First report on molecular confirmation of Nothopassalora personata causing late leaf spot of groundnut in India

Groundnut (Arachis hypogaea L.) is an important oilseed crop cultivated on approximately 5.8 million hectares, producing 10.4 million tonnes with an average productivity of 1.8 tonnes per hectare in India (GOI, Department of Agriculture, Cooperation and Farmers Welfare, 2025). Late leaf spot (LLS) of groundnut has been reported in India for several decades based on symptomatology and morphological characteristics. The causal agent has undergone several taxonomic revisions and has been successively classified under the genera Mycosphaerella, Cercosporidium/Cercospora, and Phaeoisariopsis, in earlier literature (Jenkins, 1938; McDonald et al., 1985 and Subrahmanyam et al., 1995) before being reclassified as Nothopassalora personata based on molecular phylogenetic evidence. Therefore, molecular confirmation, together with morphological characterization, has become essential for accurate identification and consistent diagnosis of the pathogen across groundnut-growing regions. During a July 2024 survey of a groundnut field at ICRISAT (17.5115°N, 78.2751°E), Patancheru, Hyderabad, Telangana, India, severe small, circular to irregular dark brown to black spots were observed mainly on the abaxial surfaces of older leaves, occasionally with a yellow halo on the TMV-2 variety, consistent with late leaf spot symptoms. The severity of the disease was assessed using the scale developed by Subramanyam et al. (1995) and reached up to 60%. Severely infected leaves turned yellow, desiccated, and dropped prematurely, resulting in complete defoliation. Similar late leaf spot symptoms have been reported from Iran (Bakhshi and Zare,2020) and Argentina (Monguillot et al.,2023), where the pathogen was confirmed through molecular characterization. The detached leaf assay was performed to confirm the pathogenicity (Deshmukh et al., 2018). Conidia were collected freshly from field-infected leaves using ROCKYVAC™ oil-free piston vacuum pump (Tarsons Products Ltd., Kolkata, India). A suspension of 3 x 107 spores/ml was prepared by suspending the spores in distilled water and used for inoculation on the leaf surface, and placed in a growth chamber at 25 ± 1℃ for 30 days. The leaves developed symptoms at 15-20 days after inoculation and pathogen was confirmed based on symptomology and microscopic observation of conidia. Conidia were elongate, cylindrical to slightly obclavate (club-shaped), hyaline with 3-7 septa, with 43.8-47.5 X 4.8-5.6 µm in size. The molecular confirmation of the pathogen was performed using the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA and the partial RNA polymerase II second largest subunit (rpb2) gene. Total DNA was extracted from conidia collected from fresh, naturally infected field-grown groundnut leaves using the DNeasy Plant Mini Kit (QIAGEN Hilden, Germany) according to the manufacturer's instructions. PCR amplification was performed in a thermocycler (Eppendorf-Vapo.Protect) using ITS1 and ITS4 primers for amplification of the ITS region and fRPB2-5F and fRPB2-7cR primers (IDT, Singapore) for amplification of the partial rpb2 region and the obtained amplicons were purified using PCR clean-up kit (Duren, Germany) as per the manufacturer’s protocol and sequenced by sanger’s dideoxy chain-termination method. The ITS and rpb2 sequences were edited in BioEdit (https://bioedit.software.informer.com/7.2/) and aligned in CLUSTALW, and the phylogenetic tree of both regions was constructed in MEGA 11 software (Tamura et al., 2021) by using the neighbor-joining method. The sequence was deposited in NCBI (rpb2 accession number PZ495589) and (ITS accession number PZ809112). Based on the symptoms, morphology, pathogenicity test and rpb2 gene sequence, the causal organism was identified as Nothopassalora personata, the pathogen causing late leaf spot of groundnut. To our knowledge, this is the first report of molecular confirmation of Nothopassalora personata causing late leaf spot of groundnut in India. These molecular baseline data will support future late leaf spot resistance screening and fungicide efficacy trials in Indian groundnut-growing regions.