Abstract
Fungicides can have a good result against phytopathogens, but alternatives, such as a biological control agents, have also been found to be effective and ecofriendly. In this experiment, Alternaria solani was isolated from rotten tomato fruit. The culture filtrates of Trichoderma harzianum gradually inhibited the radial growth of A. solani at higher concentrations, but growth was not completely inhibited until a high filtrate percentage of 75% was reached (75% by volume). The microscopic study revealed that the T. harzianum culture filtrate and its spores changed the size, number, and shape of the A. solani conidiospores. A. solani produced both cellulolytic and pectinolytic enzymes in vitro. The activity of these enzymes decreased with an increase in the T. harzianum filtrate percentage and chemical fungicide concentration. A. solani failed to produce hydrolytic enzymes, particularly pectinase, at high concentrations of fungicide (100 ppm and 150 ppm). When T. harzianum was used as a biocontrol agent, the detected hydrolytic enzyme activities increased compared with the other treatments.
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Effectiveness of a Biological Agent (Trichoderma harzianum and its culture filtrate) and a Fungicide (methyl benzimacold-2-ylcarbamate) on the Tomato Rotting Activity (growth, cellulolytic, and pectinolytic activities) of Alternaria solani
Tarek M. Abdel-Ghany,a,b,* and Marwah M. Bakri c
Fungicides can have a good result against phytopathogens, but alternatives, such as a biological control agents, have also been found to be effective and ecofriendly. In this experiment, Alternaria solani was isolated from rotten tomato fruit. The culture filtrates of Trichoderma harzianum gradually inhibited the radial growth of A. solani at higher concentrations, but growth was not completely inhibited until a high filtrate percentage of 75% was reached (75% by volume). The microscopic study revealed that the T. harzianum culture filtrate and its spores changed the size, number, and shape of the A. solani conidiospores. A. solani produced both cellulolytic and pectinolytic enzymes in vitro. The activity of these enzymes decreased with an increase in the T. harzianum filtrate percentage and chemical fungicide concentration. A. solani failed to produce hydrolytic enzymes, particularly pectinase, at high concentrations of fungicide (100 ppm and 150 ppm). When T. harzianum was used as a biocontrol agent, the detected hydrolytic enzyme activities increased compared with the other treatments.
Keywords: Alternaria solani; Fruit rot; Tomato; Cellulolytic activity; Pectinolytic activity
Contact information: a: Biology Department, Faculty of Science, Jazan University, Jazan, Saudi Arabia; b: Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Egypt; c: Academic Campus for Girls, Jazan University, Jazan, Saudi Arabia;
* Corresponding author: tabdelghany@yahoo.com
INTRODUCTION
Tomato (Lycopersicon esculentum L.) is an important fruit crop that can be infected by numerous serious diseases (Iftikhar et al. 2017). Diseases are a major impediment in the production of crops, as they can decrease productivity by up to 100%. Viruses, bacteria, fungi, and nematodes attack tomato plants at all stages of development and disseminate by means of air, soil, water, seeds, and vectors (Agrios 2005). Many phytopathogenic fungi are known to cause damage to the aerial organs of tomato plants, especially under storage conditions. Several studies have reported that the main pathogenic fungi that cause post-harvest disease in tomatoes are Rhizopus stolonifer (Schena et al. 1999), Botrytis cinerea (Lee et al. 2006), and Alternaria alternata (Feng and Zheng 2007; Kokaet al. 2017). Chaerani and Voorrips (2006) reported that the diseases appeared on the foliage (early blight), seedling basal stems (collar rot), adult plant stems (stem lesions), and fruit (fruit rot) of tomatoes as a result of A. solani infection. Chaurasia et al. (2013) stated that fruit rot caused by A. solani is the most destructive disease that occurs during storage, transportation, and selling of tomatoes. The Curvularia sp. was recently reported to be another causative agent leading to rotting of tomato fruit (Iftikhar et al.2017).
Changes in agricultural technology have been a great operator in shaping the future of agriculture. The development of natural biocontrol agents and nanomaterials could open up novel applications in plant biotechnology and agriculture (Scrinis and Lyons 2007; Abdel-Ghany et al. 2016, 2017, 2018a,b; Abdel-Ghany and Alawlaqi 2018). Numerous applications of chemical pesticides may adversely affect the soil microflora, soil enzymes, and physico-chemical characteristics such as the pH, salinity, and alkalinity, which is deleterious to soil fertility. Microorganisms are considered to be the origin of enzymatic activity in soil (Mohiuddin and Mohammed 2014). Therefore, there is a close relationship between pesticides and microorganisms because these chemicals may have a damaging effect on non-target microorganisms, and most of these pesticides could be metabolized by various microorganisms, which results in modulation of their activity (Walia et al. 2014). Various bioagents and plant extracts are reported to effectively repress several plant pathogens. The biological control of plant pathogenic fungi has received considerable attention as an alternative strategy. The utilization of the antagonistic properties of Trichoderma spp. against several plant diseases has been the subject of various studies (Wolffhechel and Jensen 1992; Ushamalini et al. 1997; Devi et al. 2017).
Hydrolytic enzymes play a great role in infecting fruit and vegetables with fungi, thus causing deterioration and spoilage. During infection, fungi and its hyphae spread in intercellular spaces and inside of the parenchyma cells of fruit and secrete enzymes that cause tissue maceration, leading to fruit corruption (Srivastava and Misra 2017). The degree to which fruits rot during infection depends on the ability of a pathogen to produce cellulolytic and pectinolytic enzymes. Various cell wall degrading enzymes have been shown to be secreted by phytopathogens (Yao et al. 1996). Pectin is a crucial ingredient for the organization of plant tissue. When it is combined with hemicellulose and cellulose microfibrils, it results in enhanced cell wall integrity and provides texture to vegetables and fruits (Pilnik and Voragen 1991). Pectinases are groups of enzymes that attack pectin and depolymerize it through hydrolysis and trans elimination, as well as by the de-esterification process, which is hydrolysis of the ester bond between the methyl and carboxyl of pectin (Gummadi and Panda 2003). Fungal pectinases are used in the food industry for clarification and to increase the production of fruit juice (Henriksson et al. 1999). In a recent study, approximately 50 fungus strains were isolated from infected fruits and tested for their pectinase production represented by polygalacturonase (PG) (Suryam and Charya 2018).
To the best of the authors’ knowledge, no fungicides have been approved for postharvest treatment of tomatoes to prevent or inhibit the development of A. solani rot; therefore, this study compared the effectiveness of a safe natural control with a commercial fungicide at controlling tomato fruit rot. This study was also done to determine the pectinolytic and cellulolytic activities of the causative agents of tomato fruit rot.
EXPERIMENTAL
Fungi Diseased Sample and Isolation of the Causal Pathogen
Tomato fruits that showed symptoms of rot were collected from agricultural fields in the Jazan region, Saudi Arabia, and brought to the laboratory. Using a sterilized blade, the diseased parts of the fruits were cut into small pieces (3 mm to 4 mm) and their surfaces were sterilized with 0.1% HgCl2. The sterilized pieces were then inoculated on a potato dextrose agar medium and incubated at 25 °C. The developed fungal culture was purified by the hyphal tip cut method, and the pure culture was identified as A. solani based on the characteristics of the hyphae, conidiophores, and conidia (Ellis 1971; Rotem 1994).
Confirmation of the Pathogenicity of the Causal Pathogen
A pathogenicity test of the isolated fungus was conducted using healthy and uninjured tomato fruits of the same type as the infected fruits. The fruits were washed thoroughly with sterilized distilled water. A conidial suspension of isolated fungus (106 spores/mL) in 50 mL of sterile water was prepared. The fruits were pricked using sterilized pins and inoculated by the spore spray inoculation method. The inoculated fruits were incubated in a moistened chamber at 25 °C for 5 d to develop disease symptoms, and fungus from the artificially induced diseased symptoms was re-isolated to confirm the pathogenicity of the fungus.
Effect of T. harzianum Culture Filtrates and Chemical Fungicide on Growth and Sporulation of A. solani in vitro
Trichoderma harzianum, used as a biological control agent, was provided by the Regional Center for Mycology and Biotechnology at Al-Azhar University (Cairo, Egypt). It was grown in 100 mL of Czapek-Dox medium (GEM Chem, Maharashtra, India) for 7 d at 25 °C, and then the metabolized medium was filtered through Whatman No. 1 paper. The filtrate was used at three concentrations (25%. 50%, and 75% filtrate volume). The Czapek-Dox agar medium components were prepared in 100 mL of filtrate metabolized medium with water. A. solani (6-d old 5-mm mycelial disc taken from an actively growing culture) was cultivated at the center of a plate containing the previously mentioned medium and incubated at 25 °C for 6 d. The Czapek-Dox agar medium was supplemented with Carbomar (methyl benzimacold-2-ylcarbamate manufactured by Gyangsoetrnal, China) as a chemical fungicide at concentrations of 50 ppm, 100 ppm, and 150 ppm. A. solani (6-d old 5-mm mycelial disc taken from an actively growing culture) was cultivated at the center of a plate containing Czapek-Dox agar medium with the different concentrations of fungicide and incubated at 25 °C for 6 d. The medium without any treatments served as the control. The inhibition percentage of the fungus was calculated after the incubation period using Eq. 1,
(1)
where C is the radial growth of the fungus in the control (cm), and T is the radial growth of the fungus in the treated sample (cm).
Effect of Chemical Fungicide on Growth T. harzianum in vitro
The Czapek-Dox agar medium was supplemented with Carbomar at concentrations of 50 ppm, 100 ppm, and 150 ppm. T. harzianum (6-d old 5-mm mycelial disc taken from an actively growing culture) was cultivated at the center of a plate containing Czapek-Dox agar medium with the different concentrations of fungicide and incubated at 25 °C for 6 d. The medium without fungicide served as the control. The inhibition percentage of the fungus was calculated according to Eq. 1
Test of Antagonism by the Dual Culture Technique
Trichoderma harzianum was used to evaluate the biocontrol potential against A. solani via the dual culture technique on Czapek-Dox agar medium. Mycelial plugs (6-d old 5-mm mycelial disc taken from an actively growing culture) of A. solani and T. harzianum antagonists were placed 3 cm apart from each other on the same Czapek-Dox agar medium plate. Each plate received two discs, one with T. harzianum mycelium and the other with A. solani mycelium. The plates were incubated at 25 °C for 6 d. After the incubation period, the antagonistic activity and morphological changes were recorded.
Test Conditions for Enzyme Production by Fruit Rot from A. solani
The medium used for enzyme production was treated with Trichoderma harzianum filtered at three concentrations (25%, 50%, and 75%), a chemical fungicide at different concentrations (50 ppm, 100 ppm, and 150 ppm), and a dual culture of T. harzianum/A. solani discs (1/2; 2/2; and 1/2 discs, each disc containing 106 spores). Media without any treatment were used as the control.
Carboxymethyl Cellulase Enzyme Production and Assay
Two 6-d old 5-mm mycelial discs taken from an actively growing culture of A. solani were cultivated in a medium consisting of 5.0 g/L NaNO3, 2.0 g/L yeast extract, 1.0 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, and 0.001 g/L FeCl3, with the addition of 1% carboxymethyl cellulose (CMCase) (Deacon 1985) and were incubated for 7 d at 25°C. At the end of the incubation period, the medium in each flask was filtered through Whatman No. 1 filter paper and the culture filtrate served as the enzyme solution. The CMCase (β-1,4-endoglucanase) activity was determined using 1.5 mL of 3,5-dinitrosalisylic acid (DNSA) (Eveleigh et al. 2009). The enzyme activity was expressed in international units (IU), which is defined as the micromoles (μmol) of glucose released per min per mL of culture filtrate.
Pectinase Enzyme Production and its Assay
Two 6-d old 5-mm mycelial discs taken from an actively growing culture of A. solani were cultivated in a medium consisting of 20 g/L NaNO3, 1 g/LK2HPO4, 50 g/L MgSO4, 50 g/L KCl, and 0.01 g/L FeSO4, with the addition of 15% pectin and were incubated for 10 d at 25 °C (Mandels 1985). A 0.1M citrate buffer containing 1% pectin (pH =5.8, and 0.5 mL) was placed in a test tube and 0.5 mL of culture filtrate (obtained by filtration through Whatman No. 1 filter paper) was added for pectinase detection. The reaction mixture was incubated at 50 °C for 30 min and terminated by adding 1.5 mL of DNSA reagent. The mixture was then cooled at room temperature. The absorbance was read at 575 nm. The enzyme activity was expressed as the mmol glucose released per min·mL of culture filtrate. One unit (U) of pectinolytic activity is defined as the amount of enzyme that catalyzed the formation of 1μmol galacturonic acid (Minjares-Carranco et al. 1997).
RESULTS AND DISCUSSION
One fungus was isolated from the rotten tomato fruits obtained from the agricultural fields (Fig. 1A), and it was identified as A. solani (Fig. 1B). This assignment agreed with numerous studies (Oladiran and Iwu 1993; Chaerani and Voorrips 2006; Chaurasia et al. 2013). When the isolated fungus from the diseased samples was artificially inoculated on healthy tomato fruits, the fruits were found to be susceptible to the development of fruit rot, which was characterized by dark brown to black lesions with concentric rings. The present findings agreed with those of Devi (2017), who confirmed A. solani as a pathogen for fruit rot disease in tomatoes. During the regular survey of a local tomato growing field, it was observed that most of the tomato fruits suffered fruit rot disease caused by several fungi, and the highest frequency of occurrences was caused by A. alternata, followed by A. solani (Sajad et al. 2017).
Fig. 1. (A) Tomato fruit rot by fungal pathogen; (B) A. solani pathogen isolate; and (C) dual culture technique of the biocontrol agent T. harzianum with A. solani
The obtained results indicated that the culture filtrates of antagonistic T. harzianum (Fig. 1C) gradually inhibited the radial growth of A. solani with an increasing filtrate concentration, but did not completely inhibit growth until a high filtrate percentage was reached (75% by volume), where there was 51.5% growth inhibition (Fig. 2). Generally, Trichoderma spp. are known to have the ability to produce some extracellular lytic enzymes that are involved in the process of antagonism against a variety of pathogenic organisms. Chohan et al. (2015) reported that T. harzianum and T. viride inhibited 67.8% and 59.6% of A. solani growth, respectively. Additionally, 79.5% of A. porri growth was inhibited by T. harzianum (Chethana et al. 2012). In contrast, the chemical fungicide inhibited 76.9% of A. solani growth (Fig. 2). To evaluate the using of chemical fungicide with the biocontrol agent T. harzianum against A. solani, these results in Fig. 3 confirmed that chemical fungicide affect the growth of the T. harzianum. Therefore, the combined application of filtrate metabolized medium of T. harzianum with the chemical fungicide is preferable than using T. harzianum spores.
In addition to the suppressive effect of the T. harzianum filtrate on A. solani growth, the microscopic study revealed that the T. harzianum culture filtrate changed the size, number, and shape of its conidiospores (Fig. 4), which are the conidial germination and sporulation of the A. solani. The chemical fungicide had a more deleterious effect on the morphological characteristics of A. solani than the T. harzianum filtrate, as the conidiospores were not developed and their longitudinal sections did not appear. Also, many chlamydospores were recorded as a result of fungicide treatment (Fig. 4). Kamala and Indira (2011) reported that Trichoderma isolates and their culture filtrates presented a high efficacy against phytopathogens. The present investigation indicated that T. harzianum was effective, and these results were in accordance with those of Ushamalini et al. (1997).
The antagonistic effects of Trichoderma spp. against pathogens might have been because of the production of non-volatile toxic/antibiotic substances that were diffused in the medium. These explanation agreement with other authors (Zhang et al. 2014; Zhang et al. 2016), reported that the influence on the microorganism development and inhibition it is associated with antibiotic production, as well as, volatile compounds.
Fig. 2. Growth of A. solani treated by the T. harzianum culture filtrate (1: control; 2: 25%; 3: 50%; and 4: 75% v/v) and chemical fungicide (1: control; 2: 50 ppm; 3: 100 ppm; and 4: 150 ppm); the data is expressed as the average ± standard deviation
Fig. 3. Effect of chemical fungicide (1: control; 2: 50 ppm; 3: 100 ppm; and 4: 150 ppm) on growth of T. harzianum; the data are expressed as the average ± standard deviation/
The results from the dual culture assay showed that T. harzianum inhibited the mycelial growth of A. solani. It actively grew as a single culture (Fig.1B), but the dual culture assays showed that T. harzianum was efficient at reducing the mycelial growth of the pathogen and it also sporulated on the pathogen colony (Fig. 1C).
The microscopic study of the dual culture assays revealed that the A. solani conidiospores were deformed compared with when it grew alone (Fig. 5).
Fig. 4. Morphology of A.solani treated by the T. harzianum culture filtrate and chemical fungicide: C: control; T: 75% (by vol.) T. harzianum culture filtrate; and F: 150 ppm chemical fungicide
Fig. 5. Morphology of the A. solani (A) and of the dual culture of T. harzianum with A.solani (A+T)
The inhibition of β-glucan synthase activity in the pathogenic fungi by Trichoderma spp. has been reported in the literature (Fogliano et al. 2002), which prevents the rebuilding of the host cell wall. Recently, 12 isolates of Trichoderma spp. exhibited remarkable biocontrol potential against pathogenic fungi, including T. harzianum (Iftikhar et al. 2017). In the present investigation, A. solani produced both cellulolytic and pectinolytic enzymes in vitro (Figs. 6 and 7).
Fig. 6. CMCase activity of A. solani treated by the T. harzianum culture filtrate (1: control; 2: 25%; 3: 50%; and 4: 75%, by vol.), chemical fungicide (1: control; 2: 50 ppm; 3: 100 ppm; and 4: 150 ppm), and T. harzianum spores/A. solani spores (1: control; 2: 1/2; 3: 2/2; and 4: 1/2 discs); the data is expressed as the average ± standard deviation
Fig. 7. Pectinase activity of A. solani treated by the T. harzianum culture filtrate (1: control; 2: 25%; 3: 50%; and 4: 75%, by vol.), chemical fungicide (1: control; 2: 50 ppm; 3: 100 ppm; and 4:150 ppm), and T. harzianum spores/A. solani spores (1: control; 2: 1/2; 3: 2/2; and 4:1/2 discs); the data is expressed as the average ± standard deviation
It is widely accepted that the production of these enzymes is a major means by which these fungi invade the host tissue, which agrees with the experimental findings of Ramachandran and Kurup (2013), who showed that pectinase-producing fungi cause spoilage of fruits and vegetables. The activity of these enzymes decreased with an increase in the T. harzianum filtrate percentage and chemical fungicide concentration. A. solani failed to produce hydrolytic enzymes, particularly pectinase, at the high fungicide concentrations (100 ppm and 150 ppm). When T. harzianum was used as a biocontrol agent, the detected hydrolytic enzyme activities increased compared with the other treatments. The enzyme activities at different spore doses did not differ remarkably. The increase in the enzyme activities in these cases (T. harzianum co-cultured with A. solani) may have been because of T. harzianum activity, rather than the A. solani. This differed from the explanation of Smitha et al. (2017), who reported that hydrolytic enzyme assays of T. viride co-cultured with pathogens showed an increased activity of cellulase, chitinase, and pectinase over a monoculture, which confirmed the positive induction of enzyme secretion by Pythium spp., Alternaria spp., and Fusarium spp. The production and activity of pectinolytic and cellulolytic enzymes detected in vitro suggested their active role in the development of disease by A. solani in tomato fruits. An earlier study by Anand et al. (2008) confirmed the role of these enzymes in fruit rot pathogenicity.
CONCLUSIONS
- Combined application of fungicide (methyl benzimacold-2-ylcarbamate) with the T. harzianum metabolites were found to be more effective than its spores in controlling phytopathogens and its hydrolytic enzymes.
- The T. harzianum metabolites were more effective than its spores at repressing hydrolytic enzymes.
Conflict of Interest
The authors declare that they have no conflict of interest.
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Article submitted: October 16, 2018; Peer review completed: January 1, 2019; Revised version received: January 6, 2019; Accepted: January 7, 2019; Published: January 10, 2019.
DOI: 10.15376/biores.14.1.1591-1602