First report of <i>Clonostachys rhizophaga</i> causing root rot of soybeans in North Dakota

Root rot occurrence has been a growing concern among soybean farmers. In 2023, a fungal isolate was collected from a soybean root symptomatic of root rot from North Dakota. The isolate was cultured in PDA, and the morphology was observed. The colony morphology and microscopic features closely matched descriptions of Clonostachys rhizophaga. For confirmation of fungal identity using molecular techniques, DNA was extracted from the pure fungal culture and its subcultures. Multiple genomic regions were used for PCR. Sequencing of the PCR products followed by BLAST analysis in the NCBI nucleotide database and alignment to the C. rhizophaga reference genome showed high similarity for all loci. Phylogenetic analysis based on beta-tubulin sequences placed the isolate within the C. rhizophaga clade. Among all loci analysed, the beta-tubulin gene provided the highest species-level resolution, clearly distinguishing C. rhizophaga from other Clonostachys taxa. The molecular evidence confirmed the isolate as C. rhizophaga. Pathogenicity tests were conducted by inoculating the soybean cultivar Barnes. Infected plants showed symptoms of taproot and lateral root necrosis, accompanied by root discoloration in two experiments. Fungal DNA reisolated from symptomatic roots, followed by PCR and sequencing confirmed the isolate as C. rhizophaga. To our knowledge, this is the first report of C. rhizophaga infecting soybean in North Dakota, representing the first record of C. rhizophaga infecting soybean in the United States.