Effect of different culture media on radial growth of potential fungal endophytes isolated from clusterbean

Introduction

Culture medium is an important factor governing the growth, development and recovery of fungi under in vitro conditions. The nutritional composition of a medium, particularly the availability and balance of carbon, nitrogen, minerals and other growth-supporting constituents, can considerably influence fungal mycelial development. This becomes particularly important when working with endophytic fungi because different taxa, and even different isolates belonging to the same taxon, may exhibit distinct nutritional requirements and growth rates. Consequently, the choice of culture medium can influence both the apparent growth potential of an isolate and its successful maintenance under laboratory conditions. Fungal endophytes associated with cluster bean represent a potentially useful group of microorganisms because some members can exhibit antagonistic, plant-growth-promoting and other beneficial characteristics. For their characterization and subsequent evaluation, it is therefore necessary to identify a suitable medium that supports vigorous and consistent mycelial growth. At the same time, dependence on a single culture medium may favour rapidly growing fungi while restricting comparatively slow-growing taxa. Evaluation of multiple media can therefore provide a more reliable understanding of the growth behaviour of selected endophytic isolates. In the present investigation, seven potential fungal endophytes, namely Fusarium falciforme JR-2, Fusarium solani BKNL-1, Colletotrichum gloeosporioides BMRL-3, Trichoderma harzianum JR-6, Trichoderma asperellum NR-1, Alternaria alternata PL-6 and Curvularia plantarum JS-1, were evaluated on six culture media. The media included Clusterbean Dextrose Agar (M1), Rose Bengal Agar (M2), Corn Meal Agar (M3), Oat Meal Agar (M4), V8-Juice Agar (M5) and Potato Dextrose Agar (M6), with PDA serving as the control. The objective was to determine the relative suitability of these media for radial growth of the selected fungal endophytes and to identify isolate × medium combinations supporting superior growth.

Materials and Methods

Experimental material

Seven potential fungal endophytes selected from the cluster bean endophyte collection were used for the study. The endophytes are tabulated below: Fusarium falciforme JR-2 2.

Colletotrichum gloeosporioides BMRL-3 4.

Trichoderma harzianum JR-6 5.

Trichoderma asperellum NR-1 6.

Alternaria alternata PL-6 7.

Culture media

Six different culture media, viz., Clusterbean Dextrose Agar (CDA), Rose Bengal Agar (RBA), Corn Meal Agar (CMA), Oat Meal Agar (OMA), V8-juice Agar and Potato Dextrose Agar (PDA), were evaluated for their suitability for the growth of selected fungal endophytes isolated from cluster bean. Potato Dextrose Agar was maintained as the control medium. The required media were prepared according to their standard composition and the pH was adjusted to the recommended level before sterilization. The prepared media were dispensed into suitable containers and sterilized by autoclaving at 121°C and 15 psi pressure for 15-20 min. After sterilization, the media were allowed to cool to approximately 45-50°C and poured aseptically into sterile Petri plates. The plates were allowed to solidify under aseptic conditions. Seven selected fungal endophytes mentioned in the table 1 were selected for the radial growth experiment on different medium. A 5-mm diameter agar disc was cut from the actively growing margin of each seven-day-old fungal culture using a sterile cork borer and transferred aseptically to the centre of each medium. The inoculated plates were incubated at 25 ± 2°C under laboratory conditions. Each treatment was maintained with three replications. Appropriate uninoculated plates were also maintained to check for contamination. The radial growth of each endophyte was recorded at regular intervals by measuring the colony diameter. Measurements were continued until the colony reached the edge of the Petri plate or until the predetermined observation period was completed. The radial growth was expressed in millimetres (mm).

Experimental design and statistical analysis

The experiment was analysed as a two-factor Completely Randomized Design (CRD), with fungal endophytes as Factor A and culture media as Factor B. The seven endophytes were evaluated on six culture media. The analysis included the main effects of endophyte, medium and their interaction (A × B). The experiment has been conducted with three replicates.

Results

Radial growth of fungal endophytes

The radial growth of the seven fungal endophytes varied considerably across the six culture media. Therefore, PDA was the most favourable medium for overall radial growth, whereas V8-Juice Agar was the least favourable among the media tested.

Interaction between endophytes and culture media

A strong interaction was observed between fungal endophytes and culture media. The A × B interaction was highly significant, with an F-value of 149.836. This indicates that the response of the fungal isolates was not uniform across the media.

The maximum colony diameter of 90.00 mm was recorded by C. gloeosporioides BMRL-3, T. harzianum JR-6 and T. asperellum NR-1 on PDA. C. plantarum JS-1 and F. falciforme JR-2 recorded 89.33 and 89.00 mm, respectively, on PDA. In contrast, A. alternata PL-6 showed the minimum colony diameter of 20.67 mm on V8-Juice Agar, followed by 33.67 mm on Rose Bengal Agar. The results demonstrate that both the identity of the fungal endophyte and the culture medium substantially affected colony development, while the significant interaction confirms that the suitability of a medium depended on the particular fungal isolate (Table 1, Fig. 1). The effect of different culture media on radial growth of potential endophytes isolated from cluster bean has been represented in the Figure 2.

Discussion

The present investigation demonstrated considerable variation in the radial growth of the seven selected fungal endophytes when cultured on different media. The significant effects of fungal endophyte, culture medium and their interaction indicate that mycelial development was influenced by both isolate-specific characteristics and the nutritional environment provided by the culture medium. Such variability is expected in culture-dependent studies because fungal taxa differ in their ability to utilize particular carbon and nitrogen sources and respond differently to the physical and nutritional properties of culture media (Dos Reis et al., 2022) [1] . Among the evaluated isolates, T. harzianum JR-6 recorded the highest overall mean radial growth of 78.50 mm. The high growth rate of this isolate may be associated with its vigorous vegetative development and efficient utilization of nutrients available in the media. Trichoderma species are generally characterized by rapid mycelial development and have considerable importance in agricultural biological control systems (Khan et al., 2022) [3] . The relatively high growth of F. falciforme JR-2, C. plantarum JS-1, T. asperellum NR-1 and C. gloeosporioides BMRL-3 further indicates that these isolates were capable of effectively utilizing the nutrients supplied by the tested media. In contrast, A. alternata PL-6 exhibited the lowest overall mean colony diameter (53.28 mm), indicating comparatively slower growth under the conditions evaluated. Among the media, PDA produced the highest overall mean colony diameter (86.10 mm). The superior performance of PDA can be attributed to its readily available nutrient and carbohydrate composition, which supports vegetative development of a broad range of fungi. PDA is also widely used for the isolation, maintenance and cultivation of fungi, including endophytic fungi (Dos Reis et al., 2022) [1] . Clusterbean Dextrose Agar ranked second, with an overall mean colony diameter of 77.86 mm. This finding is particularly relevant to the present investigation because the medium was developed/evaluated in relation to cluster bean-associated fungi and supported relatively vigorous growth of the selected isolates. Nevertheless, its performance should be interpreted specifically within the conditions of the present experiment because nutritional requirements can vary among fungal species and isolates. Corn Meal Agar and Oat Meal Agar supported intermediate levels of radial growth, whereas Rose Bengal Agar and V8-Juice Agar recorded lower overall growth. The relatively reduced growth on these media may be related to differences in nutrient availability and the ability of individual isolates to utilize the available substrates. The selective influence of culture media is an important consideration in culture-based endophyte studies because unsuitable media may favour particular taxa while limiting the development of others (Dos Reis et al., 2022) [1] . The highly significant A × B interaction provides an important interpretation of the experiment. It demonstrates that there was no single nutritional response common to all seven endophytes. For example, C. gloeosporioides BMRL-3, T. harzianum JR-6 and T. asperellum NR-1 attained the maximum recorded diameter of 90.00 mm on PDA, whereas A. alternata PL-6 recorded only 20.67 mm on V8-Juice Agar. This isolate-specific response has practical importance. Selection of an appropriate culture medium is necessary not only for obtaining rapid growth but also for maintaining and multiplying promising fungal endophytes. The strong growth of T. harzianum JR-6 is particularly noteworthy because this isolate also showed substantial antagonistic activity against Macrophomina phaseolina in the broader investigation. Rapid mycelial colonization can be advantageous for a prospective biocontrol organism because it may improve substrate occupation and competition with a pathogen. Trichoderma spp. can suppress plant pathogens through several mechanisms, including competition, antibiosis, hydrolytic enzyme production and stimulation of plant defence responses (Khan et al., 2022; Tiwari & Bae, 2022) [3,4] . Similarly, T. asperellum NR-1 showed high radial growth, with an overall mean of 75.50 mm, and reached 90.00 mm on PDA. Its combination of vigorous growth and antagonistic potential makes it a suitable candidate for further evaluation. However, radial growth should not be considered independently when selecting a biocontrol agent. Antagonistic activity, enzyme production, secondary metabolites, ecological competence and plant-growthpromoting effects should also be considered. Overall, the present findings demonstrate that PDA was the most suitable medium for supporting radial growth of the selected fungal endophytes under the experimental conditions, while V8-Juice Agar was comparatively less suitable. The significant interaction between fungal isolate and medium emphasizes that medium selection should be based on the individual isolate rather than assuming that one medium is equally suitable for all endophytes.

Conclusion

The evaluation of six culture media demonstrated significant variation in radial growth among the seven selected fungal endophytes. PDA recorded the highest overall mean colony diameter (86.10 mm) and was therefore the most suitable generalpurpose medium among those evaluated. Clusterbean Dextrose Agar was the second-best medium (77.86 mm), indicating its potential for supporting growth of cluster bean-associated endophytes. Among the isolates, T. harzianum JR-6 recorded the highest overall mean growth (78.50 mm), whereas A. alternata PL-6 showed the lowest (53.28 mm). The highly significant endophyte × medium interaction confirms that the growth response was isolate-dependent. T. harzianum JR-6 and T. asperellum NR-1 were particularly promising because of their vigorous growth and their broader potential as biological control candidates against M. phaseolina.

References

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