Quantitative PCR Assays Developed for Diaporthe helianthi and Diaporthe gulyae for Phomopsis Stem Canker Diagnosis and Germplasm Screening in Sunflower (Helianthus annuus).

Phomopsis stem canker of sunflower is caused by two fungal pathogens, Diaporthe helianthi and Diaporthe gulyae, in the United States. In this study, two quantitative PCR (qPCR) assays were developed to detect and quantify D. helianthi and D. gulyae in sunflower. The two assays differentiated the two fungi from each other, other species of the genus Diaporthe, and pathogens, and they have high efficiency (>90%). The qPCR assays detected the two pathogens on plant samples exhibiting Phomopsis stem canker symptoms sampled from commercial sunflower fields in Minnesota, Nebraska, North Dakota, and South Dakota. Furthermore, the assays were used to screen cultivated sunflower accessions for resistance to D. helianthi and D. gulyae. The disease severity index (DSI) of the accessions significantly correlated (P < 0.0001) with the amount of pathogen DNA from the qPCR assays. The qPCR assays identified PI664232 and PI561918 to be significantly less susceptible (P ≤ 0.05) to D. helianthi and D. gulyae, respectively, when compared with the susceptible check cultivar HA 288, and this was in agreement with the DSI. These results suggest that the qPCR assays for D. helianthi and D. gulyae can be used as a reliable tool to diagnose Phomopsis stem canker and screen sunflower germplasm for disease resistance.

Inoculation Method Impacts Symptom Development Associated with Diaporthe aspalathi, D. caulivora, and D. longicolla on Soybean (Glycine max).

One hundred fifty-two Diaporthe isolates were recovered from symptomatic soybean (Glycine max) stems sampled from the U.S. states of Iowa, Indiana, Kentucky, Michigan, and South Dakota. Using morphology and DNA sequencing, isolates were identified as D. aspalathi (8.6%), D. caulivora (24.3%), and D. longicolla (67.1%). Aggressiveness of five isolates each of the three pathogens was studied on cultivars Hawkeye (D. caulivora and D. longicolla) and Bragg (D. aspalathi) using toothpick, stem-wound, mycelium contact, and spore injection inoculation methods in the greenhouse. For D. aspalathi, methods significantly affected disease severity (P < 0.001) and pathogen recovery (P < 0.001). The relative treatment effects (RTE) of stem-wound and toothpick methods were significantly greater than for the other methods. For D. caulivora and D. longicolla, a significant isolate × method interaction affected disease severity (P < 0.05) and pathogen recovery (P < 0.001). Significant differences in RTEs were observed among D. caulivora and D. longicolla isolates only when the stem-wound and toothpick methods were used. Our study has determined that the stem-wound and toothpick methods are reliable to evaluate the three pathogens; however, the significant isolate × method interactions for D. caulivora and D. longicolla indicate that multiple isolates should also be considered for future pathogenicity studies.