinerea in a CORONATINE INSENSITIVE 1 (COI1)-dependent manner, and this down-regulation can be achieved by phytoprostane-A1 treatment. EXLA2 is induced significantly by salinity and cold, and by the exogenous application of abscisic acid. The exla2 mutant also showed hypersensitivity towards increased salt and cold, and this hypersensitivity required a functional abscisic acid pathway. The differential temporal expression of EXLA2 and the phenotypes in transgenic plants with altered expression of EXLA2 indicate that plant cell wall structure is an important player during Arabidopsis developmental stages. Our results indicate that EXLA2 appears to be important in response to various biotic and abiotic stresses, particularly in the pathogenesis of necrotrophic pathogens and in the tolerance to abiotic stress.