Effect of different culture media on the growth and sporulation of Macrophomina phaseolina causing dry root rot of pigeonpea

Introduction

Pigeonpea (Cajanus cajan [L.] Millsp.) is a diploid (2𝑛 = 16) grain legume of the Fabaceae family and is ecognized for its perennial growth habit, short-day flowering response and robust performance under marginal, rainfed conditions. Originally domesticated in the Indian subcontinent, the crop subsequently migrated to Eastern and Southern Africa and Central America which together with South Asia constitute the three principal production zones worldwide (Nene et al., 1990) [1] . India remains the world's leading producer of pigeonpea, with a national production of approximately 4.34 million tonnes from an area of 5.05 million hectares, corresponding to an average productivity of about 859 kg/ha (DES et al., 2022) [2] . Despite this preeminent position, average national productivity continues to lag behind the crop's genetic potential, a shortfall commonly attributed to cultivation under predominantly rainfed and marginal conditions and to exposure to an array of biotic constraints that limit realisable yield (Ghritlahre et al., 2024) [3] . Diseases constitute a particularly important biotic constraint to stable pigeon pea production. The crop is vulnerable to a wide spectrum of diseases caused by fungal, bacterial, and viral pathogens, including phytophthora blight, bacterial leaf spot, sterility mosaic disease, Alternaria blight, and Fusarium wilt (Nene et al., 1990) [1] . Among these, dry root rot associated with Macrophomina phaseolina (Tassi) Goid has emerged as a significant threat, particularly under conditions of elevated temperature and moisture stress. The pathogen is a soil-borne fungus that can remain viable and persist in soil for prolonged periods by producing microsclerotia and pycnidia, which function as significant sources of inoculum (Short et al., 1980; Gupta et al., 2012) [5,4] . Infection is initiated through the roots, where the pathogen colonizes cortical and vascular tissues, impairing the uptake and translocation of water and mineral nutrients, ultimately resulting in wilting, premature drying, and significant yield reduction (Kaur et al., 2012) [6] . Macrophomina phaseolina is distinguished by hyaline, thin-walled hyphae that may become light to dark brown and septate with age. Lateral branches typically emerge at right angles from the main hyphae and exhibit a constriction at the point of origin. Microsclerotia, which consists compact masses of hardened mycelium appears spherical, oval or oblong, initially light brown and progressively turning dark brown to black as they mature. Pycnidia, which are infrequently observed under natural conditions are larger than microsclerotia, dark brown to black, rough in texture, globose or irregular in shape, beaked and ostiolated (Lakhran et al., 2018) [7] . The pathogen infects a broad host range comprising nearly 500 species belonging to 75 plant families, and high temperature and dry climatic conditions favour disease development (Iqbal & Mukhtar, 2014; Basandrai et al., 2021) [9,8] . The successful isolation, cultivation, and characterization of M. phaseolina under laboratory conditions is fundamental to understanding its biology, nutritional requirements and pathogenic variability. Culture media serve as the primary substrate for fungal growth in vitro, providing essential nutrients consisting carbon sources, nitrogen sources, vitamins and minerals that influence mycelial development, sporulation and sclerotial formation. The nutritional composition of culture media can significantly affect growth rate, colony morphology and reproductive structures of fungal pathogens and different media may support varying degrees of growth depending on the specific physiological requirements of the organism (Dhingra et al., 1995) [10] . The present study was carried out to evaluate the effect of various culture media on the growth, sporulation along with colony charecteristics of M. phaseolina associated with dry root rot of pigeonpea.

Materials and methods

The investigation was undertaken under the facilities of the Department of Plant Pathology and Microbiology, Mahatma Phule Krishi Vidyapeeth, Rahuri.

Isolation and identification of pathogen

The pathogen was isolated from infected root tissues obtained from diseased pigeonpea plants collected from field. The recovered isolate was identified through detailed examination of its cultural and morphological attributes, particularly colony characteristics and spore morphology. Microscopic characterization was performed by preparing a temporary mount of the sporulating culture in lactophenol cotton blue stain. The preparation was examined at 400× magnification using a research microscope to record the diagnostic morphological features of the isolate. The pathogenicity of the isolated organism was subsequently established by employing the sick-soil technique.

Results and discussion

Effect of different culture media on mean colony diameter and sporulation of Macrophomina phaseolina

Significant variation in mean colony diameter of M. phaseolina was observed among the seven-culture media evaluated. The mean colony diameter ranged from 45.46 to 90.00 mm. PDA recorded the highest radial growth, recording a mean colony diameter of 90.00 mm and was significantly superior to the remaining media under the conditions of the experiment. Malt Extract Agar ranked second with a mean colony diameter of 87.20 mm. It was followed by Czapek's Dox Agar (78.20 mm), Richard's Agar (75.50 mm) and Rose Bengal Agar (75.13 mm). Yeast Extract Agar recorded a considerably lower mean colony diameter of 54.80 mm, whereas the minimum radial growth was recorded on Nutrient Agar (45.46 mm). The observed values and treatment ranking are depicted in Table 1. The difference between PDA and Malt Extract Agar was 2.80 mm, which exceeded the reported C.D. of 0.96 mm at 1 per cent probability, indicating significantly greater growth on PDA. Thus, PDA provided the most favourable conditions for radial development of the pathogen among the media evaluated. The superior performance of PDA in the present investigation is in agreement with several earlier reports. Sahi et al. (1992) [11] reported maximum growth on PDA, while Jha et al. (2000) [12] recorded the highest growth and sclerotial formation on PDA followed by Richard's medium. Suriachandraselven and Seetharaman (2003) [13] also identified PDA as the best medium among the media evaluated for growth and sclerotial formation. The repeated identification of PDA as a favourable medium suggests that it provides a suitable nutritional environment for vigorous vegetative development of M.

Colony characteristics of Macrophomina phaseolina on different culture media

Distinct differences in colony morphology were observed among the culture media. PDA produced dark grey to black, dense and velvety to compact colonies with profuse superficial mycelium and a well defined margin. Malt Extract Agar supported white to off-white, highly cottony colonies with profuse aerial mycelium, distinct radial strands and an irregular margin. Czapek's Dox Agar produced white to creamy-white colonies with dark grey to black central pigmentation, abundant aerial mycelium and slight concentric zonation. On Richard's Agar, colonies were white at the centre and progressively grey to dark grey towards the periphery, with moderately dense cottony to woolly aerial mycelium. Rose Bengal Agar produced white-centred colonies with grey to olivaceous-grey pigmentation, concentric zonation and comparatively dense central aerial mycelium. Yeast Extract Agar developed predominantly white to cream colonies with grey central pigmentation and moderate aerial mycelium, whereas Nutrient Agar produced predominantly white colonies with restricted radial development and grey to dark-grey pigmentation towards the periphery. The variation in colony colour, texture, aerial mycelium and growth pattern observed in the present study is consistent with earlier reports of cultural variability in M. phaseolina. Bairwa et al. (2023) documented differences in colony characteristics among M. phaseolina isolates, while a study of 60 groundnut isolates reported appressed, fluffy and velvety growth patterns and colony colours ranging from light grey to grey and black.

Media

Conclusion

The present study demonstrates that the growth and sporulation of Macrophomina phaseolina, the causal agent of dry root rot of pigeonpea, are significantly influenced by the composition of the culture medium. Among the seven media evaluated, Potato Dextrose Agar supported the maximum mean colony diameter (90.00 mm) along with excellent sporulation, and is therefore identified as the most suitable medium for the vegetative growth and sporulation of this pathogen under in vitro conditions, followed by Malt Extract Agar (87.20 mm). These findings confirm Potato Dextrose Agar as an effective standard medium for the laboratory isolation, maintenance and cultural characterisation of M. phaseolina.

Acknowledgement

AcknowledgementsI sincerely acknowledge the support extended by the Post Graduate Institute, Mahatma Phule Krishi Vidyapeeth, Rahuri for providing the necessary facilities and institutional assistance required for carrying out the present investigation. I express my heartfelt gratitude to my Advisor, Dr. S. V. Kolase, for his constant guidance, valuable suggestions, encouragement and careful supervision throughout the course of this research work. I am equally thankful to the members of my Advisory Committee and the faculty and staff of the Department of Plant Pathology and Microbiology, Mahatma Phule Krishi Vidyapeeth, Rahuri for their constructive suggestions, technical guidance and valuable assistance at various stages of the investigation.

References

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