Document Type

Dissertation - Open Access

Award Date

2022

Degree Name

Doctor of Philosophy (PhD)

Department / School

Agronomy, Horticulture, and Plant Science

First Advisor

Febina Mathew

Keywords

Diaporthe, Fungicides, Meta-analyses, Phomopsis, RNA sequencing, Sunflower

Abstract

Phomopsis stem canker is one of the major diseases of sunflower (Helianthus annuus L.) caused by complex of Diaporthe spp., in the U.S. This disease became of economic importance after 2010 epidemic in Minnesota, North Dakota, and South Dakota and since then the prevalence of this diseases has increased due to increased average precipitation in these major sunflower growing regions of the U.S. The two important disease management strategies involve the use of fungicides and partially resistant hybrids. However, over the years, researchers and growers have seen varied results on Phomopsis stem canker disease severity reduction and yield gains, when it comes to the application of foliar fungicides. Moreover, information on fungicide sensitivity of the two predominant pathogens causing Phomopsis stem canker (D. gulyae and D. helianthi) is lacking. Additionally, information on the resistance mechanism and the genes conferring resistance to Diaporthe species could aid future marker-assisted breeding studies. Therefore, the study was planned with the following objectives to (1) assess the potential of fungicide active ingredients and application timings in managing Phomopsis stem canker; (2) determine the fungicide sensitivity of D. gulyae and D. helianthi against fluxapyroxad, pyraclostrobin, and tebuconazole fungicides; and (3) explore the sunflower response to infection by D. helianthi using RNA-sequencing. For the first objective, we analyzed foliar fungicide efficacy trials conducted between 2009 to 2021 in Minnesota, Nebraska, North Dakota, and South Dakota. The trials served as datasets to evaluate the best fungicide application timing and active ingredient combination for managing Phomopsis stem canker and preventing yield losses. A metaanalytical approach was used to analyze 56 location-years trials using non-linear regression to determine the effect of a total of seven application timings (single and sequential) and seven active ingredients (single and combination) on the disease severity index (DSI) and yield in comparison to the non-treated control (NTC) by calculating two effect sizes (Cohen’s f or Hedges’s g) corresponding to the differences in DSI and yield between the treated and NTC. The pooled effect was significant for both fungicide application timings [DSI (f=-0.43), yield (f=0.26)] and active ingredients [DSI (f=-0.51); yield (f=0.39)] on DSI and yield (P < 0.0001). Based on the 95% confidence intervals (CI) around g, fungicide applications made at R1 growth stage (miniature floral head development) did not contain zero indicating a significant disease reduction (g=-0.57), and yield gain (g=0.33), in comparison to NTC. In addition, a split application of pyraclostrobin at R1 + R5 [DSI (g=-0.55)] outperformed NTC in managing the disease. Similarly, based on the 95% CI, the active ingredients fluxapyroxad + pyraclostrobin [DSI (g=-0.55), yield (g=0.36)], mefentrifluconazole + pyraclostrobin + fluxapyroxad [DSI (g=-2.23), yield (g=0.85)], and pyraclostrobin [DSI (g=-0.65), yield (g=0.43)] were able to significantly reduce the disease and prevent yield losses. This study suggests that use of pyraclostrobin based fungicides at R1 can reduce Phomopsis stem canker severity and increase yield when initial disease symptoms start to appear in the field. The second objective involved a proactive fungicide sensitivity monitoring program to determine the sensitivity of D. gulyae and D. helianthi to fluxapyroxad, pyraclostrobin, and tebuconazole fungicides. The assay was conducted under lab conditions (22±2⁰C in dark) using a total of 103 isolates collected from Minnesota, Nebraska, North Dakota, and South Dakota. In addition, three baseline isolates [one isolate of D. gulyae (ex-type BRIP 54025) from Australia and two D. helianthi isolates (ATCC 201540 and 52763), obtained from ATCC collection] were used in the study. Technical grade products of the three fungicides were dissolved in acetone to prepare stock solutions. Following which, water-agar media was serially amended with different concentrations of fluxapyroxad, pyraclostrobin + SHAM (salicylhydroxamic acid – 20 μg ml-1), and tebuconazole, using poison food technique. A 0.6-cm inverted mycelial plug of D. gulyae or D. helianthi was placed onto the center of each fungicide amended Petri-dish and a fungicide concentration of 0 μg ml-1 or acetone served as the control. For each fungus and fungicide combination, the experiment was arranged in a completely randomized design, with four replications per fungicide concentration and was repeated once. The plates were then incubated in dark at 22±2°C for 5 days (D. gulyae) and 7 days (D. helianthi), after which the radial growth of the fungus was measured at right angles using a scale. The percent mycelial growth inhibition was used to calculate the effective concentration that inhibited the growth of fungus by half (EC50) through non-linear regression. A significant effect of EC50 for the three fungicides on all isolates (P < 0.0001) was observed using ANOVA Type Statistics (ATS). For D. gulyae, the mean EC50 values were 6.234, 0.919, and 0.245 μg ml-1 for fluxapyroxad, pyraclostrobin, and tebuconazole, respectively where six, 22, and 21 isolates for each fungicide had significantly greater EC50 (P < 0.0001) than the baseline. While the mean EC50 values for D. helianthi, were 5.999, 0.171, and 0.127 μg ml-1 for fluxapyroxad, pyraclostrobin, and tebuconazole, respectively where three, three, and 13 isolates for each fungicide had significantly greater EC50 (P < 0.0001) than the two baselines. The results indicate a possible decline in the sensitivity of Phomopsis species to fluxapyroxad, pyraclostrobin, and tebuconazole fungicides. This study establishes the first fungicide sensitivity monitoring of D. gulyae and D. helianthi against fluxapyroxad, pyraclostrobin, and tebuconazole fungicides program and the generated information from this study will be critical for future sensitivity assays. The last objective was to investigate the host pathogen interaction between D. helianthi isolates and sunflower genotypes using transcriptome analysis. For this, we carried out RNAseq analysis of one susceptible (PI 552934) and two partially resistant (PI 561918 and PI 509062) accessions at 0 and 72 hrs. post-inoculation (hpi) on infection of three D. helianthi isolates (DIA-59, DIA-130, DIA-197) to spot the sunflower transcriptome response and figure out the mechanisms underlying Phomopsis stem canker causing fungal disease resistance. The results showed a total of 249, 277, and 264 differentially expressed genes (P ≤ 0.001; log2FC >2 or <-2) in genotypes PI 552934, PI 561918, and PI 509062, respectively at 72 hpi. On exploring the functions of the uniquely expressed genes in the tolerant genotypes, PI 561918, and PI 509062, in response to D. helianthi infection the genes belonged to several major disease resistance associated families like the basic leucine zipper (bZIP), WRKY transcription factors, SNARE family proteins, pectinesterase inhibitors, and GDSL esterase/lipase family. These gene functions are well known for their increased expression in response to abiotic and biotic stresses, thus providing increased plant tolerance. Thus, our findings highlight the diversity of transcriptional responses to D. helianthi in different sunflower genotypes that might play a crucial role in plant defense. Overall, our study showed that only a few groups of fungicides (QoI based) might be effective in managing Phomopsis stem canker. The application of such fungicides appears to be effective in managing the disease and increasing grain yield if applied at R1 (miniature floral head formation) growth stage in presence of disease. Moreover, the result from this study suggests that the stem canker causing fungi might be developing resistance against the fungicides labelled to manage foliar diseases of sunflower including fluxapyroxad (SDHI), pyraclostrobin (QoI), and tebuconazole (DMI). In addition, investigation into the genetic management aspect showed that diverse genes are expressed in sunflower genotypes in response to infection by stem canker pathogen (D. helianthi), which can help Phomopsis stem canker disease resistance breeding programs.

Number of Pages

293

Publisher

South Dakota State University

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Rights Statement

In Copyright