First Report of Rhizopus arrhizus (syn. R. oryzae) causing Sunflower Head Rot in Arizona, USA

Sunflower (Helianthus annuus) is an ornamental, edible-seed, and important oil source plant in the USA. In June 2023, head rot was observed in sunflowers grown in an experimental field at Yuma County Cooperative Extension, University of Arizona, AZ (32°42'35.5"N, 114°42'25.0"W). The disease incidence was of >70%. Head lesions were dark-brown and extended through the head to the bracts and stem. White to gray mycelia and black sporangia-like structures were also observed on sunflower heads. Symptomatic plants (n =10) were sampled to determine the disease causing agent. Five symptomatic tissues for each plant (0.5 to 1 cm) were submitted to surface sterilization by dipping in 75% ethanol 2min, 1% NaOCl for 2 mins and rinsing with sterile water. Once sterilized, the tissues were plated on potato dextrose agar (PDA) plates and incubated at 25±0.2 °C. After two days, hyaline mycelia were observed on PDA which turned white after 7 days. A total of 50 isolates were obtained, of which ten were randomly selected and purified by the hyphal-tip method and later used for morphological analysis. Microscopic observations revealed hyaline and aseptate hyphae, sporangiophores measuring 900 to 1.2000 µm in length and dark-brown sporangium (72 to 144 µm, mean = 90). The columella was sub-globose, and the sporangiospores ranged from 7.29 to 9.37 µm in size (mean = 7.5 µm). The morphological characteristics described above were similar for the ten isolates and were in accordance with the species R. arrhizus as described by (Gryganskyi et al. 2018). Genomic DNA was extracted from three randomly chosen isolates using The DNeasy Plant kit (Qiagen) and used for further molecular identification. The internal transcribed spacer (ITS) region was amplified using ITS1/ITS4 primers (White et al. 1990) and then Sanger sequenced. The sequences shared 100% nucleotide identity with each other (GenBank accession numbers PP747852, PP747853 and PP747854) and shared 100% identity to R. arrhizus GenBank accessions (MT316366.1, MN547407.1). One isolate, YPHC-94-A, was randomly selected for Phylogenetics and Pathogenicity analyses. Phylogenetics analysis based on sequence data of ITS showed that the isolated YPHC-94-A clustered together with R. arrhizus species (Zhang. 2023). Pathogenicity test was conducted by inoculating four sunflower varieties (American giant, Lemon queen, Solar eclipse and Mammoth) (n = 12 for each variety). Plant heads were inoculated with a disc of mycelia (0.5 cm2) and incubated for 24 h at 30 ±2 °C and 94% RH. Five uninoculated plants of each variety were used as controls. Head rot symptoms were observed within 3-5 days on inoculated plant post inoculation depending on the variety, whereas the control plants stayed asymptomatic. R. arrhizus was re-isolated from all the inoculated plants and was morphologically and molecularly identical to the field isolates, thus fulfilling Koch’s postulates. R. arrhizus has already been reported in different US regions (Sanogo et al. 2010), however, to the best of our knowledge this is the first report in Arizona. Due the high disease incidence and pathogen aggressiveness found in the environmental conditions of the U.S southwest desert, we consider sunflower Head rot a potential risk for sunflower production in Arizona as well as the large population of wild sunflowers in the State.