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

Endophyte, fungicide, meta analysis, Phomopsis stem canker, QoI, sunflower

Abstract

Phomopsis stem canker (PSC), caused by species of Diaporthe, is a major disease of sunflower (Helianthus annuus L.) in the U.S., which can cause yield loss over 40%. The disease prevalence increased since 2010 in the major sunflower producing U.S. states of Nebraska, Minnesota, North Dakota, and South Dakota. Previous studies on the use of foliar fungicides in managing Phomopsis stem canker indicated mixed results on their efficacy in disease reduction and yield gain. Additionally, we do not have sufficient information on asymptomatic colonization by endophytic Diaporthe on sunflower tissue and how foliar fungicide and weather parameters affect the prevalence and diversity of Diaporthe and other fungi. Therefore, these objectives were developed to (1) study the diversity of endophytic fungi and association of endophytic Diaporthe with weather variables; (2) assess the effect of QoI fungicide on endophytic fungal community in sunflower; and (3) assess the fungicides for management of PSC in sunflower and profitability of fungicide application. Field trials were conducted in Nebraska, North Dakota, and South Dakota from 2019 to 2021 to determine the presence of endophytic Diaporthe in asymptomatic sunflower plants at different growth stages. Sunflower hybrids that were susceptible to causal fungi of PSC were planted in eight large plots in each location. Plants were sampled at two to three weeks after planting at V2-V4 (two to four leaves) stage from each plot and repeated twice at every two to three weeks until flowering stage. A culture-dependent characterization method was used to identify endophytic fungal community. Putative fungal isolates were identified by morphological characteristics, sequencing of the internal transcribed spacer (ITS) genes or the translation elongation factor 1-alpha (EF1-𝛼) genes, and the quantitative polymerase chain reaction (qPCR) assays. A total of 2174 fungal isolates belonging to 17 genera were recovered where Alternaria had the highest frequency of isolation (57.7%). Relative importance for Fusarium and Diaporthe were 40.5% and 6.31%, respectively. Endophytic Diaporthe (D. helianthi, D. gulyae, D. longicolla and D. caulivora) was identified from V2 to flowering stage of sunflower. To identify the association of weather variables with the prevalence of endophytic Diaporthe, a binary logistic regression model was developed using weather variables (temperature, relative humidity, precipitation) as predictors and presence of endophytic Diaporthe as response variable. The number of precipitation days during 30 days prior to sampling showed significant association with the presence of endophytic Diaporthe in sunflower. Greenhouse experiments were conducted to assess the aggressiveness of two randomly selected endophytic isolates each of D. helianthi, D. gulyae, D. longicolla and D. caulivora on a susceptible sunflower hybrid along with a non-inoculated control. A single mycelial plug of freshly prepared cultures of the isolates was inoculated at internode of stem using the mycelial contact inoculation method and stem-wound inoculation method. PSC severity was scored using a 0-5 rating scale 20 days after inoculation (DAI). A significant isolate by inoculation method interaction was observed for D. helianthi (P = 0.03) and D. gulyae (P = 0.03). Isolate DIA-190 and DIA-205 of D. gulyae and isolate DIA-178 of D. helianthi had significantly lower relative treatments effect (RTE) in mycelium contact method than the stem wound method but isolate DIA-177 of D. helianthi did not differ in RTE between the inoculation methods. A significant effect of inoculation methods (P < 0.001) was observed to affect the RTE for D. longicolla and D. caulivora. The RTE for stem-wound method was significantly greater than that of the mycelial contact method for D. longicolla and D. caulivora. This indicated that the PSC symptom development is impacted by the inoculation methods. Re-isolation of D. helianthi and D. gulyae from stem tissue near inoculation point for both methods proved that the endophytic Diaporthe have potential to cause PSC under controlled conditions. However, re-isolation of D. longicolla (in both methods) and D. caulivora (in mycelium contact method) was not successful to complete the Koch’s postulate. During 2019 to 2021, field trials were conducted in Nebraska, North Dakota, and South Dakota to study the effect of a quinone outside inhibitor (QoI) fungicide on a diversity of endophytic fungi in sunflower. Trials were conducted in a randomized complete block design with four replications and the effect of pyraclostrobin, a QoI fungicide, was compared with non-treated control (NTC). The pyraclostrobin fungicide was sprayed at R1 (miniature floral bud initiation) growth stage of sunflower at an application rate of 438.5 ml/ha. Stem samples were collected two to three weeks after fungicide application and endophytic fungi were isolated by culture dependent method. Putative fungal isolates were identified by morphological characteristics, sequencing of ITS or EF1-𝛼 gene region and the qPCR assays. Alternaria was dominant in both Pyraclostrobin treatment and NTC. Overall relative importance (RI) for Diaporthe and Fusarium was 4.1% and 18.3% in NTC but 3.2% and 27.5% in pyraclostrobin application, respectively. Application of pyraclostrobin did not show significant effect on prevalence of endophytic Alternaria, Fusarium and Diaporthe in sunflower. Field trials were conducted from 2009 to 2020 in Minnesota, Nebraska, North Dakota, and South Dakota to evaluate the effect of foliar fungicides on PSC and yield of sunflower. A total of 49 location-years were analyzed using non-linear regression to determine the effect of PSC severity index (DSI) on yield where a four-parameter logistic model described the yield loss with increase in DSI. The model indicated 10% increase in DSI can cause yield reduction up to 440 kg ha-1. Further, meta-analysis was conducted to evaluate the effect of six fungicide groups against PSC, when compared with no-fungicide control (NTC). Effect sizes corresponding to DSI and yield (Cohen’s f or Hedges’s g) were expressed in standard deviation units between the fungicide and NTC treatments. Pooled Cohen’s f for DSI (f= -0.35) and yield (f= 0.35) indicated that fungicides had a significant effect on DSI reduction and yield gain compared to NTC (p

Number of Pages

174

Publisher

South Dakota State University

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Rights Statement

In Copyright