Black gram (Vigna mungo L.) is an important pulse crop cultivated widely in India for its high protein content and role in improving soil fertility through biological nitrogen fixation (Anonymous, 2022). It serves as a major source of dietary protein for a large section of the population and contributes significantly to sustainable cropping systems, particularly in rainfed and semi-arid regions. Despite its agronomic and nutritional importance, the productivity of black gram remains low due to several biotic and abiotic constraints, among which soil-borne diseases pose a serious threat to crop establishment and yield stability.
       
Dry root rot, caused by the necrotrophic fungus Macrophomina phaseolina (Tassi) Goid. is one of the most devastating soil-borne diseases affecting black gram and other pulse crops (Elmerich et al., 2022). The pathogen infects about 500 plant species and is commonly found in tropical and subtropical locations around the world. Macrophomina phaseolina survives in soil and crop residues primarily in the form of microsclerotia, which can persist for extended periods in the absence of a host. These survival structures enable the pathogen to withstand adverse environmental conditions and contribute to the recurrence of the disease in successive cropping seasons (Pamala et al., 2023). The survival and persistence of M. phaseolina in soil through microsclerotia have been documented by earlier studies (Dhingra and Sinclair, 1978; Songa and Hillocks, 1998; Su et al., 2001). Infected plants often exhibit bark shredding, wilting, yellowing of the leaves and subsequent drying of the entire plant. The root system becomes dry, brittle and discoloured, with cortical shredding, resulting in premature plant mortality. High temperatures, moisture stress and poor soil conditions, all of which are common in many pulse-growing areas of India (Yamunarani et al., 2023) often make the disease worse.
       
Field surveys play a crucial role in determining the distribution, incidence, and severity of plant diseases and provide essential baseline information for designing effective disease management strategies. Several studies have reported variable incidence of dry root rot of black gram across different agro-climatic regions, influenced by factors such as soil type, cropping system, cultivar susceptibility, and environmental conditions (Patel et al., 2023). Surveys conducted in black gram-growing regions have also reported variation in dry root rot incidence caused by M. phaseolina (Israel and Prakash, 2013). Region-specific data on the occurrence and pathogenic behaviour of M. phaseolina in black gram-growing areas of Tamil Nadu remain limited. The management of dry root rot is particularly challenging due to the soil-borne nature of the pathogen, its broad host range, and the limited availability of resistant cultivars. The broad host range and complex epidemiology of M. phaseolina require integrated disease management strategies for effective control (Sinclair, 1982; Gupta et al., 2012). Chemical control options are often ineffective under field conditions, emphasizing the need for accurate identification of the pathogen and an understanding of its pathogenic variability. Host resistance evaluation and eco-friendly management approaches using bio-agents and organic amendments have been studied for reducing M. phaseolina infection in pulse crops (Kumar et al., 2024).
       
Isolation and characterization of the causal pathogen are essential for confirming disease etiology and understanding variability among pathogen populations. Morphological and cultural characterization, including colony growth pattern, pigmentation and microsclerotia formation, have traditionally been used for the identification of M. phaseolina (Patel et al., 2023). Morphological and cultural variability among M. phaseolina isolates has been reported from different geographical regions (Raut and Nimbalkar, 2017). In addition, pathogenicity testing is critical for establishing the virulence of isolates and fulfilling Koch’s postulates. Sorghum seed-based inoculum is commonly employed for pathogenicity assays under controlled conditions, as it provides uniform and reliable infection. Molecular characterization using DNA-based techniques has further strengthened the accurate identification of M. phaseolina and has revealed genetic diversity among isolates from different hosts and geographical regions. PCR-based detection methods and ITS-based molecular approaches have been successfully used for accurate identification and characterization of fungal pathogens (Babu et al., 2007). Such variability in pathogenicity and genetic makeup has significant implications for disease management, resistance breeding and epidemiological studies (Manjunatha and Saifulla, 2023). Variations in morphological, genetic and pathogenic characteristics among M. phaseolina isolates have been reported from different hosts (Islam and Ali, 2011; Kaur et al., 2010; Kaur and Chauhan, 2012).
       
In this context, the present investigation was undertaken to assess the incidence and distribution of dry root rot of black gram in selected districts of Tamil Nadu, isolation and characterization of M. phaseolina associated with the disease and evaluation of pathogenicity of different isolates under controlled conditions. Particular emphasis was placed on identifying virulent isolates to support the molecular characterization.

Survey and assessment of dry root rot incidence in black gram
 
The present investigation was carried out at SRM College of Agricultural Sciences, SRM Institute of Science and Technology, Baburayanpettai, Chengalpattu District, Tamil Nadu, India. A roving survey was conducted from September to November 2025 to assess the incidence of dry root rot of black gram in four major black gram-growing districts of Tamil Nadu following the survey method described by Kamala and Kennedy (2021). In each district, representative fields were selected at random. Disease incidence was recorded by examining plants along a zig-zag (W-shaped) transect in each field. A minimum of 100 plants per field was observed for typical dry root rot symptoms such as sudden wilting, yellowing of foliage, drying of plants without leaf drop and dry, brittle roots with shredding of the cortical tissues.
 
Disease incidence was calculated using the following formula:

 
The incidence data were summarized district-wise to understand the distribution and prevalence of dry root rot in the surveyed regions.
 
Collection of diseased samples
 
Black gram plants showing typical symptoms of dry root rot were carefully uprooted from the surveyed fields. The root systems were gently cleaned to remove adhering soil particles and then packed in sterile paper bags. Each sample was properly labelled with details such as location, district and date of collection. The samples were transported to the laboratory and processed for pathogen isolation within 24 hours of collection.
 
Isolation and purification of Macrophomina phaseolina
 
Isolation of the pathogen was done by using the tissue segment method followed by Meena et al. (2020). Diseased root portions were washed thoroughly under running tap water and chopped into small pieces (approximately five mm in length) from the junction of healthy and diseased tissues. The root segments were surface sterilized with 70% ethanol for one minute and then rinses three times in sterile distilled water. The sterilized tissues were blotted dry with sterile filter paper and aseptically placed on potato dextrose agar (PDA) plates.
       
The inoculated plates were incubated at 25±2°C under dark conditions and monitored daily for fungal growth. Hyphal tips originating from the tissue segments were transferred to fresh PDA plates to obtain pure cultures. A total of five fungal isolates were obtained from various locations and maintained on PDA slants at 4°C for future research.
 
Morphological and cultural characterization
 
The purified isolates were initially identified based on cultural and morphological characteristics following the description by Sarr et al., (2014). Each isolate was inoculated at the centre of PDA plates using a  five mm mycelial disc and incubated at 25±2°C. Colony characteristics such as colour, texture, growth pattern and rate of radial growth was recorded at regular intervals. Microscopic observations were made by mounting small portions of mycelium in lactophenol cotton blue and examining under a compound microscope to observe hyphal characteristics and microsclerotia formation.
 
Molecular characterization of M. phaseolina
 
The CTAB method was used to extract genomic DNA from the representative isolate (VPM) of actively growing mycelium. The internal transcribed spacer (ITS) region of ribosomal DNA was amplified by universal primers ITS1 and ITS4 (Schoch et al., 2012). PCR amplification was performed in a thermal cycler under standard conditions. The amplified PCR products were visualized and purified by agarose gel electrophoresis and the sequencing was carried out at PAR Laboratory, Tiruchirappalli, Tamil Nadu, India. The acquired ITS sequence was compared with sequences available in the NCBI database using BLAST analysis. The sequence of the isolate was deposited in the GenBank database with accession number PZ054021.1.
 
Pathogenicity test of M. phaseolina
 
Preparation of sorghum seed inoculum
 
Sorghum seeds were soaked overnight in water then drained and transferred into 250 mL conical flasks at the rate of 100 g per flask and sterilized at 121°C for 20 minutes on two consecutive days. After cooling, each flask was inoculated with actively growing mycelial discs of M. phaseolina and incubated at 25±2°C for 10-12 days, with recurrent shaking to ensure uniform colonization of the seeds (Kapadiya et al., 2024).
 
Pot culture experiment
 
The pathogenicity of five isolates of M. phaseolina was tested under pot culture conditions using a Completely Randomized Design (CRD) with five replications per isolate including control. Earthen pots were filled with sterilized potting mixture (soil: sand: FYM in the ratio of 2:1:1). In each pot 20 healthy blackgram seeds were sown in sterilized soil without the addition of pathogen inoculum. The pots were maintained under natural environment conditions and irrigated as required (Kumar et al., 2024). Sorghum seed inoculum of M. phaseolina was mixed thoroughly with the potting mixture at 5% (w/w) at 20th day after sowing.
 
Disease assessment and re-isolation
 
Plants were observed daily for the development of disease symptoms and disease incidence was recorded from 10 to 20 days after inoculation of pathogen. Disease incidence was calculated as the percentage of plants showing typical dry root rot symptoms in each pot.
       
The pathogen was re-isolated from infected plants of all isolates and its identity was confirmed based on cultural and morphological characteristics, thereby fulfilling Koch’s postulates (Patel et al., 2023).

Incidence of black gram dry root rot in surveyed districts
 
The roving survey conducted during September to November 2025 revealed the widespread occurrence of dry root rot of black gram in all the surveyed locations across four districts of Tamil Nadu. The disease was recorded in five locations of the districts viz., Villupuram, Cuddalore, Kallakurichi and Chengalpattu district (two locations). Typical symptoms of root rot were observed in the field included sudden wilting, yellowing of foliage, drying of plants and dry, brittle roots with shredding of cortical tissues.
       
Disease incidence varied among the surveyed locations. In Chengalpattu district, disease incidence ranged from 13.33% to 21.33% across two locations. An incidence of 14.00% was recorded in Kallakurichi district, whereas Cuddalore district recorded the highest disease incidence (28.00%) among all surveyed locations. In Villupuram district, dry root rot incidence was recorded as 20.00%. The variation in disease incidence indicated the differences in local environmental conditions and crop management practices. The presence of dry root rot in all surveyed districts confirmed the widespread distribution of the disease in black gram-growing areas of Tamil Nadu. The disease incidence recorded from different surveyed locations is presented in Table 1 and the field symptoms observed during the survey are illustrated in Fig 1 and 2.






 
Isolation and identification of M. phaseolina
 
Isolation of the pathogen from diseased root samples resulted in the recovery of five fungal isolates. All isolates produced fast-growing colonies on potato dextrose agar (PDA), which were initially grey to whitish and later turned dark grey to black. The cultures produced abundant microsclerotia, a characteristic feature of M. phaseolina. Based on cultural and morphological characteristics, all five isolates were tentatively identified as M. phaseolina.
 
Cultural and radial growth characteristics of M. phaseolina isolates
 
The five isolates of M. phaseolina showed variation in colony growth rate on PDA medium. Radial growth was recorded from 24 hours after inoculation (Day 2) continuously. All isolates exhibited rapid mycelial growth however, differences in growth rate based on days to cover the plate were observed among the isolates.
       
The isolate of Villupuram district (VPM) exhibited fast radial growth and covered the entire Petri plate (90 mm) on fifth day of inoculation followed by the isolates of Kallakurichi (KK), Chengalpattu (CGP1) and Cuddalore (CDE) and Chengalpattu (CGP2) isolates covered the plates on sixth day of inoculation.
       
Culture of all the isolates initially produced thin, hyaline to light grey mycelium with a cottony to slightly fluffy texture. With increased incubation time, the colonies became dense and greyish to dark grey, followed by profuse microsclerotia formation. The radial growth pattern of different isolates of M. phaseolina on PDA medium is presented in Table 2, while the cultural characteristics of the isolates are shown in Fig 3.




 
Pathogenic variability among M. phaseolina isolates
 
All five isolates of M. phaseolina were found to be pathogenic to black gram under pot culture conditions, producing typical dry root rot symptoms, such as poor seedling emergence, yellowing, wilting and dry, brittle root symptoms. No disease symptoms were observed in the uninoculated control plants.
 
Disease progression of dry root rot
 
Disease incidence was recorded at regular intervals from 10 to 20 days after inoculation (DAI) and the results are presented in Table 3. The disease symptoms were first observed at 10 DAI and increased progressively with time in all isolates. The pathogenic variability and symptom development caused by different isolates under pot culture conditions are illustrated in Fig 4.




       
Among the isolates, VPM showed rapid disease development and recorded the highest disease incidence (55.00%) at 20 DAI, indicating its high virulence. The isolates CDE (32.00%) and KK (26.00%) showed moderate disease incidence, whereas CGP-2 (23.00%) exhibited intermediate pathogenicity. The lowest disease incidence was observed in CGP-1 (19.00%).
       
To further validate the observed variation among isolates, the final disease incidence recorded at 20 days after inoculation (DAI) was subjected to statistical analysis. The data were analyzed using analysis of variance (ANOVA) under a completely randomized design (CRD) and the significance of differences among the isolates was determined using the critical difference (CD) at 5% level.
       
The pathogen was successfully re-isolated from the infected root tissues of inoculated plants showing typical dry root rot symptoms. The re-isolated cultures exhibited morphological and cultural characteristics similar to the original isolates of M. phaseolina. No pathogen was isolated from the uninoculated control plants. This confirmed the pathogenicity of the isolates and fulfilled Koch’s postulates.
       
Following the pathogenicity test, the isolate VPM was identified as the most virulent based on the severity of symptoms produced in the host plants. Hence, the isolate was selected for further morphological and molecular characterization. The observed pathogenic variability among isolates highlighted the existence of diverse pathogen populations in different agro-climatic regions. Similar differences in virulence and disease development among  M.phaseolina isolates have been reported in several crops (Khan, 2007; Sharma et al., 2010; Mengistu et al., 2011). Such variability has important implications for disease management.
       
Microscopic observation of the cultures revealed typical morphological characteristics of the M. phaseolina. The fungus produced hyaline, septate hyphae and numerous dark microsclerotia. The microsclerotia were spherical to irregular in shape and were distributed throughout the mycelial network. These structures serve as survival propagules of the pathogen. The microscopic characteristics of M. phaseolina including microsclerotia, hyphae and pycnidiospores are presented in Fig 5.



Molecular identification of M. phaseolina
 
The ITS rDNA region of the representative isolate VPM was successfully amplified using ITS1 and ITS4 primers. The PCR amplification produced a fragment of approximately 600 bp, confirming successful amplification of the ITS region. The amplified product was sequenced using Sanger sequencing at PAR Laboratory, Tiruchirappalli.
       
BLAST analysis of the obtained sequence revealed a high similarity with M. phaseolina sequences available in the NCBI GenBank database. The sequence of the present isolate was deposited in the GenBank database with the accession number PZ054021.1.
       
Phylogenetic analysis based on ITS sequences demonstrated that the isolate VPM clustered closely with other reference strains of M. phaseolina, confirming its molecular identity. PCR amplification of the ITS region and phylogenetic relationship of the isolate VPM with reference strains are presented in Fig 6 and 7, respectively.

The present investigation clearly demonstrated the widespread occurrence of dry root rot of black gram in major growing regions of Tamil Nadu, with disease incidence varying significantly across locations. The successful isolation and identification of M. phaseolina from infected samples confirmed its role as the causal agent of the disease. Morphological, cultural, and molecular characterization validated the identity of the pathogen and revealed noticeable variability among the isolates. Among the isolates studied, the Villupuram isolate (VPM) exhibited higher radial growth and maximum disease incidence under pot culture conditions, indicating its highly virulent nature.
       
Overall, the findings emphasize the need for region specific and integrated disease management strategies to effectively control dry root rot in black gram. Further studies focusing on host resistance and eco-friendly management approaches are essential for sustainable crop protection and improved productivity.

The present study was supported by SRM College of Agricultural Sciences, Baburayanpettai.  
 
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The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.