Isolation, pathogenicity and cultural variability of Stagonosporopsis cucurbitacearum

Introduction

Cucurbitaceous vegetables are economically important crops, but their productivity is severely constrained by gummy stem blight (GSB), one of the most destructive fungal diseases causing leaf spots, stem cankers with gummy exudation, vine wilting, and fruit rot under warm and humid conditions. The disease is caused by Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae), a seed-, soil-, and residue-borne pathogen. Isolation and pathogenicity testing are essential for confirming disease etiology and fulfilling Koch's postulates. In addition, in vitro evaluation of different culture media provides valuable information on mycelial growth, colony morphology and fructification which are crucial for accurate identification and maintenance of the pathogen. Therefore, the present study was undertaken to isolate and confirm the pathogenicity of S. cucurbitacearum and to assess the effect of different nutrient media on its growth and fructification under laboratory conditions.

Material and Methods

The pathogen was isolated from infected leaf and stem tissues of ridge gourd using the tissue segment method. Surface-sterilized tissue bits (2-5 mm) from the diseased-healthy junction were plated on potato dextrose agar amended with streptocyclin (30 mg L⁻¹) and incubated at 27±1 °C. The fungus was purified by the single hyphal tip method and maintained on PDA slants at 5 °C for further studies. Pathogenicity was confirmed by spray inoculation of ridge gourd seedlings (3-4 leaf stage) with a spore suspension (1×10⁶ spores mL⁻¹). Typical symptoms developed under high humidity, and the pathogen was successfully re-isolated, fulfilling Koch's postulates. For cultural studies, five different solid media viz., potato dextrose agar, corn meal agar, V 8 juice agar, oat meal agar and host leaf extract agar were evaluated with respect to their support for growth of the pathogen. Culture discs of 5 mm diameter were lifted with a sterilized cork borer from a 10 days old culture and were aseptically transferred to a petri plate (90 mm diameter) containing test medium and incubated at 25±1 °C. Colony growth and fructification were recorded after 7 and 15 days of incubation respectively.

Results and Discussion

The pathogen was confirmed as S. cucurbitacearum. Pathogenicity of the isolate was confirmed by fulfilling Koch's postulates. Typical gummy stem blight symptoms appeared 5-7 days after inoculation, beginning as light brown leaf spots that later coalesced, while control plants remained symptomless. The pathogen was successfully reisolated on PDA and found to be identical to the original culture, confirming its pathogenic nature.

Comparative studies were conducted on five different media viz., potato dextrose agar, corn meal dextrose agar (CMDA), oat meal agar (OMA), V-8 juice agar (V-8A), host leaf dextrose agar (HLDA) to determine the ideal media for attaining the optimum growth and fructification of Didymella sp. Average colony diameter and fructification (per 5 mm culture disc) in each medium were recorded after 7 and 15 days of incubation respectively at 25±1 °C. The results revealed that on potato dextrose agar (PDA) and host leaf dextrose agar (HLDA) the growth of the fungal mycelium was maximum (90 mm) after 7 days of incubation. This was followed by V-8 agar (81 mm) and oat meal agar media (67 mm). Minimum mycelial growth was observed on corn meal agar (62 mm). On potato dextrose agar media, pigmentation varied from white to greyish white and cottony aerial growth was observed. On host leaf dextrose agar media, white to grayish white coloured fluffy mycelial growth was observed. On V-8 agar media, growth of the fungus was fluffy and white in colour. Colour of the fungus on oat meal agar media was white and the growth pattern was fluffy. On corn meal agar media, white and semitransparent growth was observed. The present study confirmed Stagonosporopsis cucurbitacearum as the causal agent of gummy stem blight of ridge gourd, based on symptom expression, pathogenicity tests and successful re-isolation of the pathogen, thereby fulfilling Koch's postulates. The appearance of typical gummy stem blight symptoms within 5-7 days after inoculation and the absence of symptoms in control plants clearly established the pathogenic nature of the isolate. The study demonstrated that culture media exert a significant influence on the growth, morphology and fructification of Stagonosporopsis cucurbitacearum. The findings are in agreement with earlier reports which indicated PDA as the most suitable medium for culturing the gummy stem blight pathogen. The superior performance of HLDA suggests that host-specific nutrients play an important role in enhancing fungal growth and sporulation. Variations in colony characteristics observed across different media highlight the adaptive nature of the pathogen and emphasize the importance of selecting appropriate media for laboratory studies.

Acknowledgement

AcknowledgementThe authors immensely thank the Dean of Post Graduate Studies, for providing the necessary facilities to conduct the research. The author is also thankful to the Professor and Head, Department of Plant Pathology, Dr. YSRHU, Andhra Pradesh, for providing facilities throughout the research work and time required to pursue Ph.D. degree. Competing interestsAuthors have declared that no competing interests exist.

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