Molecular characterization of Fusarium oxysporum f.sp. dianthi and evaluation of fungicides against Fusarium wilt of carnation under protected cultivation

Fusarium wilt, caused by Fusarium oxysporum f.sp. dianthi (FOD), is one of the most devastating carnation diseases globally, resulting in significant crop loss (40-70%). The purpose of this study was to determine the efficacy of several fungicides against Fusarium wilt of carnation in vitro and in vivo under protected cultivation. The disease infected plant samples were collected, and pathogen isolates were confirmed through morphological and molecular techniques. The rDNA sequences of isolates had 97-100% similarity with other sequences of FOD available in Genbank and the isolates were identified as Fusarium oxysporum f.sp. dianthi. Phylogenetic analysis of rDNA sequences revealed 62-99% sequence similarity among the isolates. Further, we evaluated nine different fungicides against the virulent pathogenic isolate CFODTNAU1 under in vitro and in vivo. Three different concentrations such as 500, 1000 and 1500 ppm were evaluated on mycelial growth of the pathogen under in vitro and percent inhibition over control was calculated. Among the fungicides, three of them namely, tebuconazole 50% + trifloxystrobin 25% WG, tebuconazole 25.9% SC and azoxystrobin 23% EC completely inhibited (100% inhibition) the mycelial growth of the fungus in vitro. Under protected cultivation, application of tebuconazole 50% + trifloxystrobin 25% WG through root dipping and soil drenching @ 1.0 g/L recorded 11.11% of wilt incidence compared to control (29.71% wilt incidence), which was 59.99% reduction over control. Besides, this fungicide also increased the stem length, earliness of flowering and yield than the control and other treatments. Thus, the fungicide tebuconazole 50% + trifloxystrobin 25% WG can be used for amelioration and control of wilt disease in carnation

Molecular characterization of Fusarium oxysporum f.sp. dianthi and evaluation of fungicides against Fusarium wilt of carnation under protected cultivation

770-775Fusarium wilt, caused by Fusarium oxysporum f.sp. dianthi (FOD), is one of the most devastating carnation diseases globally, resulting in significant crop loss (40-70%). The purpose of this study was to determine the efficacy of several fungicides against Fusarium wilt of carnation in vitro and in vivo under protected cultivation. The disease infected plant samples were collected, and pathogen isolates were confirmed through morphological and molecular techniques. The rDNA sequences of isolates had 97-100% similarity with other sequences of FOD available in Genbank and the isolates were identified as Fusarium oxysporum f.sp. dianthi. Phylogenetic analysis of rDNA sequences revealed 62-99% sequence similarity among the isolates. Further, we evaluated nine different fungicides against the virulent pathogenic isolate CFODTNAU1 under in vitro and in vivo. Three different concentrations such as 500, 1000 and 1500 ppm were evaluated on mycelial growth of the pathogen under in vitro and percent inhibition over control was calculated. Among the fungicides, three of them namely, tebuconazole 50% + trifloxystrobin 25% WG, tebuconazole 25.9% SC and azoxystrobin 23% EC completely inhibited (100% inhibition) the mycelial growth of the fungus in vitro. Under protected cultivation, application of tebuconazole 50% + trifloxystrobin 25% WG through root dipping and soil drenching @ 1.0 g/L recorded 11.11% of wilt incidence compared to control (29.71% wilt incidence), which was 59.99% reduction over control. Besides, this fungicide also increased the stem length, earliness of flowering and yield than the control and other treatments. Thus, the fungicide tebuconazole 50% + trifloxystrobin 25% WG can be used for amelioration and control of wilt disease in carnation

Expression Kinetics of Regulatory Genes Involved in the Vesicle Trafficking Processes Operating in Tomato Flower Abscission Zone Cells during Pedicel Abscission

The abscission process occurs in a specific abscission zone (AZ) as a consequence of the middle lamella dissolution, cell wall degradation, and formation of a defense layer. The proteins and metabolites related to these processes are secreted by vesicle trafficking through the plasma membrane to the cell wall and middle lamella of the separating cells in the AZ. We investigated this process, since the regulation of vesicle trafficking in abscission systems is poorly understood. The data obtained describe, for the first time, the kinetics of the upregulated expression of genes encoding the components involved in vesicle trafficking, occurring specifically in the tomato (Solanum lycopersicum) flower AZ (FAZ) during pedicel abscission induced by flower removal. The genes encoding vesicle trafficking components included soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), SNARE regulators, and small GTPases. Our results clearly show how the processes of protein secretion by vesicle trafficking are regulated, programmed, and orchestrated at the level of gene expression in the FAZ. The data provide evidence for target proteins, which can be further used for affinity purification of plant vesicles in their natural state. Such analyses and dissection of the complex vesicle trafficking networks are essential for further elucidating the mechanism of organ abscission

Role of the KNOTTED1‐LIKE HOMEOBOX protein (KD1) in regulating abscission of tomato flower pedicels at early and late stages of the process

The KNOTTED1-LIKE HOMEOBOX PROTEIN1 (KD1) gene is highly expressed in flower and leaf abscission zones (AZs), and KD1 was reported to regulate tomato flower pedicel abscission via alteration of the auxin gradient and response in the flower AZ (FAZ). The present work was aimed to further examine how KD1 regulates signaling factors and regulatory genes involved in pedicel abscission, by using silenced KD1 lines and performing a large-scale transcriptome profiling of the FAZ before and after flower removal, using a customized AZ-specific microarray. The results highlighted a differential expression of regulatory genes in the FAZ of KD1-silenced plants compared to the wild-type. In the TAPG4::antisense KD1-silenced plants, KD1 gene expression decreased before flower removal, resulting in altered expression of regulatory genes, such as epigenetic modifiers, transcription factors, posttranslational regulators, and antioxidative defense factors occurring at zero time and before affecting auxin levels in the FAZ detected at 4 h after flower removal. The expression of additional regulatory genes was altered in the FAZ of KD1-silenced plants at 4-20 h after flower removal, thereby leading to an inhibited abscission phenotype, and downregulation of genes involved in abscission execution and defense processes. Our data suggest that KD1 is a master regulator of the abscission process, which promotes abscission of tomato flower pedicels. This suggestion is based on the inhibitory effect of KD1 silencing on flower pedicel abscission that operates via alteration of various regulatory pathways, which delay the competence acquisition of the FAZ cells to respond to ethylene signaling

The Tomato Hybrid Proline-rich Protein regulates the abscission zone competence to respond to ethylene signals.

The Tomato Hybrid Proline-rich Protein (THyPRP) gene was specifically expressed in the tomato (Solanum lycopersicum) flower abscission zone (FAZ), and its stable antisense silencing under the control of an abscission zone (AZ)-specific promoter, Tomato Abscission Polygalacturonase4, significantly inhibited tomato pedicel abscission following flower removal. For understanding the THyPRP role in regulating pedicel abscission, a transcriptomic analysis of the FAZ of THyPRP-silenced plants was performed, using a newly developed AZ-specific tomato microarray chip. Decreased expression of THyPRP in the silenced plants was already observed before abscission induction, resulting in FAZ-specific altered gene expression of transcription factors, epigenetic modifiers, post-translational regulators, and transporters. Our data demonstrate that the effect of THyPRP silencing on pedicel abscission was not mediated by its effect on auxin balance, but by decreased ethylene biosynthesis and response. Additionally, THyPRP silencing revealed new players, which were demonstrated for the first time to be involved in regulating pedicel abscission processes. These include: gibberellin perception, Ca2+-Calmodulin signaling, Serpins and Small Ubiquitin-related Modifier proteins involved in post-translational modifications, Synthaxin and SNARE-like proteins, which participate in exocytosis, a process necessary for cell separation. These changes, occurring in the silenced plants early after flower removal, inhibited and/or delayed the acquisition of the competence of the FAZ cells to respond to ethylene signaling. Our results suggest that THyPRP acts as a master regulator of flower abscission in tomato, predominantly by playing a role in the regulation of the FAZ cell competence to respond to ethylene signals

Microarray Analysis of the Abscission-Related Transcriptome in the Tomato Flower Abscission Zone in Response to Auxin Depletion1[C][W][OA]

The abscission process is initiated by changes in the auxin gradient across the abscission zone (AZ) and is triggered by ethylene. Although changes in gene expression have been correlated with the ethylene-mediated execution of abscission, there is almost no information on the molecular and biochemical basis of the increased AZ sensitivity to ethylene. We examined transcriptome changes in the tomato (Solanum lycopersicum ‘Shiran 1335’) flower AZ during the rapid acquisition of ethylene sensitivity following flower removal, which depletes the AZ from auxin, with or without preexposure to 1-methylcyclopropene or application of indole-3-acetic acid after flower removal. Microarray analysis using the Affymetrix Tomato GeneChip revealed changes in expression, occurring prior to and during pedicel abscission, of many genes with possible regulatory functions. They included a range of auxin- and ethylene-related transcription factors, other transcription factors and regulatory genes that are transiently induced early, 2 h after flower removal, and a set of novel AZ-specific genes. All gene expressions initiated by flower removal and leading to pedicel abscission were inhibited by indole-3-acetic acid application, while 1-methylcyclopropene pretreatment inhibited only the ethylene-induced expressions, including those induced by wound-associated ethylene signals. These results confirm our hypothesis that acquisition of ethylene sensitivity in the AZ is associated with altered expression of auxin-regulated genes resulting from auxin depletion. Our results shed light on the regulatory control of abscission at the molecular level and further expand our knowledge of auxin-ethylene cross talk during the initial controlling stages of the process

Identification of defense-related genes newly associated with tomato flower abscission

The current abscission model suggests the formation of a post-abscission trans-differentiation of a protective layer as the last step of the process. The present report expands the repertoire of genes activated in the tomato flower abscission zone (AZ), which are likely to be involved in defense responses. We identified four different defense-related genes, including: Cysteine-type endopeptidase, α-Dioxygenase 1 (α-DOX1), HopW1-1-Interacting protein2 (WIN2) and Stomatal-derived factor-2 (SDF2), that are newly-associated with the late stage of the abscission process. The late expression of these genes, induced at 8–14 h after flower removal when pedicel abscission was already in progress, was AZ-specific, and was inhibited by treatments that prevented pedicel abscission, including 1-methylcyclopropene pretreatment or IAA application. This information supports the activation of different defense responses and strategies at the late abscission stages, which may enable efficient protection of the exposed tissue toward different environmental stresses