First Report and Molecular Characterization of Phytophthora megasperma Causing Root Rot on Alfalfa in Waterlogged Fields

Abstract

Severe root rot symptoms characterized by root browning, cortical decay, plant stunting, chlorosis, and progressive stand decline were observed in alfalfa (Medicago sativa) fields subjected to waterlogged soil conditions, leading to substantial forage yield losses. Diseased plants collected from affected fields were used for pathogen isolation and characterization through morphological, pathogenicity, and molecular approaches. Oomycete isolates consistently produced cottony white colonies with coenocytic hyphae and abundant sporangia typical of Phytophthora megasperma. Considerable variation in colony growth rate and sporangial morphology was observed among isolates obtained from different field locations. Pathogenicity tests conducted on healthy alfalfa seedlings reproduced characteristic root rot symptoms, including root necrosis, reduced root length, and plant collapse, whereas control plants remained symptom-free. Re-isolation of the pathogen from inoculated tissues fulfilled Koch’s postulates, confirming its causal role. Molecular characterization was performed using amplification and sequencing of internal transcribed spacer (ITS) regions and additional conserved loci relevant to oomycete phylogeny. Sequence analysis revealed high similarity with authenticated P. megasperma isolates available in international databases. Phylogenetic analysis grouped field isolates within the P. megasperma clade, confirming species identity and indicating limited genetic variation among regional populations. Waterlogged field conditions significantly favored zoospore production, motility, and root infection, thereby accelerating disease development and spread. Continuous saturation of soil promoted pathogen survival and increased inoculum density in the rhizosphere. The study documents the first confirmed occurrence of P. megasperma causing root rot on alfalfa in the surveyed regions. The integration of morphological and molecular tools provided accurate identification and improved understanding of pathogen epidemiology.