Melatonin delays ABA-induced leaf senescence via H2 O2 -dependent calcium signalling.

Precocious leaf senescence can reduce crop yield and quality by limiting the growth stage. Melatonin has been shown to delay leaf senescence; however, the underlying mechanism remains obscure. Here, we show that melatonin offsets abscisic acid (ABA) to protect photosystem II and delay the senescence of attached old leaves under the light. Melatonin induced H2 O2 accumulation accompanied by an upregulation of melon respiratory burst oxidase homolog D (CmRBOHD) under ABA-induced stress. Both melatonin and H2 O2 induced the accumulation of cytoplasmic-free Ca2+ ([Ca2+ ]cyt ) in response to ABA, while blocking of Ca2+ influx channels attenuated melatonin- and H2 O2 -induced ABA tolerance. CmRBOHD overexpression induced [Ca2+ ]cyt accumulation and delayed leaf senescence, whereas deletion of Arabidopsis AtRBOHD, a homologous gene of CmRBOHD, compromised the melatonin-induced [Ca2+ ]cyt accumulation and delay of leaf senescence in Arabidopsis under ABA stress. Furthermore, melatonin, H2 O2  and Ca2+ attenuated ABA-induced K+ efflux and subsequent cell death. CmRBOHD overexpression and AtRBOHD deletion alleviated and aggravated the ABA-induced K+ efflux, respectively. Taken together, our study unveils a new mechanism by which melatonin offsets ABA action to delay leaf senescence via RBOHD-dependent H2 O2 production that triggers [Ca2+ ]cyt accumulation and subsequently inhibits K+ efflux and delays cell death/leaf senescence in response to ABA.

Comparing Stagonosporopsis spp. Fungicide Resistance Profiles in Florida and East China Cucurbit Production Systems.

Gummy stem blight, caused by Stagonosporopsis spp., is a major disease of cucurbits in the United States and China that is managed primarily through the use of fungicides. The objective of this study was to monitor and compare the recent fungicide resistance profiles of Stagonosporopsis spp. in Florida open-field and East China protected-structure production systems. Isolates of Stagonosporopsis spp. were evaluated for sensitivity to the commonly used fungicides azoxystrobin, boscalid, tebuconazole, and thiophanate-methyl at discriminatory rates of 0.096, 0.034, 0.128, and 100 mg/liter, respectively. Isolates were collected from Jiangsu, Jiangxi, Zhejiang, and Anhui provinces in China (n = 69), 12 counties in Florida (n = 89), and one county in Georgia (n = 6). More than 50% of isolates from Florida and East China were resistant to thiophanate-methyl. Boscalid resistance was detected in both isolate collections but was two times more frequent in China. Resistance to azoxystrobin was detected in 66% of isolates in Florida but only 7% in China. Tebuconazole was effective in controlling the mycelia growth of Stagonosporopsis spp. in both collections. The results indicate that both production systems currently face similar challenges related to the development of fungicide resistance in Stagonosporopsis spp. However, the resistance profiles are unique for both production systems.

Construction of a high-density DArTseq SNP-based genetic map and identification of genomic regions with segregation distortion in a genetic population derived from a cross between feral and cultivated-type watermelon.

Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] is an economically important vegetable crop grown extensively worldwide. To facilitate the identification of agronomically important traits and provide new information for genetic and genomic research on this species, a high-density genetic linkage map of watermelon was constructed using an F2 population derived from a cross between elite watermelon cultivar K3 and wild watermelon germplasm PI 189225. Based on a sliding window approach, a total of 1,161 bin markers representing 3,465 SNP markers were mapped onto 11 linkage groups corresponding to the chromosome pair number of watermelon. The total length of the genetic map is 1,099.2 cM, with an average distance between bins of 1.0 cM. The number of markers in each chromosome varies from 62 in chromosome 07 to 160 in chromosome 05. The length of individual chromosomes ranged between 61.8 cM for chromosome 07 and 140.2 cM for chromosome 05. A total of 616 SNP bin markers showed significant (P < 0.05) segregation distortion across all 11 chromosomes, and 513 (83.3 %) of these distorted loci showed distortion in favor of the elite watermelon cultivar K3 allele and 103 were skewed toward PI 189225. The number of SNPs and InDels per Mb varied considerably across the segregation distorted regions (SDRs) on each chromosome, and a mixture of dense and sparse SNPs and InDel SDRs coexisted on some chromosomes suggesting that SDRs were randomly distributed throughout the genome. Recombination rates varied greatly among each chromosome, from 2.0 to 4.2 centimorgans per megabase (cM/Mb). An inconsistency was found between the genetic and physical positions on the map for a segment on chromosome 11. The high-density genetic map described in the present study will facilitate fine mapping of quantitative trait loci, the identification of candidate genes, map-based cloning, as well as marker-assisted selection (MAS) in watermelon breeding programs.

Genetic Characterization of Didymella bryoniae Isolates Infecting Watermelon and Other Cucurbits in Florida and Georgia.

Gummy stem blight caused by Didymella bryoniae (anamorph Phoma cucurbitacearum) is a major fungal disease of watermelon (Citrullus lanatus) and other cucurbits. Thirty-five isolates of Didymella and Phoma spp. associated with symptoms of gummy stem blight on watermelon, Canary melon (Cucumis melo), muskmelon (C. melo), and winter squash (Cucurbita maxima) from Florida and Georgia were characterized based on morphology on agar media, pathogenicity on 'Melody' watermelon, the internal transcribed spacer (ITS) sequence of ribosomal DNA (rDNA), random amplified polymorphic DNA (RAPD) analysis, and polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) analysis. All of the isolates were pathogenic on watermelon but differed in virulence. RAPD and ITS sequence analysis indicated genetic variability among the isolates but PCR-RFLP analysis did not show any variability. ITS sequence phylogenetic analysis identified two isolates, DB-05 and DB-33, which had a greater identity to that of D. bryoniae isolates from China (98 to 100% sequence homology) than other isolates from Florida and Georgia (95 to 98%). These two isolates possessed a single nucleotide substitution of A to G at position 131 of the ITS1 region. The study characterized the genetic profile of a collection of D. bryoniae isolates from Florida and Georgia in relation to isolates from other U.S. states and countries.

CmRBP50 protein phosphorylation is essential for assembly of a stable phloem-mobile high-affinity ribonucleoprotein complex.

RNA-binding proteins (RBPs) form ribonucleoprotein (RNP) complexes that play crucial roles in RNA processing for gene regulation. The angiosperm sieve tube system contains a unique population of transcripts, some of which function as long-distance signaling agents involved in regulating organ development. These phloem-mobile mRNAs are translocated as RNP complexes. One such complex is based on a phloem RBP named Cucurbita maxima RNA-binding protein 50 (CmRBP50), a member of the polypyrimidine track binding protein family. The core of this RNP complex contains six additional phloem proteins. Here, requirements for assembly of this CmRBP50 RNP complex are reported. Phosphorylation sites on CmRBP50 were mapped, and then coimmunoprecipitation and protein overlay studies established that the phosphoserine residues, located at the C terminus of CmRBP50, are critical for RNP complex assembly. In vitro pull-down experiments revealed that three phloem proteins, C. maxima phloem protein 16, C. maxima GTP-binding protein, and C. maxima phosphoinositide-specific phospholipase-like protein, bind directly with CmRBP50. This interaction required CmRBP50 phosphorylation. Gel mobility-shift assays demonstrated that assembly of the CmRBP50-based protein complex results in a system having enhanced binding affinity for phloem-mobile mRNAs carrying polypyrimidine track binding motifs. This property would be essential for effective long-distance translocation of bound mRNA to the target tissues.

Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay.

Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB) in Cucurbitaceae crops (e.g., cantaloupe, muskmelon, cucumber, and watermelon). GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated amplification (LAMP) assay for D. bryoniae detection based on sequence-characterized amplified regions (GenBank accession nos GQ872461 and GQ872462) common to the two random amplification of polymorphic DNA group genotypes (RGI and RGII) of D. bryoniae; ideal conditions for detection were optimized for completion in 45 min at 63°C. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with those of a conventional polymerase chain reaction (PCR). The sensitivity of the LAMP assay was 1000-fold higher than that of conventional PCR with a detection limit of 0.1 fg µL(-1) of targeted DNA. The LAMP assay could be accomplished in about 45 min, with the results visible to the naked eye. The assay showed high specificity in discriminating all D. bryoniae isolates from seven other fungal pathogens that occur in Cucurbitaceae crops. The LAMP assay also detected D. bryoniae infection in young muskmelon leaves with suspected early symptoms of GSB disease. Hence, the technique has great potential for developing rapid and sensitive visual detection methods for the D. bryoniae pathogen in crops and seeds. This method has potential application in early prediction of disease and reducing the risk of epidemics.

Brassinosteroids-Induced Systemic Stress Tolerance was Associated with Increased Transcripts of Several Defence-Related Genes in the Phloem in Cucumis sativus.

Brassinosteroids (BRs), a group of naturally occurring plant steroidal compounds, are essential for plant growth, development and stress tolerance. Recent studies showed that BRs could induce systemic tolerance to biotic and abiotic stresses; however, the molecular mechanisms by which BRs signals lead to responses in the whole plant are largely unknown. In this study, 24-epibrassinosteroid (EBR)-induced systemic tolerance in Cucumis sativus L. cv. Jinyan No. 4 was analyzed through the assessment of symptoms of photooxidative stress by chlorophyll fluorescence imaging pulse amplitude modulation. Expression of defense/stress related genes were induced in both treated local leaves and untreated systemic leaves by local EBR application. With the suppressive subtractive hybridization (SSH) library using cDNA from the phloem sap of EBR-treated plants as the tester and distilled water (DW)-treated plants as the driver, 14 transcripts out of 260 clones were identified. Quantitative Real Time-Polymerase Chain Reaction (RT-qPCR) validated the specific up-regulation of these transcripts. Of the differentially expressed transcripts with known functions, transcripts for the selected four cDNAs, which encode an auxin-responsive protein (IAA14), a putative ankyrin-repeat protein, an F-box protein (PP2), and a major latex, pathogenesis-related (MLP)-like protein, were induced in local leaves, systemic leaves and roots after foliar application of EBR onto mature leaves. Our results demonstrated that EBR-induced systemic tolerance is accompanied with increased transcript of genes in the defense response in other organs. The potential role of phloem mRNAs as signaling components in mediating BR-regulated systemic resistance is discussed.