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Molecular characterization and vegetative growth of pathogenic seed-borne fungus, Curvularia lunata of tomato and its in vitro control measures

Present studies were conducted to isolate and identify the seed-borne pathogenic fungus from the selected tomato variety through morphological and molecular techniques based on the sequencing of internal transcribed spacer (ITS) region of 18S rDNA. According to the colony and conidial features, the fungus was identified as Curvularia sp. The obtained ITS sequencing showed above 99% similarity with Curvularia lunata in the NCBI database. The sequence of the fungus was deposited in NCBI GenBank under the accession number: ITS, MH382879.1. Besides, the phylogenetic tree further confirmed the taxonomic position of the studied fungus. Growth characteristics of the fungus on nine different fungal culture media were evaluated, in which Honey peptone agar, Carrot agar, Potato sucrose agar, and Kauffman’s agar were found the most suitable. The maximum vegetative growth of the fungus was recorded at 30°C temperature and pH conditions. The bio-control potential of five different antagonists against the studied fungus was assessed, in which Trichoderma harzianum showed the better performance to restrict mycelial growth. Three ethanolic plant extracts were also evaluated, in which Lowsonia inermis L. exhibited above 60% mycelial growth inhibition of the fungus. Among three tested fungicides, Tilt 250 EC was found as an excellent fungicide to inhibit mycelial growth of C. lunata under in vitro conditions.


Introduction
Tomato (Lycopersicon esculentum Mill.) is a popular vegetable crop belonging to the family Solanaceae. It is especially honoured due to its high nutritive value, taste, and versatile use as vegetable, food, and preparation of various food items. It is widely grown in almost all countries of the world due to its adaptability to a wide range of soils and climatic conditions. The quality of seed plays an important role in crop production. However, seeds are known to be vulnerable to attack by seed-borne fungi either saprophytically or within the tissues of the embryo (Vijendra and Sinclair, 1997). Infected seeds play an important role in the spreading of plant pathogens and disease establishment (Agarwal, 1981). The disease-causing fungal pathogens might be involved in shrunken seed, seed abortion, seed rot, seed discoloration, and causes of reduction of germination capacity (Hamim et al., 2014).
Curvularia spp. are internally and externally seed-borne pathogenic fungi, affecting seed germination and seedling vigour, known to be responsible for pre-and post-emergence seedling mortality (Gupta et al., 2017). They cause seed disclouration, even produce toxins that may be injurious to man and domestic animals.
Seed treatment is the safest and the cheapest way to control seed-borne fungal diseases and to prevent bio-deterioration of grains (Bagga and Sharma, 2006). However, uses of fungicides are not considered as sustainable solutions due to their harmful effects on human beings as well as soil health. Therefore, nowadays the focus is shifting in the direction of exploitation of biological organisms and natural products for the control of plant diseases as an alternative way to synthetic fungicides. Environmental factors such as temperature, pH, and water activity play a key role in fungal development (Yadav et al., 2014). Carbon and nitrogen sources available in the fungal culture media besides changes in environmental factors, incubation time, and shaking have a significance influence on the growth of microbial pathogen (Tyagi and Paudel, 2014). Hence, the present studies were undertaken to isolate and identify of seed-borne tomato fungal pathogen using morphological and molecular techniques; to study growth characteristics of the fungus; to evaluate the efficacy of biological agents, plant extracts, and chemical fungicides against the isolated fungus.

Pathogen isolation and identification
For isolation of the fungal pathogen from tomato seeds of selected varieties, a direct isolation method was performed. This method consisted of incubation of surface sterilized tomato seeds in a humid chamber following the paper towel method. For the identification of isolated fungus, we used a standard manual-Dematiaceous hyphomycetes by Ellis (1971), and further confirmation was done via molecular techniques.
The Maxwell® 16 DNA Purification Kit was used to purify the PCR products (Promega, USA). The purified PCR product of approximately 650 bp was sequenced in First BASE Laboratories Sdn Bhd (Malaysia). Sequencing data was blastn searched and compared with similar DNA sequences after retrieving from NCBI Genbank. The phylogenetic analysis was conducted by the multiple sequence alignment tools using MEGA 6 software (Tamura et al., 2013).

Effect of fungal culture media, temperature, and pH on the mycelial growth of Curvularia lunata
Nine different culture media i.e. Potato Dextrose Agar (PDA), Yeast Extract Agar (YEA), Honey Peptone Agar (HPA), Hansen's Agar (HA), Sobouraud's Glucose Agar (SGA), Kauffman's Agar (KA), Potato Sucrose Agar (PSA), Richard's Agar (RA) and Carrot Agar (CA) media were evaluated on the mycelial growth of the fungus . To find out an optimum temperature for the mycelia growth of the fungus, inoculated plates were incubated at 10℃, 15℃, 20℃, 25℃ and 30℃ for 7 days . The fungal culture media were adjusted to pH 5, 6, 7, and 8 with the addition of 1N NaOH or HCl before autoclave, and inoculated petri-plates were incubated at 25℃ for 7 days .

Efficacy of bio-control agents, plant extracts, and fungicides against Curvularia lunata
The two rhizobacterial isolates Bacillus subtilis and Pseudomonas fluorescens and three fungal biocontrol agents-Trichoderma harzianum, Trichoderma reesei isolate 1 and Trichoderma reesei isolate 2 were assessed against the isolated fungal pathogen using duel culture technique .
The ethanolic plant extracts were prepared from leaves of Tagetes erecta L., Azardirachta indica L., and Lowsonia innermis L. and each plant extract of different concentrations (10%, 20% and 30%) were mixed with PDA as different treatment combinations (Rahman et al., 2015). Inoculated petri-plates were kept in an incubation chamber at 25±2°C. Radial growth of fungal mycelium was measured at 7 days postincubation (dpi).
The effect of fungicides, namely Jazz 80 WP (Active ingredient: Ethylene bisdithiocarbamate), Amister Top 325 SC (active ingredients: Azoxystrobin + Difenoconazole), and Tilt 250 EC (active ingredients: Propiconazole) were assessed against C. lunata. A requisite quantity of fungicides was added to the medium to get concentration of 250 ppm, 500 ppm, and 750 ppm; inoculated plates were incubated at 25±2°C . The fungal colony diameter was recorded at 7 dpi.
Percentage inhibition of the fungus was calculated by using the following formula: I = ((C-T)/C) × 100. Here, I = percentage of mycelium growth inhibition; C = growth of mycelium in control; T = growth of mycelium in treatment.

Statistical analysis
Data generated in the experiment was checked for normality and homogeneity of variance; and analyzed using the SPSS program (SPSS Inc., Chicago, IL, USA). All parameters for inter-group differences were analyzed by Oneway ANOVA followed by post-hoc test.

Morphological and molecular identification of the fungus
Fungal colonies were fast-growing, brown to blackish brown with dark reverse. Conidiophores were erect, septate, unbranched, flexuose in the apical part, with flat, dark brown scars (Figure 1).
Conidia were septate, smooth-walled, olivaceous brown, end cells somewhat paler, obovoidal to broadly clavate shaped, curved at the subterminal cell ( Figure 1). Based on morphological features, the studied organism was identified as Curvularia sp.   To identify the fungus at species rank, sequencing of the ITS region was performed. In blast search, our fungus (MH382879.1: Curvularia lunata) showed above 99% sequence similarity with previously identified fungus-KX610322.1: C. lunata, MF380920.1: C. lunata, KT309032.1: C. lunata, and KY859790.1: C. lunata. The phylogenetic analysis was conducted via the multiple sequence alignment tools using MEGA 6 software ( Figure 2). Phylogenetic tree confirmed the studied organism as a C. lunata, which formed a separate cluster with other C. lunata.

Effect of culture media on the mycelial growth of C. lunata
To find out the most suitable solid culture media for the growth and development of the fungus, the pathogen was grown on nine different solid media (Figure 3). Most of the culture media contains dextrose as a carbon source and peptone as a nitrogen source for mycelial growth and development. Our results revealed that Carrot agar, Kauffman's Agar, Honey peptone agar, and Potato sucrose agar were supported the maximum mycelial growth of C. lunata while yeast extract agar and potato dextrose agar media were less suitable to the fungus. Shabana et al. (2015) found the maximum colony diameter and growth rate of Curvularia prasadii on malt extract agar, followed by carrot agar and potato dextrose agar. Kumar et al. (2018) reported significantly higher mycelial growth of C. lunata on Sabouraud´s agar compared to PDA while other studies reported that C. lunata showed best vegetative growth on the PDA medium (Bhatt and Kumar, 2018). Similarly, Potato dextrose agar, Sabouraud's agar, and Host extract agar were best for the vegetative growth and sporulation of C. lunata (Sumangala and Patil, 2010). Our results suggest that HPA, KA, PSA, and CA are the most suitable fungal culture media of C. lunata.

Effect of temperature on the mycelial growth of C. lunata
Among the environmental factors, temperature plays a vital role in the growth, development, and reproduction of fungi. Each fungus has its optimum temperature requirement. We aimed to know the effect of different incubation temperatures on the growth of C. lunata on PDA media (Figure 4). The highest mycelial growth of the fungus was recorded at 30⁰C temperature, which was statistically different compare to other temperature conditions. With the decreasing temperature, the mycelial growth of C. lunata was also decreased and the lowest vegetative growth was recorded at 10⁰C. Present findings are in agreement with the previous results. The most preferable temperature ranges was between 25 and 30⁰C for vegetative growth of C. lunata, Curvularia clavata, Curvularia pallescens, Curvularia trifolii and Curvularia aeria (Almaguer et al., 2013;Sumangala and Patil, 2010;Bhatt and Kumar, 2018;Lal et al., 2014). Likewise, Shabana et al. (2015) also reported the best mycelial growth of CPO 1 and CPO 3 isolates of Curvularia prasadii was obtained at an incubation temperature of 30⁰C. Figure 4. Effect of different temperatures on the mycelial growth C. lunata at 7 dpi. The value represents as mean ± standard error (SE) of three replications. Means followed by a common letter (s) do not differ significantly at the 5% level by DMRT.

Effect of pH on the mycelial growth of C. lunata
Different fungal pathogens require a particular pH for their growth and development. The growth rate of the different pathogens changed with the pH conditions of the fungal culture media. In the present study, an effort was made to investigate an optimum hydrogen ion concentration requirement for the C. lunata. Our results revealed that the maximum mycelial growth was obtained at pH 6, followed by pH 7 at 7 dpi ( Figure 5). It was observed that C. lunata preferred more acid than alkali conditions. Presents study are supported by other researchers who reported the maximum growth of C. lunata (Sumangala and Patil, 2010) and Curvularia pallescens (Sonia et al., 1998) at pH 6. In another study showed that the pH levels between 6 and 8 induced the highest mycelial growth of Curvularia pradasii (Shabana et al., 2015). Figure 5. Effect of pH on the mycelium growth of C. lunata at 25±2℃ temperature at 7 dpi. The value represents as mean ± standard error (SE) of three replications. Means followed by a common letter (s) do not differ significantly at the 5% level by DMRT.

Efficacy of plant extract on the mycelial growth inhibition of C. lunata
Results on the effect of plant extract on mycelial growth inhibition of C. lunata have been presented in Figure 6. The maximum mycelial growth inhibition was obtained due to the higher dose of Lowsonia inermis, followed by Tagetes errecta and Azardirachta indica at 7 dpi. Present findings are in conformity with the results of Barupal et al. (2019) who found that L. inermis inhibited the mycelial growth of 65% of C. lunata and chemical analysis revealed abundant quantities of volatile oils, flavonoids, steroids, and tannins contents in plant leaves. Similar mycelial inhibition of C. lunata was also observed by Mohana et al. (2011).

Efficacy of bio-control agents on the mycelial growth inhibition of C. lunata
In our study, C. lunata was shown sensitivity to the antagonistic bio-control agents (Figure 7 and 8). The maximum inhibition of mycelium growth of C. lunata was exhibited by Bacillus subtilis whereas Pseudomonas fluorescens was ineffective against the fungus. Importantly, Trichoderma sp. showed good results against C. lunata, in which 63.18%, 69.57%, and 54.89% mycelial inhibition was recorded due to Trichoderma reesei isolate 1, Trichoderma reesei isolate 2 and Trichoderma harzianum, respectively ( Figure 8). Our results are supported by a study, in which sorghum rhizosphere originated Bacillus sp. showed higher antagonistic activity against C. lunata. Besides, scanning electron microscopic observations revealed hyphal lysis and degradation of the fungal cell walls (Basha and Ulaganathan, 2002). Tapwal et al. (2011) cited that T. viride showed the utmost mycelial inhibition of fungal pathogens including C. lunata. Anwar et al., (2008) reported that Trichoderma spp. inhibit pathogenic invasion through phenomena of mycoparasitism, antibiosis and competition and lysis of pathogenic hyphae. Figure 7. Effect of different antagonists on the mycelium growth inhibition of C. lunata at 25±2℃ temperature.

Efficacy of fungicides on the mycelial growth inhibition of C. lunata
The different concentrations of three commercial fungicides (Jazz 80 WP, Amister Top 325 SC, and Tilt 250 EC) were evaluated on the mycelium growth inhibition of C. lunata under in vitro conditions. Among the three concentrations (250 ppm, 500 ppm, and 750 ppm) of fungicides, Tilt 250 EC showed the maximum inhibition of mycelium growth resulting in 100% inhibition due to all three concentrations at 7 dpi (Figure 8  and 9). Besides, Amister Top 325 SC also showed better performance compared to Jazz 80 WP against C. lunata with the inhibition 48.54%, 57.50%, and 64.41% due to 250 ppm, 500 ppm, and 750 ppm, respectively (Figure 8 and 9). Our results are agreement with the previous findings of Mamun et al. (2016) who also reported that Tilt 250 EC (Propiconazole) completely inhibited the radial growth of C. lunata at all concentrations (100, 200, 300, 400 and 500 ppm). Likewise, Chowdhury et al. (2015) evaluated Tall 25 EC (Propiconazole) fungicides at 100, 200, 300, 400 and 500 ppm against several pathogenic fungi viz. Alternaria alternata, Curvularia lunata, Drechslera oryzae, and Pestalotiopsis guepinii and found very promising against tested fungi at all the concentrations.   Hossen et al. (2017) reported that Tilt 250 EC was known to reduce the incidence of fungal pathogens namely-Aspergillus spp., Colletortichum capsici, Curvularia lunata, and Fusarium spp., on chilli seeds. Besides, Tekade et al. (2017) cited that among all the systemic and non-systemic, and combination products, complete inhibition of C. lunata causing blight of coleus was achieved in propiconazole, carbendazim + mancozeb, tricyclozole + mancozeb, and zineb + hexaconazole.