Cotton, often called the king of fibres, is a vital commercial crop that supplies natural fibre for the textile industry. India ranks first by area and second by production among the world’s cotton-growing countries. In India, cotton is grown on 114.47 lakh hectares, with production of 294.25 lakh bales and productivity of 436.99 kg/ha during 2024-25 (https://caionline.in). Several pests and diseases affect cotton from seedling to maturity, leading to yield loss and poor fibre quality. Cercospora leaf spot, caused by Cercospora gossypina, is one of the important leaf spot diseases of cotton. It impedes cotton production and productivity in the major cotton-growing states of South India. Symptoms include small, numerous reddish to brownish spots with a whitish centre. Cercospora leaf spot in cotton appears as concentric ring spots, similar to those of target spot and conidia appear as long, thin, whip-like structures with horizontal septation (Kelly, 2021). Disease severity of Cercospora leaf spot ranged from 20.37% in Raichur district, followed by 17.58% in Yadagir district and 14.65% in Koppal district of Karnataka (Indira et al., 2019). Cercospora is expected to cause 5-10% yield losses under severe conditions. It is important to study the cultural and morphological characters, as well as pathogenic diversity. Cercospora leaf spot occurs in all cotton-producing areas of the U.S. and when plants are under stress or in combination with Alternaria or Stemphylim leaf spots, yield and fibre quality reductions are recorded (Kelly, 2021).
       
The incidence of Cercospora leaf spot in cotton fields is slowly increasing. Cultural and morphological characteri-sation, together with pathogenic diversity, provide insights into the dynamics of the pathogen population and variability among isolates. Dida et al. (2019) studied the cultural and morphological characteristics of 52 Cercospora zea-maydis isolates from maize. Colony colour varied from grey, light brown, corn silk to white on the top and from grey, dark grey, brown to corn silk on the reverse, with round to irregular shape and complete or wavy, raised mycelium. Conidia were slightly curved or straight, with 4 to 6 septations.
       
Variability among pathogen populations is characterised by cultural, morphological and pathogenic differences among isolates. The present study was carried out with the following objectives: (1) collection of Cercospora leaf spot samples from major cotton-growing states in South India and pathogen isolation; (2) cultural and morphological variation among isolates; and (3) pathogenic variation and virulence diversity among isolates.

Collection of leaf spot samples and isolation of the pathogen
 
Cercospora leaf spot samples were collected during the 2021-22 and 2022-23 crop seasons by survey in South India. Fifty-five Cercospora leaf spot samples were collected from nine major cotton-growing districts of Karnataka, namely Dharwad, Haveri, Gadag, Belagavi, Raichur, Vijayapura, Yadagiri, Kalaburagi and Ballari. Four districts in Telangana were covered, with 15 samples collected from Ranga Reddy, Warangal, Nirmal and Adilabad. Ten leaf spot samples were collected from the Kurnool district Andhra Pradesh. Twenty leaf spot samples were collected from the Coimbatore, Annur and Kinathukadavu blocks of Coimbatore district, Tamil Nadu. The pathogen was isolated from diseased samples using the tissue segment method (Dhingra and Sinclair, 1995) on potato dextrose agar (PDA). Isolates were named according to the states of origin. The single-spore isolation method was used to purify the isolates. Pure cultures were stored in 80% glycerol for long-term storage and on PDA slants for short-term storage.
 
Cultural characterization of Cercospora isolates
 
Cultural characters such as colony colour, margin, growth pattern, mycelial characters, texture, conidial and conidiophore characters and conidial size were studied on PDA. Sporulation capacity was measured on a 15-day-old culture grown on PDA medium.
 
Morphological characterization of Cercospora isolates
 
Morphological characterisation was performed on fifteen-day-old Cercospora isolates cultured on PDA. Morphological characters, including colony shape, conidiophore charact- eristics and conidial size and shape, were examined on PDA. Conidial size was measured using a compound microscope. Thirty conidia were measured for size and the average was calculated for each isolate.
 
Species identification of Cercospora isolates through conidial characters
 
Morphological characteristics of conidia, such as length, breadth and shape, were used to identify the Cercospora isolates.
 
Pathogenicity and pathogenic diversity of Alternaria isolates
 
Pathogenicity and virulence characterisation of the isolates were carried out on a 25-day-old susceptible cotton cultivar, LRA5166. A spray inoculation method was used under glasshouse conditions. PDA broth cultures of C. gossypina were harvested separately, ground with sterile water and adjusted to a concentration of 2 × 10⁶ Cfu/ml. Mild pin-pricking of leaves was performed before inoculation and plants were covered with polybags for 24 hours after inoculation to maintain humidity and enhance infection. Three replicates were maintained for each isolate. After 10, 20 and 30 days of inoculation, PDI was calculated using the 0-4 disease rating scale according to Sheoraj (1988). Isolates were classified as Highly Virulent (> 40.01 PDI), Moderately Virulent (20.01 to 40.00 PDI) and Less Virulent (0.00 to 20.00 PDI) based on symptom expression during artificial inoculation.

Collection and isolation of Cercospora from leaf spot samples from the South Zone
 
A survey was conducted in cotton-growing states of South India and Cercospora leaf spot samples were collected. The pathogen was isolated on PDA. Previous researchers also isolated the Cercospora spp. on PDA (Zia Hassan et al., 2018; Dida et al., 2019; Mahapatra et al., 2023). Out of fifty-five Cercospora leaf spot samples collected from nine major cotton-growing districts of Karnataka during the survey, 29 Cercospora isolates were isolated on PDA from Karnataka. Seven isolates from Telangana, six from Andhra Pradesh and 9 from Tamil Nadu were isolated from the collected samples.
       
Different leaf spot diseases of cotton were recorded from 8.5-19.8 PDI, including Cercospora leaf spot, across six major cotton-growing districts of Tamil Nadu (Rajaswaminathan et al., 2021). Cercospora leaf spot disease incidence in cotton has been widely reported recently. The Cercospora leaf spot incidence ranged from 3.5 to 12.0 PDI among surveyed cotton fields in four states of South India (Sampathkumar et al., 2023).
 
Cultural and morphological characterisation of Cercospora isolates
 
Cultural and morphological characters of C. gossypina isolates were studied on PDA (Table 1). Several workers have studied the cultural and morphological characters of Cercospora species (Dida et al., 2019; Kumar et al., 2021).



In the present study, colony colour varied from ashy to dark grey or greyish black among the isolates. Most colonies were dark grey, followed by ashy grey and greyish black. 51 per cent of the colonies were recorded as dark grey, 31.4 per cent as ashy grey and 17.6 per cent as greyish black. All three colour types were observed across all four study states (Table 1). These results are consistent with those of others across different crops. Cercospora tezpurensis from chilli produced greyish colonies with a white margin and less sporulation on PDA (Meghvansi et al., 2013). Two types of colony margins were observed in this study: irregular and smooth. Ashy grey colonies recorded with a smooth margin; dark grey and greyish black colonies were observed with an irregular margin, irrespective of the place of collection. Mycelial characters recorded as raised for Ashy grey cultures, medium raised for dark grey cultures and flat to greyish black colonies. Fluffy mycelial growth was observed in raised and medium-raised cultures. Ashy grey colonies produced a villous texture with a smooth margin, dark grey cultures exhibited a cottony texture with an irregular margin and greyish black colonies produced a velvety texture with an irregular margin (Table 1). Colonies of Cercospora cf. flagellaris from melons were pale pinkish to light grey, with cottony aerial mycelium on PDA (Park et al., 2020). The colony colour of C. canescens isolated from mung bean varied from grey to light greyish white to brownish, with convex, fluffy growth, a smooth or zig-zag margin and a dense centre (Kumar et al., 2021). Cultural characters of C. canescens from mung bean varied from white, fluffy to less fluffy, cottony, pale cream colour, with red to radish brown margins (Mahapatra et al., 2023). Cultures of C. dispori isolated from Disporum spp. were olivaceous to pale olivaceous in colour, with an irregularly folded surface and an entire or slightly undulate white margin on PDA (Cho et al., 2025).
       
In the present study, short, unbranched, tufted, pale olivaceous brown, septate conidiophores were observed. They were mostly slightly curved, fasciculate and pale towards the apex. Conidia were thin-walled, hyaline and multi-septate. They were needle-shaped, slender and slightly curved, appearing cylindrical and truncated to subtruncate at the base. The tips of the conidia were straight or variously curved. They were acicular, with an acute base and a thickened, darkened hilum (Table 1). Bakhshi et al. (2018) also described the morphology of C. gossypii from G. herbaceum, with findings similar to those of the present investigation. They reported that conidiophores were aggregated into dense fascicles, pale brown to brown, simple, straight or flexuous. Conidia were solitary, smooth, obclavate to subcylindrical, 1-7 septate, hyaline, straight or mildly curved and tapering towards the apex. Zia Hasan et al. (2018) isolated Cercospora sp. from Okra on PDA and found that conidiophores were pale brown and septate. Conidia were long, straight to slightly curved, thin-walled, multi-septate and pale olivaceous brown. Cercospora canescens from mung bean produced pale olivaceous conidiophores and straight to slightly curved, multi-septate conidia (Mahapatra et al., 2023). 
       
In this investigation, conidia measured 55.72 - 165.10 × 3.0 - 5.0 µm, with 9-19 horizontal septa across different isolates (Fig 1, 2, 3, 4 and 5; Table 1). These results align with the study of Bakhshi et al. (2018), who reported that C. gossypii isolates from cotton varied from 30 - 160 × 2-4 μm. The maximum and minimum conidia lengths were recorded for Karnataka isolates. Conidia from C. canescens were hyaline, straight to sub-straight or slightly curved, cylindrical, 2-12 septate and measured 10-300 × 1.3-5.0 μm (Kumar et al., 2021). Conidiophores were solitary, dark brown and pale towards the apex. Conidia were solitary, hyaline, acicular to cylindrical, slightly curved, 2- 12 septate and measured 60-210 × 3.5-5 μm (Cho et al., 2025).










 
Species identification of Cercospora isolates based on conidial characters
 
An isolated pathogen was identified as Cercospora gossypina based on long, slender, hyaline and multicellular conidial characters (Fig 1, 2, 3, 4 and 5).
 
Pathogenicity and virulence characterization of Cercospora isolates
 
Pathogenicity and virulence of Cercospora gossypina isolates were assessed on 25-day-old susceptible genotype LRA 5166 under greenhouse conditions (Fig 6 and 7 and Table 2). Earlier researchers also studied the pathogenic diversity of Cercospora isolates from other crops (Kumar et al., 2021; Mahapatra et al., 2023).






       
PDI ranged from 11.0 to 34.0 among 29 Karnataka isolates. Of these, 3 were highly virulent (>40.0 PDI), 13 moderately virulent (20.01 to 40.00 PDI) and 13 less virulent (0.00 to 20.00 PDI). PDI among 7 Telangana isolates ranged from 14.0 to 23.5, with 3 moderately virulent and 4 less virulent. PDI ranged from 12.0 to 21.5 among 6 Andhra Pradesh isolates. Of these, 2 were moderately virulent and 4 were less virulent. Tamil Nadu isolates showed PDI ranging from 15.0 to 26.0 across 9 isolates. Five were moderately virulent and 4 less virulent (Table 2). Isolates from Karnataka were found to be more virulent than those from other states. Likewise, Moretti et al., (2004) tested the virulence of C. beticola isolates on a 28-day-old susceptible sugar beet cultivar, Roberta and reported that 3 isolates were the most virulent, 2 were less virulent and the remaining 5 were moderately virulent. Kumar et al., (2021) studied the pathogenic variability of ten C. canescens isolates on 40-day-old mung bean plants and reported that PDI ranged from 35.30 to 54.48. Isolates from the Nagaur region were found to be more virulent than those from Jaipur and Tonk. Likewise, identifying the resistance source is crucial for a resistance breeding programme. Prasad et al. (2024) screened 200 mungbean genotypes against Cercospora leaf spot diseases for two seasons and found that four genotypes, viz., PDM 04-123, PDM 54, EC520034-1 and EC 520022, were resistant to the disease. Integrating different management strategies are important for effective disease control. Foliar applications of garlic extract at 10% and Hexaconazole (0.1%) were effective in controlling the Cercospora leaf spot of mung bean caused by Cercospora canescens (Kumar et al., 2023).
               
Variation within the pathogen population is a continuous process. A study of pathogen variability is paramount for assessing pathogen status and devising a management strategy. In the present investigation, three dark or ashy grey colonies with raised or moderately raised mycelium and a cottony or villous texture, isolated from Karnataka, were found to be highly virulent. Although isolates with these characters were observed in other parts of Karnataka and in states such as Telangana, Andhra Pradesh and Tamil Nadu, they were recorded as either moderately or less virulent. This indicates that the virulence of the isolates depends purely on the environmental factors prevailing in the region, together with a susceptible host and a source of inoculum. Moreover, the genetic makeup of isolates also contributes to their highly virulent nature. These highly virulent isolates were isolated from samples collected from rainfed cotton fields. Under rainfed conditions, the pathogen’s adaptability to harsh environments will always be higher because of stronger selection pressure at high temperatures. These could have played a critical role in pathogen virulence and disease spread. 

In the present study, colony colour varied from ash to dark grey or greyish black among the isolates. Short, unbranched, tufted, olivaceous, pale brown and septate conidiophores were observed. Thin-walled hyaline conidia and multi-septation were observed. Horizontal septa ranged from 9 to 19 among isolates. PDI ranged from 11.00 to 34.0 and Karnataka isolates were more virulent than those from other states.  A study of pathogen population structure and continuous monitoring of pathogen variability are necessary for devising a disease management strategy, including a resistance-breeding program. This study mapped the pathogen’s virulence across cotton-growing areas in the southern zone. Alternate wetting and drying conditions,along with nutrient and water stress, are conducive to Cercospora infection in cotton. Fungicide sprays and potassium applications can help control Cercospora leaf spot disease in cotton. Further study of fungicide resistance and cultivar screening of the identified highly virulent isolate will help to control the disease efficiently. Molecular characterisation of the isolates will reveal the pathogen’s genetic makeup. The influence of weather parameters on disease development should also be studied to support effective disease management.

 

I am thankful to Dr J P Nidagundi, Cotton Breeder - AICRP on Cotton, UAS, Raichur, for coordinating sample collection in the Raichur region. I thank Dr Venkatesh Kulkarni, Cotton pathologist, AICRP on Cotton, UAS, Dharwad, for coordinating sample collection in the Dharwad region. This research was supported by the Indian Council of Agricultural Research, Department of Agricultural Research and Education, Government of India.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsor- ship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.