First Report of Catenaria anguillulae Infecting Rotylenchulus renifonnis and Heterodera glycines in Alabama

Rotylenchulus reniformis and Heterodera glycines are plant pathogenic nematodes that affect a wide variety of commercially grown crops worldwide. Infection of vermiform life stages of R. reniformis and H. glycines was first observed in stock cultures of the nematodes at the Plant Science Research Center in Auburn, Alabama. Symptoms of the infection begin with sporangia forming inside the body of the nematodes. The sporangia produce zoospores that are released outside the body of the nematode through formation of an exit tube. This differs from the Catenaria auxiliaris reported by Castillo and Lawrence (2011); C. auxiliaris does not form an exit tube, but sporangia erupt through the cuticle to release zoospores. R. reniformis and H. glycines were extracted from stock cultures; infected vermiform nematodes were separated by hand, washed in sterile water, and placed onto 0.4% beef extract agar (BEA) and allowed to grow for 5 days. A sample of fungal mycelium was aseptically transferred from the 5-day-old cultures using a compound microscope to ensure fungal isolate purity, and transferred onto a new BEA plate. Cultures were then incubated at room temperature for 14 days to increase mycelium mass for molecular species identification. DNA was extracted and prepared for PCR using a ZR Fungal/Bacterial DNA MiniPrep kit. ITS1 and ITS4 primers were used to amplify the internal transcribed spacer (ITS) region and then sequenced by Eurofins Genomics (Louisville, KY). The results of sequencing were aligned using BioEdit, BLAST showed a 95% shared identity with C. anguillulae (KY606231 and KY606232). Koch’s postulates were performed through in vitro testing; infected nematodes were placed onto 0.4% BEA plates to grow fungal cultures that were morphology identified as C. anguillulae. Healthy, uninfected vermiform R. reniformis and H. glycines J2 nematodes were exposed to one C. anguillulae infected nematode of the respective species in 96 well plates. The plates were incubated at room temperature. The first infected nematodes were observed after 3 days, and colonized nematodes were evaluated by light microscopy to confirm C. anguillulae infestation. To confirm Koch’s postulates, the infected nematodes were cultured on BEA plates and the resulting fungal growth was morphologically identified as C. anguillulae. Greenhouse tests were also conducted by inoculating R. reniformis and H. glycines cultures growing on cotton and soybean with 2 ml of a C. anguillulae slurry. Three slurries of the C. anguillulae were used, which had OD600 values of 0.377, 0.566, or 0.754, to compare different rates of inoculation, and they were compared with two chemical controls (Poncho/VOTiVO and ILeVO applied as seed treatments) and an untreated control. This test contained five replications and was arranged in an RCBD; data were analyzed in SAS 9.4 using PROC GLIMMIX and means were compared using the Tukey-Kramer’s method with a significance level of P ≤ 0.1. The C. anguillulae at any of the inoculation rates did not reduce R. reniformis eggs per gram of root compared with the untreated control. However, low to high inoculation rates of C. anguillulae significantly (P ≤ 0.1) reduced H. glycines cyst numbers by 44, 40, and 52%, respectively, compared with the control. Population reductions were similar to those induced by the Poncho/VOTiVO chemical control, which reduced cyst numbers by 54% as compared to the control. C. anguillulae has been observed in some Alabama soils and it is important to consider that biological interaction between nematodes, such as R. reniformis and H. glycines, and C. anguillulae may occur. © 2017, American Phytopathological Society. All rights reserved.