First Report of Clonostachys rhizophaga Causing Root Rot on Lentil in France and Cross-Pathogenicity on Pea.

Root rot affects legumes such as lentil (Lens culinaris subsp. culinaris Medik.) and pea (Pisum sativum L.) (Chatterton et al. 2019). In France, legume root rot occurs in 65% of cultivated areas and cause up to 60% yield loss (Augagneur et al. 2021; Moussart el al. 2011). Soil was sampled from plots where root rot disease was previously observed. Samplings were conducted in April 2019 from two plots in central France (46.59 N, 2.05 E; 46.57 N, 2.3 E). Pre-germinated lentil (cv. Anicia) seeds were transplanted in both soils and symptomatic plants were collected after two months. They showed yellowing and wilting symptoms associated with necrotic spots on the root system. The light brown color on the roots turned to dark brown as the necrotic spots progress. Diseased root fragments were surface-disinfected by soaking for 30 s in 70% ethanol, rinsed with sterile water and placed on malt extract agar (MEA). Fungal colonies growing from the root fragments were purified by single-spore sub-culturing and preserved in the Microorganisms of Interest for Agriculture and Environment (MIAE) collection (INRAE Dijon, France). They were transferred to potato dextrose agar and carnation leaf agar for macroscopic and microscopic observations. The colonies developed a dense and cottony aerial mycelium, pearly in color with pink reflections (Fig. S1). From below, the colonies were orange-pink. Under the microscope, Verticillium-like conidiophores arising on the same whorl released asymmetrical ovoid single-celled conidia, from 4 to 7 µm long and 2.5 to 3 µm wide, similar to the description of Clonostachys rhizophaga by Schroers (2001). Isolates MIAE08192 and MIAE08209 were identified as Clonostachys sp. based on their internal transcribed spacer (ITS) sequences (GenBank accessions OR902030 and OR902031). They were further identified based on their sequence of the translation elongation factor 1-α gene (TEF-1α) (Moreira et al. 2016) (GenBank accessions OR947648 and OR947649). A BLAST search identified the two isolates as C. rhizophaga based on 100% identity of their sequences to the published sequence KX184992 (382 bp out of 382 bp and 377 bp out of 377 bp, respectively). A phylogenetic analysis combining ITS and TEF-1α sequences confirmed that the two isolates clustered within the C. rhizophaga species (Fig. S2). Two strains MIAE07881 and MIAE07884 of C. rhizophaga have been reported to cause root rot on pea (Gibert et al. 2022). Due to equivalent symptoms on lentil and pea, and the fact that the strains belonged to the same taxon led to test cross-pathogenicity on the two plant species with the two strains collected from lentil and the two strains collected from pea. Surface-disinfected lentil (cv. Anicia) and pea (cv. Firenza) seeds were placed on MEA, and after 72 h, the germinated seeds were transferred into sterile glass tubes containing 30 mL of Hoagland's No. 2 basal salt mixture at 1.6 g.L-1 added with agar 8 g.L-1. Three days later, they were inoculated with 1 mL of a conidial suspension at 105 conidia.mL-1 or sterile deionized water for the control plantlets. Twelve tubes per strain and twelve negative control tubes were prepared. The tubes were incubated in a growth chamber at 22°C day/18°C night and 12 h light for four weeks. The bioassay was performed twice. The control plants were asymptomatic. Whatever their origin, the four isolates inoculated on lentil or pea caused necrotic areas on 100% of the plants leading to strongly degraded root system. Koch's postulates were verified by reisolating the inoculated fungi from one symptomatic plant among the twelve replicates. Our results confirm the absence of specificity of this interaction and suggests the possible pathogenicity of C. rhizophaga on other legumes (Abang et al. 2009). To our knowledge, this is the first report of C. rhizophaga as a causal agent of root rot on lentil and of its cross-pathogenicity on lentil and pea.