Received date: July 01, 2015; Accepted date: July 09, 2015; Published date: July 12, 2015
Citation: Abdel Ghany TM, Shater ARM, Negm ME, Al Abboud MA, Elhussieny NI (2015) Efficacy of Botanical Fungicides against Curvularia lunata at Molecular Levels. J Plant Pathol Microb 6:289. doi:10.4172/2157-7471.1000289
Copyright: © 2015 Abdel Ghany TM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Juniperus procera and Avicennia marina extracts were tested for their antifungal activity against Curvularia lunata isolated from rice (Oryza sativa L.). Eextracts were able to supress C. lunata at high concentration 3 mg/ml. J. procera extract retarted the growth C. lunata by 88.42% while A. marina extract was less effective (37.50%). Nucleic acids content of C. lunata was reduced by both plant extracts especially J. procera extract in a distinct reverse proportion relationship when compared with the untreated samples. The antifungal effect of J. procera and A. marina extracts were explored at the molecular level, using random amplification of polymorphic DNA (RAPD). The results demonstrated polymorphic banding pattern. Secondary metabolites analysis of C. lunata revealed that both plants extracts were capable of blocking biosynthesis of some secondary metabolites including curvulalic acid and lunatin.
About 738 million metric tons rice are produced annually , therefore Gautam et al.  suggests that it is necessary to check the rice grains before allowing distribution for public use. Different fungi are significant destroyers of seeds during storage and crops at seedling stage. They are also remain dormant in seed and transmit to seedlings and mature plant showing different symptoms . Fungi including, Curvularia oryzae, and C. lunata, have been isolated from seeds of different varieties of rice [4-6]. Curvularia phytopathogenic species produce spots in grains and seeds, as well as damage to plant leaves, their species are saprophytes, occur mostly in tropical and subtropical areas, and are isolated from soil, air, organic matter, plants and animals, including humans . This genus of filamentous fungi colonizes soil and vegetation and spreads by airborne spores. Some of the 40 Curvularia species are phytopathogens. Plant diseases range from seedling failure to leaf blight, . Curvularial growth on stored grain, thatch, and other dead plant material looks like smudges of blackish dust. Culture filtrate of Curvularia lunata caused reduction in seed germination, radicle, plumule length and low vigour index of Trigonella foenumgraecum .
It is now realized that chemical fungicides cause serious environmental problems and are toxic to non-target organisms [10-13]. The toxic effect of synthetic chemicals can be overcome, only by persistent search for new and safer pesticides accompanied by wide use of pest control methods, which are eco-friendly and effective. Plant metabolities and plant based pesticides appear to be one of the better alternatives as they known to have minimal environmental impact and danger to consumers in contrast to the synthetic pesticides [14,15]. Rahman  observed that bishkatali, garlic, ginger and neem extract were effective against seed-borne Curvularia lunata. Garlic extract was superior in terms of reducing seed-borne infections by Alternaria spp., Bipolaris sorokiniana, Curvularia lunata, Fusarium spp. of wheat to other extracts followed by ginger and neem [17,18]. Methanolic extracts of Bridelia montana and Scoparia dulcis showed the high antifungal activity against C. lunata and recorded minimum inhibitory concentration below 50 mg/ml. The overall reports provide promising baseline information for the potential use of the crude extracts from medicinal plants in the treatment of fungal diseases caused by Curvularia lunata . The efficacy of leaf extracts of Gliricidia sepium, Tithonia diversifolia, Phyllanthus amarus and Morinda lucida were assessed in vitro  to control C. lunata. The extracts of the four plants suppressed the growth of C. lunata in vitro. Mangroves are medicinal plants and extracts from different parts of them are widely used through the world. These are considered as rich sources of steroids, triterpenes, saponins, tannins, alkaloids and flavonoids. Antiviral, antibacterial and antifungal activity of the extracts from these plants have been shown in former studies [21,22]. A. marina and A. officinalis were used as test plants due to the presence of much evidence that prove their therapeutic value against microbial infections . Also preliminary studies have been demonstrated that the mangrove plant extracts have antibacterial activity against pathogenic bacterial strains; Staphylococcus sp., E. coli and Pseudomonas sp. . Juniperus species have been extensively investigated as a source of natural products with potential antibacterial, antifungal and insecticidal activities [25-27]. Mariana and Camelia,  reported that Juniper oil has inhibition action against Aspergillus niger, Fusarium oxysporum, Monascus purpureus and Penicillium hirsutum. The aim of this study was to determine antifungal activity J. procera and A. marina extracts as safe alternative for controlling C. lunata and their mechanisms on molecular levels.
Isolation and identification
Stored grains of rice (Oryza sativa L.) were surface sterilized with 0.1% mercuric chloride for two minutes followed by washings with sterilized water. The surface sterilized grains were transferred on three layered filter paper beds in 9 cm diameter sterilized Petri plates containing Czapek-Dos growth medium. The growing fungus was identified according to the cultural characteristics and spore morphology  as Curvularia lunata.
Juniperus procera and Avicennia marina leaves were collected from Jazan region, KSA. Freash leaves (500 g) of plants were air-dried at room temperature, ground into powder using an electric grinder, then extracted with methanol in a Soxhlet. The solvent was removed using rotary evaporator under reduced pressure at temperature below 50°C. The resulting crude extracts were stored at 4°C in dark until used. Carbomar (Methyl Benzimacold-2-ylcarbamate) from (Gyangsoetrnal, China) was used as chemical fungicide.
Poisoned food technique was used. Potato dextrose agar medium (PDA) suplemented with different concentrations of each palnt extract and carbomar were prepared separately. About 25 ml of the growth medium was poured into each petri-dish and allowed to solidify. Five mm disc of 5-day-old culture of the Curvularia lunata was placed at the center of the petridish and incubated at 27°C for 7 days, the growth was measured in millimeter. For each treatment three replicates were maintained. PDA medium without the methanolic extract served as control . The toxicity eacg\h treatment in terms of inhibition percentage of mycelial growth was calculated by using the formula:
Where Dc=average increase in mycelial growth in untreated samples, Dt=Average increase in the treated samples .
Secondary metabolites detection
After 10 days of incubation period, the growth medium ( PDA broth containing 1 and 2mg/ml of J. procera and A. marina; 5 and 25 μg/ ml of Carbomar) of C. lunata containing extracellular metabolites was extracted twice with Chloroform/methanol (2:1 v/v), then concentrated and separated using High-Performance Thin-Layer Chromatography (HPTLC) techniques. Twenty microliters of the samples, with or without treatment, were applied to HPTLC plates (10 cm × 10 cm, 0.2 mm silica gel Merck 60 F 254 precoated plate; Merck Darmstadt, Germany) using CAMMAG LINOMAT 5 application system. The TLC plates were eluted for the detection of extracellular metabolites in toluene/ethyl-acetate/90% formic acid 5:4:1 (TEF). Once the runs had finished, the plates were observed under visible and ultraviolet at 254 and 365 nm illumination. Griseofulvin dissolved in chloroform/ methanol 2:1 was used as standard in all cases and relative retention factor value (Rf value) to griseofulvin were calculated as Rfg. To identify the metabolites the absolute Rf and the relative Rf to griseofulvin (Rfg) were measured. Secondary metabolites were identified by descriptions in literature and comparison with the available standard was made [32-35]. Retention factor value (Rf value): The distance that the spot of a particular compound moved up on the TLC plate relative to the distance moved by the solvent front is called the retention factor or Rf value. The Rf values of individual secondary metabolites were calculated by following, Rf=Distance traveled by the compound/Distance travelled by the solvent. Qualitative determination of secondary metabolites were done by comparing the Rf values calculated using CAMMAG TLC scanner at Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University Cairo, Egypt.
DNA from each treated and untreated C. lunata mycelia was extracted using DNeasy kit (Qiagen-Germany). PCR amplification was conducted using 5 random primers listed in Table 1 . The amplification was performed in a thermal cycler programmed as follow: Cycle-1: 94°C for 10 minute, Cycle-2 : 97°C for 15 minute, 36°C for 1 minute, 72°C for 2 minute Repeat for 40 times, Cycle-3: 72°C for 10 minute and Cycle-4: 4°C for 30 minute. PCR amplification products were separated by electrophoresis using 1.5% agarose gel and 100 bp ladder. The products were visualized and photographed under UV transilluminator.
|Primer code||Nucleotide sequence 5`-3`|
Table 1: Ten-mer random primers.
The banding patterns of RAPD were scored using Gel analyzer 2010a software and data fed to the computer as 1 and 0 for the presence and absence of bands, respectively. Pair-wise comparisons of genotypes, based on the presence or absence of monomorphic and polymorphic fragments, were used to generate percentage of polymorphism.
Curvularia lunata was found associated with the contaminated seeds of rice (Oryza sativa L.). Under microscopic examination, Conidia were smooth-walled, olivaceous brown, end cells somewhat paler, conidia obovoidal to broadly clavate, curved at the subterminal cell, 25- 30 × 10-14 μm.Conidiophores were erect, septate, unbranched, flexuose in the apical part, with flat, dark brown scars., 3-septate. Morphological characteristics of the isolated fungus were in agreement with Ellis, . Earlier study analyzed 25 rice samples that were contaminated with one or more fungal genera like Aspergillus, Fusarium, Curvularia, Cladosporium and Alternaria . Curvularia trifolii, C. lunata and C. brachyspora were isolated from 40 different paddy samples . Isolated Curvularia lunata colonies showed morphological variation on the two used media (Czapek-Dox and potato dextrose agar media). C. lunata colony appeared light-brown to grey on Czapek-Dox medium while it turned to buff light when grown on PDA medium (Figure 1). The fungus failed to sporulate on PDA medium but heavy sporulation occurred on Czapek-Dox agar. The differences in media composition particularly, the carbon and nitrogen sources might be the cause of these phenomena. Lanisnik Rizner and Wheeler,  and Lanisnik Rizner and Romih,  observed that C. lunata was dark brown when grown in malt extract broth, however, it remained white or very pale brown when grown in yeast nitrogen base broth medium.
C. lunata was completely inhibited by 50 μg/ml of chemical fungicide Carbomar. Similar results were obtained by Shikha et al.  who used Carbendazim (10 mg/ml) to inhibit C. lunata growth. Topsin and mencozeb suppressed the growth of Curvularia sp. by 50% . Even though effective and efficient control of seed borne pathogenic fungi can be achieved by the use of synthetic fungicides, they cannot be applied to grains because of their toxicity [12,43]. Thus, there is a need for safe, nontoxic, eco-friend and cheap alternative approaches to store grains/cereals for human consumption. Exploration of safe alternative antifungal agents, especially the plant extracts has intrinsic worth. Plant extracts as potential antifungal compound has been explored against several fungal species. In this study, two plants (J. procera and A. marina) extracts reduced C. lunata mycelium growth (Table 2). Although none of these plants extracts inhibited the mycelial growth 100%, these plants have been reported to possess antifungal properties against C. lunata. J. procera extract was more effective against C. lunata than A. marina. C. lunata growth was inhibited by 88.42 and 37.50 % when growth medium was treated with J. procera and A. marina extracts respectively. Recently, Abdelghany  reported the antifungal effect of J. procera crude extract against growth of Aspergillus flavus and Fusarium oxysporum. Surprisingly, A. marina extract at low concentrations induced the vegetative growth of C. lunata (Figure 2), but it suppressed the fungal sporogenisis. This may be due to A. marina extract component(s) reduced the activity of sporogenesis enzymes which saved energy for mycelial growth. Antifungal activity of A. marina was investigated Nayak et al.,  who concluded that the leaf extract has both antifungal and antibacterial activities. Antifungal metabolites of A.marina include alkaloids, flavonoids and related compounds, fatty acids, oxygen heterocyclics, proanthocyanidins, quinones, stilbenes, terpenoids and triterpenoid, saponins .
|Plant extract Concentration (mg/ml)||J. procera||A. marina||Carbomar|
|Colony radius(mm)||Inhibition (%)||Colony radius(mm)||Inhibition (%)||Concentration (µg/ml)||Colony radius(mm)||Inhibition (%)|
|0.0||5.60 ± 0.10||0.00||5.60 ± 0.10||0.00||0.00||5.60 ± 0.10||0.00|
|1.0||5.20 ± 0.10||41.89||6.70 ± 0.12||0.00||5.00||4.30 ± 0.02||23.21|
|1.5||3.90 ± 0.12||48.64||6.70 ± 0.15||0.00||10.0||3.20 ± 0.14||42.85|
|2.0||3.20 ± 0.15||59.86||5.50 ± 0.10||8.00||25.0||1.70 ± 0.12||69.64|
|2.5||2.50 ± 0.12||76.08||4.30 ± 0.12||23.21||50.0||0.00 ± 0.00||100|
|3.0||0.80 ± 0.06||88.42||3.50 ± 0.10||37.50||100||0.00 ± 0.00||100|
Table 2: Effect of plant extracts and Carbomar different concentrations on the growth of Curvularia lunata.
Natural antifungal agents can be potential exploited in controlling the growth of fungi consequently inhibiting secondary metabolites production [46,47]. Therefore in recent years, much attention has been given to the preservation of grains by natural products so that the growth and mycotoxin production may be effectively retarded. Curvularia lunata secondary metabolites qualitative profile (Table 3 and Figure 3) indicates the ability of J. procera and A. marina extracts and carbomar to alter its metabolic pathways. Curvularia sp. secondary metabolites profile has been investigated by Trisuwan and his team , curvulapyrone, curvulalide and curvulalic acid, modiolides A and B, pyrenolide A, stagonolide E, mycoepoxydiene, and deacetylmycoepoxydiene were identified. J. procera extract low concentration (1.0 mg/ml) was capable of blocking the biosynthesis of curvulapyrone and radicinin while higher concentration (2 mg/ml) blocked the synthesis of lunatin and curvulalic acid in addition to curvulapyrone and radicinin. Deacetylmycoepoxydiene, mycoepoxydiene, cytochalasin B, radicinin and curvulapyrone were not synthesised by C. lunata in both media treated with low and high concentration of A. marina extract, while lunatin was not synthesised only in A. marina high concentration media. Except of cytoskyrins, all investigated metabolites were blocked by both carbomar concentrations. C. lunata produces cytochalasin B , that is a mycotoxins acts on the cytoskeleton and may show other bioactivities . A. marina extract and carbomar inhibited cytochalasin B.
|Secondary metabolite||Control||J. procera extract concentration (mg/ml)||A. marina extract concentration(mg/ml)||Carbomar Concentration (μg/ml)|
Table 3: C. lunata secondary metabolites profiles at different concentration of plant extracts and Carbomar.
Al-Qurainy and Abdel-Megeed  stated that some antifungal agents have the capacity to alter the genetic material. This statement pushed this research to investigate nucleic acids content in both treated and untreated C. lunata samples (Table 4). Both plants extracts induced an increase in total DNA and RNA content of C. lunata especially at relatively low concentrations 1 and 2 mg/ml growth medium. On the other hand, it was found that DNA and RNA content decreased with the increase plant extracts specially with using J. procera. Carbomar sharply decreased the nucleic acids content of C.lunata compared with the control and plant extracts used.
|Extract Conc. (mg/ ml)||J. procera extract||A. marinaextract||Carbomar|
|0.0||8.75 ± 0.26||60.50 ± 0.10||8.75 ± 0.26||60.50 ± 0.10||0.00||8.75 ± 0.26||60.50 ± 0.10|
|1.0||10.20 ± 0.15||65.25 ± 0.12||9.30 ± 0.16||75.25 ± 0.10||5.00||4.05 ± 0.20||45.00 ± 0.20|
|1.5||9.10 ± 0.25||69.10 ± 0.20||11.50 ± 0.20||79.10 ± 0.09||10.0||4.15 ± 0.20||35.00 ± 0.10|
|2.0||6.15 ± 0.10||40.22 ± 0.20||10.00 ± 0.05||60.22 ± 0.10||25.0||3.05 ± 0.15||31.00 ± 0.09|
|2.5||6.25 ± 0.15||40.30 ± 0.25||7.05 ± 0.10||50.30 ± 0.20||50.0||No fungal growth|
|3.0||4.30 ± 0.20||40.00 ± 0.20||5.15 ± 0.26||42.00 ± 0.08||100|
Table 4: Effect of different concentrations of J. procera, A. marina extracts and Carbomar on DNA and RNA concentrations (mg/g mats dry weight of mass) of Curvularia lunata.
Alternation in growth, sporulation, secondary metabolites pathways and nucleic acids content of C. lunata treated with J. procera extract, A. marina extract, and carbomar drive this study to investigate the potential genotoxic effects of the J. procera and A. marina extracts at molecular level using RAPD analysis. Two levels in the form of two different concentrations were tested for each extract against wild type (untreated sample). Carbomar treated samples were the positive control. RAPD analysis was extensively used as molecular analysis tool for Curvularia sp. [52-55]. Five 10-mer random primers were tested for RAPD analysis. Four primers (P1, P2, PE7 and PE20) produced considerable bands (Figure 4) while the primer P14 produced no distinct bands. A total of 52 amplified fragments were observed, 33 of them were monomorphic bands while 19 were polymorphic that reflect high degree of polymorphism (36.5%) among all samples. Comparison of each treatment with the untreated sample (wild type) showed that C. lunata treated with A. marina extract showed the highest degree of polymorphism (32%) among its 50 amplified fragment which recommend that A. marina has high mutagenic efficacy; followed by the carbomar which induced 14 polymorphic band (29.2% polymorphism) from 48 total amplified fragments. These molecular markers can demonstrate similarities and dissimilarities between different isolates of same species even when a morphological description is severely limited. J. procera comes finally as its treated C. lunata samples showed the lowest degree of polymorphism with 19% polymorphism and 42 amplified fragments (Table 5). Studying the effect of concentration on mutagenic rate in terms of percentage of polymorphism indicated that for J. procera and A. marina it was clear that there is a proportion between the concentration of the treatment and the percentage of polymorphism in the treated C. lunata samples. A. marina extract low concentration result in 35 monomorphic fragments and 8 polymorphic that represent 18.6% of polymorphism in the treated fungal samples. The high concentration showed higher level of polymorphism with 33 monomorphic fragments and 12 polymorphic fragments (26.7% polymorphism). The same phenomena was noticed while studying J. procera different concentrations (Table 6).This observation gives good evidence to the ability of these plant extracts and chemical fungicide Carbomar to induce mutation as a result of compromising at least one nucleotide as revealed by the disappearance of many genetic bands and change in restriction endonucleases sites as compared with untreated C. lunata. Generally in all cases, polymorphisms were due to the loss and/or gain of amplified bands in the treated samples compared with the control. This result s were agreement with Sameer  in their study on the effect of Calotropis procera latex on Aspergillus terreus. Our findings support this claim that DNA polymorphisms detected by RAPD can be considered as a powerful biomarker assay for detection of the level of DNA damage in various treated C. lunata and to extracts of J. procera and A. marina, as suggested by similar results obtained by Hajar and Nehad  in their study on the effect of Moringa peregrine extract on bacteria and fungi.
|Treatment||Amplification Products||Ten mer random primers codes||Total|
Table 5: Amplification products polymorphism of treated C. lunata with plant extracts and chemical fungicide carbomar. TAF: total amplified fragments; Poly: Polymorphic fragments; Mono: Monomorphic fragments.
Table 6: Polymorphism of C. lunata treated with plant extracts and chemical fungicide carbomar.
Thanks to Regional Center of Mycology and Biotechnology, Al Azhar University, Cairo, Egypt for providing laboratory facilities.