In vitro assay of different fungicides and bio control agents against fungal pathogens of dragon fruit (Hylocereus spp.) plantation in Assam

Introduction

Like many other crops, dragon fruit is also attacked by several of economically important diseases, the majority of which causes fungal diseases. Although dragon fruit is a lowmaintenance crop, there is a significant and numerous new and first country reports of dragon fruit diseases and their associated pathogen. Disease incidence has arisen with significant impact, likely due to increases in planting acreage and the consequent buildup of pathogen inoculums. Anthracnose is the most destructive and frequently reported fungal disease of dragon fruit. The disease is caused by different species of Colletotrichum such as C. truncatum, C. gloeosporioides, C. siamense, C. fructicola, C. aenigma, C. karstii and C. boninense (Masyahit et al.,2009; Vijaya et al., 2014; Palmateer et al., 2007; Awang et al., 2010; Abirami et al., 2019; Ma et al., 2014, Pegu et al., 2025) [1,2,3,4,5,6] . The fungus C. gloeosporioides is the most widely reported and is hosted by H. undatus, H. monacanthus and H. megalanthus. The abundance of viable spores is a huge challenge with diseases caused by Colletotrichum spp. In India, 30 per cent of dragon fruit plants were found to be infected by anthracnose and caused yield reduction (Abirammi et al., 2019). Fusarium spp. associated with dragon fruit stem rot (Wright et al., 2007; Rita et al., 2016; Hawa et al., 2017 and Mohd et al., 2019, Pegu et al., 2025) [6,7,8,9,10] and of basal rot (Wright et al., 2007) [7] has been reported. Shakirah et al. (2021) [11] reported gray blight on the stem of H. polyrhizus in Malaysia caused by Diaporthe spp. such as D. phaseolorum, D. tectonendophytica, D. arecae, D. eugeniae and D. hongkongensis.

Materials and Methods

The present investigation entitled "In vitro assay of different fungicides and bio control agents against fungal pathogens of Dragon fruit (Hylocereus spp.) plantation in Assam" was carried out in the Department of Plant Pathology, Assam Agricultural University, Jorhat, Assam during the year 2023-2024. The details of materials used, procedures followed and techniques adopted for carrying out the experiments are presented in this chapter

In vitro assay

The pathogens isolated from dragon fruit plantations and used for the in vitro assay are presented in the

In vitro assay of fungicides

Three fungicides viz., combination Tebuconazole 50% + Trifloxystrobin 25% WG, Azoxystrobin and Copper oxychloride were used to evaluate its in vitro efficacy against the pathogens.

The fungicides used and their concentrations are presented in Table 2.2 Standard poison food technique (Nene and Thapliyal, 1979) [12] was followed to assess the efficacy of the fungicide.

Preparation of Culture and culture media

The pathogens were cultured on potato dextrose agar (PDA) medium in 9 cm diameter Petri plates and incubated at 25±2 ºC temperature for 7 days. Standard PDA medium (30ml) was autoclaved at 121 ºC for 15 minutes, cooled to 40±5 ºC and then amended with 0.3g/l of Streptomycine sulphate. The required concentrations of the fungicides (30ml) were prepared and added to the media while it was warm and agitated thoroughly to get a uniform distribution of fungicides in the media. For control plates appropriate amount of Streptomycin sulphate (0.3g/l) alone was added. Approximately 15 ml of each of the amended media was poured aseptically into 9 cm sterilized Petri plates. The same amount of medium without the fungicide was used as control for comparison under the same conditions. Mycelial disc of 5 mm diameter of the fungal pathogen was cut with sterile cork borer and placed aseptically in the centre of each Petri plates containing the poisoned medium as per treatment combination presented in Table 2.3. Inoculated Petri plates were than incubated at 25±2 °C. For each treatment three replications were maintained. The study was arranged in a completely randomized design consisting of 10 treatments and 3 replications.

Bio control agents used for the in vitro assay

Three bio control agents used to evaluate its in vitro efficacy against the pathogens were collected from the Mycology department of Plant Pathology, AAU, Jorhat and are presented in Table 4. Dual culture method (Skidmore and Dickson, 1976) [13] was followed for the assay as per the treatment combinations presented in Table 2.5 Mycelial disc of the pathogen from seven day old culture grown on PDA were placed on one side of the plate and T. viride (KF439055) were placed on the other side of the plate. In case of B. subtilis (ON386193) and P. fluorescens (KT258013), a line of streak was made on both sides of the Petri plate with the pathogen at the center and incubated at 25±2ºC. Three replications were maintained for each tested pathogens. The pathogen grown as monoculture served as control. The plates were observed daily after 24 h of inoculation till the pathogen grew and covered the plate in the control.

Observation recorded

The colony diameter of the pathogens were measured and recorded and percent inhibition of mycelial growth was calculated using formula given by Vincet (1947) [14] as-

Where, MGI = Mycelial growth inhibition MGC = Mycelial growth in control MGT = Mycelial growth in treatment

Statistical analysis

Completely Randomized Design (CRD) and Fischer's method of analysis of variance (ANOVA) was employed for statistically analysis of the experimental data. Multiple comparisons amongst treatment means were undertaken using DMRT.The analysis of variance (ANOVA) was used to compare the significant difference with the t-test at p =0.05. The standard error of difference between the treatment means (S.Ed.) was calculated using the formula:

The CD (Critical Difference) was performed to test the significance of the difference between pairs of means for various observations by using the following formula: CD = S.Ed. × t 0.05 Where, S.Ed stands for Standard Error of Difference and t0.05 denotes table value of "t" at a 5% probability level.

Results and Discussions 3.1 Effect of different fungicides on radial growth of C. gloesporoides OQ274997

Results presented in Table 3.1 showed that all the three fungicides tested against C. gloesporoides OQ274997 inhibited 100 per cent at higher concentrations. Among the fungicides, significantly lowered mycelia growth of the pathogen at lower concentration was recorded in treatment T1 (Tebuconazole 50% + Trifloxystrobin 25% WG @ 0.05%) with a per cent radial inhibition of 82.59 followed by treatment T5 (Azoxystrobin @ 1%), T8 (Copper oxychloride @ 0.1%), T4 (Azoxystrobin @ 0.5%) and T7 (Copper oxychloride @ 0.1%) with a per cent radial inhibition of 66.66, 64.07, 55.92 and 40.00 respectively. Comparatively Copper oxychloride was found significantly less effective at a concentration of 0.1 per cent. Combination fungicide Tebuconazole 50% +Trifloxystrobin 25% WG resulted in maximum per cent radial growth inhibition of C. gloesporoides OQ274997 and found most superior over all the fungicides tested at its lowest concentration and hence selected for further studies. The superior effect of the combination fungicide may be due to the fact that Tebuconazole a triazole fungicide is dimethylase inhibitor which interferes in process of building the structure of fungal cell wall thereby inhibiting the reproduction and further growth of fungus. Moreover, Trifloxystrobin a strobilurin fungicide that blocks the mitochondrial electron transport chain and inhibits energy production in fungi. Azoxystrobin, a strobilurin group of fungicide and it works on the respiratory system of the fungus, thus depriving the fungus of the energy required to grow and killing the fungus. The results of the present study were in line with the in vitro evaluation of Tebuconazole 50% + Trifloxystrobin 25% WG against C. gloeosporioides by Ann et al. (2017) [15] who reported 84.62 per cent mycelia inhibition at 0.2 g/l and complete inhibition of the fungus at 0.4 g/l. 4.44 *Data are mean of three replications In column mean followed by common letter do not differ significantly at 5% level of probability.

Effect of different fungicides on radial growth of F. oxysporum OQ274999

Result presented in Table 3.2 showed that treatment T1 (Tebuconazole 50%+Trifloxystrobin 25% WG @ 0.05%) completely inhibited the radial growth of F. oxysporum OQ274999. This was followed by T7 (Copper oxychloride @ 0.1%) and T4 (Azoxystrobin @ 0.5%) with a per cent inhibition of 77.03 and 70.37 respectively. Hence it is evident from the data presented in Table 3.2 that maximum per cent inhibition of F. oxysporum OQ274999 was caused by Tebuconazole 50%+Trifloxystrobin 25% WG @ 0.05 per cent and found superior over other two tested fungicides. Results of present findings were in close proximity to the in vitro evaluation of Tebuconazole 50%+Trifloxystobin 25% WG carried out by Bhimani et al. (2018) [16] that significantly inhibited the radial growth of F. oxysporum. Patra and Biswas (2016) [17] also reported the combination fungicide Tebuconazole 50% + Trifloxystobin 25% WG as effective fungicides against F. oxysporum f.sp. ciceri. Tebuconazole 50%+Trifloxystrobin 25% at @ 0.07 per cent evaluated in vitro against F. oxysporum f.sp. lycopersici on PDA significantly inhibited mycelial growth (Ahmad et al., 2021) [18]

Effect of different fungicides on radial growth of F. dimerum OQ274998

Results pertaining to Table 3.3 showed that all the three tested fungicides completely inhibited the growth of F. dimerum OQ274998 at higher concentrations. However treatment T1 (Tebuconazole 50%+Trifloxystrobin 25% WG @ 0.05%) showed 100 per cent mycelial inhibition of the pathogen at its lower dose as compared to other fungicides. These was followed by T7 (Copper oxychloride @ 0.1%) and T4 ([email protected]%) with a per cent inhibition of 74.81 and 60.74. Hence, the data presented in Table 3.3 showed that maximum per cent inhibition of F. dimerum OQ274998 was recorded in Tebuconazole 50%+Trifloxystrobin 25% WG @ 0.05 per cent and found superior over the other two tested fungicides. Trichoderma restrict further growth of the pathogen upon contact and it continued its growth over the pathogen. Observation on mycelium from the zone of interaction of T. viride KF439055 and pathogen under light microscope revealed lysis of the pathogen hyphae. Our results were in line with the in vitro evaluation carried out by Valenzuela et al. (2015) [19] who reported that T. viride inhibited the growth of C. gloeosporioides by 60 per cent. The results from the present findings showed that the fast growing T. viride KF439055 caused more growth inhibition of the pathogens which might be due to mycoparasitism and competition for space and nutrients. Some contrasting interactions were noticed in the experiments in which T. viride KF439055 showed antagonism by overgrowing the pathogen and reducing its growth although P. fluorescens KT258013 and B. subtilis ON386193 were still growing in treatment T4, T5 and T7. This might be due to chitinolytic enzymes produced by Trichoderma spp. which are reported biologically more active than enzymes of other microorganism and more effective against a larger spectrum of fungi (Lori et al., 1993) [20] an is well known biological agents that have significant antagonistic activity against several plant pathogenic fungi. 2.19 *Data are mean of three replications In column mean followed by common letter do not differ significantly at 5% level of probability 4.2 Effect of different bio control agents on radial growth of F. oxysporum OQ274999 at 7 days after inoculation The result presented in Table 4.2 showed that treatment T5 (T. viride KF439055 + B. subtilis ON386193) and T7 (T. viride KF439055 + P. fluorescence KT258013 + B. subtilis ON386193) showed the highest inhibition per cent of 72.59 followed by T4 (T. viride KF439055 + P. fluorescence KT258013), T6 (P. fluorescence KT258013+ B. subtilis ON386193) and T3 (B. subtilis ON386193) with a per cent inhibition of 70.74, 63.33 and 61.11 whereas the lowest radial growth inhibition of F. oxysporum OQ274999 was in T2 (P. fluorescens KT258013) with a per cent mycelia inhibition of 50.37 respectively. However, treatment T1 (T. viride KF439055 alone) showed radial inhibition per cent of 70.00 and were at par with T4, T5 and T7 and hence selected for further studies. The formation of inhibition zones between T. viride KF439055 and F. oxysporum OQ274999 was evident in the present study which might be due to production of secondary metabolites. The result of the present investigation was in accordance with the results reported by Abhiram and Masih (2018) [21] who stated that T. viride inhibited the growth of F. oxysporum by 71 per cent in dual culture plate technique. Trichoderma spp. act against pathogens through a combination of hyperparasitism, competition for space and nutrients and antibiosis (Whipps and Lumsden, 1991; [22] Calistru et al., 1997) [23] . Similarly Rehman et al. (2013) [24] also showed the efficacy of T. viride against F. oxysporum f. sp. ciceris with a per cent mycelia inhibition of 81 respectively. Cherkupally et al. (2017) [25] have also evaluated the efficacy of T. viride against F. oxysporum f.sp. melongenae with a per cent inhibition of 78.88 respectively. 5.46 *Data are mean of three replications In column mean followed by common letter do not differ significantly at 5% level of probability

Effect of different bio control agents on radial growth of F. dimerum OQ274998 at 7 days after inoculation

The results presented in Table 4.3 showed that the tested bio control agent and their combination had significant effect in reducing the radial growth of the pathogen. Treatment T7 (T. viride KF439055+P. fluorescence KT258013+B. subtilis ON386193) showed the highest per cent inhibition of 85.55 followed by T5 (T. viride KF439055+ B. subtilis ON386193), T4 (T. viride KF439055+ P. fluorescence KT258013) and T1 (T. viride KF439055) with a per cent inhibition of 82.96, 82.59 and 81.85 respectively. Among the three bio control agents T. viride KF439055 was found most promising because it alone showed radial growth inhibition of 81.85 per cent of the pathogen and were at par with T4 and T5. The clear zone of inhibition observed in between T. viride KF439055 and F. dimerum OQ274998 in dual culture indicates that T. viride KF439055 restricted further growth of F. dimerum OQ274998. The hyphae of the antagonist along with the pathogen at the meeting point

Conclusion

In vitro evaluation of three different fungicides viz., Tebuconazole 50% +Trifloxystrobin 25% WG, Azoxystrobin and Copper oxychloride at three different concentrations were conducted for their efficacy against the pathogens through poisoned food technique and result revealed that combination fungicide Tebuconazole 50%+Trifloxystrobin 25% WG @ 0.05% was found to significantly lowered mycelia growth of C. gloeosporioides OQ274997, F. oxysporum OQ274999, F. dimerum OQ274998 and D. phaseolorum OQ275000 pathogens at lower concentration. Similarly in vitro screening of three bio control agents viz., T. viride KF439055, P. fluorescens KT258013 and B. subtilis ON386193 against C. gloeosporioides OQ274997, F. oxysporum OQ274999, F. dimerum OQ274998 and D. phaseolorum OQ275000 were carried out through dual culture method and result revealed that T. viride KF439055 resulted in maximum per cent radial growth inhibition of the tested pathogens and appeared to be most superior over individual effect of P. fluorescens KT258013 and B. subtilis ON386193. Recent researches on this crop in different parts of the country are particularly focusing on its physico-chemical characteristics, great potential health benefits and nutritional value. However, its scientific report of various diseases of the crop is still lacking. Moreover dragon fruit growers in Assam are importing planting materials from various part of the country and there is a need for disease monitoring and pathogen identification to ensure plant bio security as pest and disease incidence can have significant constraints in production of the crop. Accurate pathogen identification is important for formulations of effective disease management strategies. The findings of the present research investigation revealed that Tebuconazole 50% + Trifloxystrobin 25% WG @ 0.05 per cent and T. viride KF439055 were superior in inhibiting the growth of the tested pathogens and can be incorporated in the future management strategies against the pathogens in the field condition.

Acknowledgement

AcknowledgementWe are thankful to the Director of Post Graduate Studies, and Head, Department of Plant Pathology, AAU, Jorhat for the facilities provided for the experiment.

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