First Report of Globisporangium heterothallicum Causing Seedling Disease on Guayule in Arizona.

Guayule (Parthenium argentatum A. Gray) is known for producing low-allergenic latex that is used in high end rubber products for medical use such as rubber gloves, catheters, and condoms. Currently, there are growing efforts from tire industry to commercialize guayule for rubber production in Arizona. During May 2019, wilting and death of c. 25% of seedling plants were observed in direct-seeded guayule fields in central Arizona. Symptoms of root rots and hypocotyl constriction were observed on affected seedling plants. To identify the causal agent, four symptomatic plants were collected to isolate the putative pathogen. Small pieces of symptomatic root (2-5 mm) were surface sterilized in 0.6% sodium hypochlorite for 1 min, rinsed copiously in sterile distilled water, blotted dry, and plated on 10% clarified V8-PARP (Jeffers and Martin 1986). Four oomycete-like isolates with abundant hyphal swellings were purified by transferring tips of single hypha onto new 20% CV8 plates and incubating at 23°C for one week. Sporangia were formed abundantly, globose or lemon-shaped (average 20 ± 4 × 20 ± 4 µm, n = 15). Isolates did not produce oospores (heterothallic). Genomic DNA was extracted from the mycelia of two isolates using DNeasy Plant Pro Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. The internal transcribed spacer (ITS) region of rDNA and mitochondrially encoded cytochrome c oxidase 1 (cox 1) gene were amplified with primers ITS1/ITS4 (White et al., 1990) and OomCoxI-Levup/OomCoxI-Levlo (Martin and Tooley, 2003; Robideau et al., 2011) and the resulting amplicons were sequenced (GenBank Accession No. OL514636 and OL539842). A BLASTn search of 808-bp amplicon (OL514636) revealed 100% match with ITS sequences MT039880 which was G. heterothallicum causing root and crown rot of pepper in Turkey. BLAST analysis of the 658-bp amplicon (OL539842) showed 99.39 % identity with the COX 1 sequence of G. heterothallicum from tomato in Australia (MT981128). To fulfill Koch's postulates, pathogenicity tests were conducted twice on 2-week-old 'Az 2' guayule plants grown in 1.9-liter pots filled with a steam-disinfested potting mix. Pots were placed in a plastic container and watered three times a week by flooding, to create waterlogged conditions. Plants were maintained in a greenhouse and fertilized weekly with a 20-20-20 fertilizer at 1mg/ml. Twenty plants in 5 pots (4 plants/pot) were challenged with a G. heterothallicum isolate by drenching pot with 50 ml of a 1×106 zoospore/ml suspension. Twenty plants in 5 pots, serving as a control, received each 50 ml of distilled water. Symptoms of wilting and water-soaked root rot, and plant death were observed 2 weeks afterward, whereas control plants remained asymptomatic. G. heterothallicum was reisolated from necrotic roots of inoculated plants but not from control plants. G. heterothallicum has been increasingly reported as a pathogen of damping-off or root and crown rot on hosts such as alfalfa in Minnesota (Berg et al., 2017), soybean in Pennsylvania (Coffua et al., 2016), spinach in Sweden (Larsson, 1994), corn in China (Gan, et al., 2010), pepper in Turkey (Dervis, et al., 2020). To our knowledge, this is the first report of G. heterothallicum causing guayule seedling diseases in the United States. The presence of broad-host-range pathogen G. heterothallicum suggests that new strategies are needed for managing this pathogen to increase stands in direct-seeded guayule production system.