First report of Diaporthe ueckeri and Diaporthe longicolla as the causal agent of pod and grain rot of soybean in Brazil

Epidemics of pod and grain rot (PGR) of soybean (Glycine max (L.) Merr.), popularly referred to as "pod anomaly", have economically impacted Brazilian farmers, especially in Mato Grosso (MT), Brazil's largest producer state, where incidence varies from 10 to 40%. Although Diaporthe ueckeri was reported causing soybean seed decay in Santa Catarina state (SC), a disease similar to PGR, no pod inoculation was attempted and no rot symptoms reproduced. Here, we report Diaporthe ueckeri and D. longicolla as the causal agents of PGR. A total of 118 isolates were obtained from symptomatic grains, pods, and stems, across five Brazilian states other than SC: Mato Grosso (n = 62), Paraná (n = 20), Minas Gerais (n = 19), Goiás (n = 14), and Rio Grande do Sul (n = 3) during the 2022/23 and 2023/24 seasons. Infected grains and pods usually had necrotic lesions and were poorly developed. Symptomatic plant parts (stems, pods, and grains) were collected and maintained under high relative humidity conditions in the laboratory. Pycnidia or cirri were transferred from infected tissues onto PDA at 25 °C and monoconidial isolates were obtained, thereafter. Colonies were light gray with a dense and fluffy appearance. Pycnidia were erumpent, with elongated necks and cirri were observed. Alpha conidia were aseptate, hyaline, and biguttulate, measuring 6.6 to 8.6 μm length × 1.6 to 2.2 μm width, similar to those of Diaporthe spp. (Udayanga et al. 2015). Isolates were considered pathogenic when depressed necrotic lesions with or without pycnidia were formed after inoculation of 'Williams 82' at the V2 growth stage by the toothpick method which consisted of the insertion of a toothpick colonized by the fungus 1 cm below the cotyledonary node. Insertion of a non-infested, sterilized toothpick, was used as control. Partial sequences of the internal transcribed spacer (ITS) (White et al. 1990), translation elongation factor 1-α (TEF) (Carbone and Kohn 1999), calmodulin (CAL) (Carbone and Kohn 1999), and beta-tubulin (TUB) (Glass and Donaldson 1995) were amplified. GenBank accessions codes for UFVDUS-20 and UFVDLS-67 are ITS: PQ331841 and PQ331842; TEF: PQ37542 and PQ375427; CAL: PQ375426 and PQ375428; TUB: PQ375429 and PQ375430, respectively. Sequences of TEF of UFVDUS-20 and UFVDLS-67 were 100% similar to D. ueckeri strain 17-DIA-079 (MK941298.1) and D. longicolla D15.1 (MN584786.1), respectively. Bayesian inferences with the concatenated dataset grouped UFVDUS-20 and UFVDLS-67 with the reference strains of D. ueckeri or D. longicolla, respectively. Isolates UFVDUS-20 (D. ueckeri from MT) and UFVDLS-67 (D. longicolla from Goiás) were used for inoculation of soybean plants and detached pods with a 1×106 conidia/mL suspension. Sterile distilled water with Tween was used as control. Five pots (replicates) each with two 'L93-3312' plants at the V4 and R5 stages were used for inoculation until the runoff point and incubated at 28 °C, > 95% RH, and 12h-photoperiod. Plants were assessed until R8, and the experiment was conducted twice. Pod rot symptoms and pycnidia were observed in plants inoculated in both phenological stages. Moreover, detached pods at the R5 stage of 'L93-3312' were submerged in the conidial suspension while those of 'BMX Olimpo IPRO' were drop-inoculated with 10 μl of the conidial suspension. The treatments (immersion or drop-inoculation) were applied to 10 replicates, each composed of a humid chamber (11 x 11 x 3 cm plastic box) containing 5 pods. Rot symptoms with erumpent pycnidia with cirri were visible in all inoculated pods after seven days. Fungal colonies were reisolated, and identity was confirmed by morphological and molecular analysis. This is the first report fulfilling Koch's postulates, unequivocally establishing D. ueckeri and D. longicolla as the causal agents of soybean PGR. This establishes the correct etiology of PGR in Brazil, providing valuable information for disease management strategies.