Ginger (Zingiber ofcinale) essential oils against Phytophthora colocasiae -

Ginger (Zingiber ofcinale) essential oils against Phytophthora colocasiae

This study is intended to appraise the fungicidal properties of ginger EOs to combat leaf blight disease of taro, which threatens global taro production. Farmers often hinge on extremely toxic synthetic fungicides to manage diseases, but the residual efects and resistance of chemicals are unavoidable. The microwave-assisted hydrodistillation method was used for ginger EOs extraction and an FTIR (ATR) spectrometer was used to evaluate their chemical composition and citral was identifed as most abundant compound (89.05%) in oil. The pathogen isolated from lesions of diseased taro plants was identifed as and used as test fungus in the present study. Ginger EO was evaluated in-vitro for antifungal properties against mycelium growth, sporangium production, zoospore germination, leaf, and corm necrosis inhibition. Repeated experiments have shown that the concentration of ginger essential oil (1250 ppm) proved to be the lowest dose to obtain 100% inhibition of fungal growth and spore germination, sporangia formation and leaf necrosis assessment. These results are derived from this fungal species and a hypothesis that involves further research on other plant pathogens to demonstrate the overall potency of essential oils. This study references the easy, economic, and environmental management and control of plant diseases using essential oils and byproducts.

Materials and methods

Extraction of essential oil. Fresh ginger rhizomes were harvested and  rhizomes were dried at room temperature and cut into small pieces with a kitchen knife before extraction. Essential oils were extracted with a modified microwave-assisted hydrodistillation machine designed with 800-Watt microwave power, highest power input, (1000 Watt) resonant frequency (2.400 GHz) internal microwave generator voltage (420 VAC) and a working temperature range of 100–300 °C. Equipped with a 5-L glass container for heating plant material mounted with a Clevenger system connected with leak-proof water circulation pipes. The instrument provides a self-water cooling technology and a condensation loop to avoid excessive use of tap water. A device for mechanical mixing is also installed that homogenizes the plant material during extraction. PLC with a color touch screen, convenient for use, and a closed-loop PID automatically controls microwave power, temperature, and working duration, with programming for different plant extractions. For extraction, 200 g of ginger pieces were presoaked for 1 h in 300 mL distilled water and boiled at 100 °C for 30 min and then 150 °C for the next 30 min, and the process was repeated three times to obtain the maximum oil yield. The oil floating on surface of the aqueous distillate was parted from the latter and dried over anhydrous sodium sulfate. EO was kept in 2.5 ml opaque glass bottles enfolded with aluminum foil and stored at 4 °C in the refrigerator for further application.

Essential oil chemical analysis.

A PerkinElmer USA Spectrum Two™ FT-IR spectrometer with analytical equipment was used to perform FTIR spectroscopy with the ATR (attenuated total reflectance) technique to identify the major components of EO. A small drop of EO sample was placed on ATR crystal of FTIR spectrometer using a glass dropper, and the spectra and Peaks were identified and observed on a computer screen. The data were taken three times for authenticated results.

Pathogen isolation.

The pathogen was cultured from lesions of infected leaves with typical blight symptoms, i.e., silvery rings of sporangia and plant sap exudates near necrotic leaf areas, petioles, and corms. The leaves showing disease symptoms were sliced into 2–5 mm² pieces from margins of infected parts adjacent to healthy tissues. The leaf pieces were surface sterilized with 0.1% mercuric chloride for 1 min and then washed twice with sterile distilled water. After drying on sterilized blotting paper, the leaf pieces were then transferred to petridish containing fresh potato dextrose agar (PDA) medium and kept for incubation at 25 °C. Immediately after mycelia appeared on culture plates, it was inoculated to new PDA culture plates to maintain pure culture of fungus. The isolated fungus was identified as Phytophthora colocasiae based on colony morphology, sporangia shape, zoospores, and microscopic observation of mycelium. Pathogenicity established by recapitulation of isolated pathogens to satisfy Koch’s postulates. Pathogen isolates were maintained at 5 °C in a refrigerator and sub-cultured periodically on fresh PDA medium. Sporangium production was induced by inoculation of five culture slants (5 mm) with 10 ml sterilized distilled water in test tubes for 3 days under a bright fluorescent lamp. For the release of zoospores, sporangia were chilled at 4 °C for half an hour and then shifted to 25 °C for 15 min. Zoospores separated from sporangia by sieving with thin filter paper. The volumes of Sporangia and zoospores were calibrated to 103 /ml.

Fungicidal assessment of ginger EO.

Mycelium growth inhibition (MGI) assay. Mycelial growth inhibition was evaluated using the poisoned food technique as described by Ali53 with little modification. The oil was diluted at a Tween 80 to 90:10 (V/V) ratio as a stock solution; a final concentration of 5000, 2500, 1250, 625, 312, and 156 ppm was supplemented with PDA at 50 °C before pouring in Petri dishes. Ten milliliters of PDA medium was poured into every 9 cm sterilized petridish, and each treatment was repeated four times. PDA medium containing only Tween 80 served as a negative control, and a minimum concentration of 156 ppm metalaxyl was added to PDA as a positive control. In the control treatment, only PDA was retained, which was used to compare the growth in all treatments. A 5 mm disc from the edge of actively growing mycelium of test fungus was inoculated and kept for incubation at 25 °C. When the controlled Petri dishes were fully grown, the antifungal potential of the essential oils was evaluated. Mycelial growth inhibition (MGI) was determined by reducing the radial colony growth diameter of the treatment plate (Xt) from the negative control petri dish (Xck). (MGI= Xck − Xt ). From complete or partial mycelial inhibition treatments, mycelium discs already inoculated on treatment plates were transferred to fresh unamended PDA plates to confirm viability of inoculated mycelium after treatment with EO

Efect of ginger EO on sporangia production.

The production of sporangia was measured by liquid dilution method using eight vegetable juice broth (V8) medium amended with EO concentrations as described before for mycelium inhibition assays. Ten discs of 5 mm from 7 days actively growing culture of the test fungus were mixed in (10 ml) sterilized distilled water and centrifugated at 4000 rpm (5 min) for homogenization. A hemocytometer was used to count spores from 10 μL of precipitation. The metalaxyl treatment was retained as an active control and treated with Tween 80, and media without treatment served as the negative control

Inhibition of sporangia and zoospore germination

The essential oils as per concentrations described earlier were dissolved in tween 80 whereas metalaxyl was dissolved in sterile distilled water, and homogenized with (V8) broth, and transferred to test tubes. One-week-old pure culture was inoculated into tubes with 500 μL pathogen suspension pre-adjusted to 106 cells/mL and incubated at 25 °C. Each treatment was repeated three times, and this process was repeated twice. Tree hours later, the percentage of zoospore germination was counted with a hemocytometer under a microscope, and sporangia were observed the day after inoculation.

Assessment of necrosis and sporangia production on leaves

Mature taro leaves detached from healthy plants were inoculated under controlled sterile conditions in an incubator. Before inoculation, leaves were sprayed with essential oil concentrations as described earlier. The metalaxyl used as an active control and treated with only sterile distilled water served as the negative control. After 1 h of treatment, 5 mm discs cut from active growing mycelium culture of fungus was inoculated on the adaxial surface of leaves. Four leaves were inoculated for each treatment, and the experiment was repeated twice. Inoculated leaves were immediately covered in black polythene bags to ensure moisture, and incubated in 90% humidity for 3 days and monitored every day until characteristic symptoms appeared in negative control. Disease evaluation was based on observation of latency period (time until symptoms appeared), diameter of necrotic area and sporangia count on the leaves.

Antifungal assay of EO on taro corms.

Selected taro corms of uniform shape, size, (approximately 200 gm each) bearing no visible signs of damage or substantial distortion were used for the trial. The corms were rinsed under tap water, and immersed in 1% sodium hypochlorite (NaOCl) for 1 min and then washed twice with sterile distilled water for 5 min for surface sterilization. The corms were then submerged to EO for 1 h at different concentrations as previously described, and metalaxyl was used as a control. Taro corms were inoculated by removing three 5 mm deep plugs from the upper, middle, and lower sites of corms. The boreholes were inoculated with 5 mm PDA plugs obtained from margin of active growing culture of P. colocasiae. The boreholes were sealed with the same plugs removed from same site of corm. Each treatment consisted four replications whereas Metalaxyl, served as positive control and no EO as negative control, and the trial repeated twice. Taro corms were kept in a dark humid incubator at 25 °C for one week; then, the corms were cut vertically at inoculation site to check fungus infection.

Results

Chemical analysis of ginger essential oil. The essential oil was extracted from ginger rhizomes by the MAHD method with a yield of 2.5%. EO was analyzed, and found Citralz+e; 3,7-dimethyl 1-2,6 octadienal C10H16O1 (95%) as the major oil compound. Ginger essential oil is widely used as an antifungal and antibacterial agent in numerous ways, such as synergistic effects when supplemented with other biological compounds. Therefore, ginger oil has many characteristics, which determine its importance in different industries. Effect of ginger EO on radial colony growth inhibition. The results showed that increasing the concentration of ginger essential oil significantly inhibited the radial growth of the fungal mycelium. The minimal inhibitory concentration (MIC) for the growth of the radial fungal colony was 1250 ppm and beyond. At the same time, metalaxyl inhibited 100% growth of the fungal colony at a 0.15 mg/mL concentration under the similar conditions. Pre-inoculated mycelium plugs from treatments causing complete mycelia inhibition were removed and transferred onto new PDA plates to confirm the viability of the mycelium, which was not able to grow again and was considered as dead. In this study, citral was evaluated as the most important constituent of ginger EO. At the same time, in this sense, the volatile compounds in ginger may inhibit the growth of the fungi.

Ginger EO against sporangia and zoospore germination. As per our results, with a consistent increase in EO concentration, a subsequent decrease in the germination of zoospores and sporangia. The minimum inhibitory concentration for sporangia and zoospores was 625 ppm. However, the morphology of the sporangia and zoospores was slightly deformed, as observed under the microscope. Alternatively, the trace components of essential oils inhibit germination through a synergistic effect because their hydrophobic properties allow them to penetrate cell membranes. EO can disturb some enzyme functions involving spore germination by prolonging the lag phase in the course of spore germination and cytoplasmic disruption in fungal hyphae, causing thinner, distorted and damaged hyphal walls; the cell wall was observed in Cladosporium cladosporioides, Trichoderma viride, and two other molds from Alternaria.

Reduction of leaf necrosis by ginger essential oil.

Afer a 3-day incubation time, some visible symptoms of necrosis were observed on leaves in the negative control treatments, and certain concentrations of ginger oil were applied at 156, 312, and 625 ppm. In contrast, inoculated leaves sprayed with 156 ppm metalaxyl and essential oil concentrations (1250 ppm) or higher did not show any noticeable symptoms. The results shown that an increase in essential oils concentration significantly decrease the leaf necrosis symptoms. An outcome of fungicidal components present in ginger essential oil, which lyse zoospores or hinder their germination. It also prevents mycelial development and the production of afatoxin, which leads to irreparable changes in the mycelium structure, reduction in cytoplasm and mitochondria dysfunction. These compounds also interfere in electron transmission and interact with ionic channel proteins on the surface of mycelial membrane which may consequently modify the membranes permeability leading to cytoplasm leakage. The results show that curative treatment is more effective in preventing leaf necrosis with EO application.

The maximum inhibition of sporangia was recorded at 1250 ppm with ginger essential oil and metalaxyl treatment as 100% inhibition. Furthermore, when the oil concentration was elevated, some deformities in the morphology of the sporangia witnessed. Ginger EOs contain phytochemicals that have antifungal properties against a wide range of phytopathogenic fungi. The lipoids components of EO enable them to enter the biological membranes, interfering with enzymes that ultimately malfunction and produce uncontrolled cell wall synthesis. Moreover, EO contains several volatile chemicals that may induce cell lysis, impairing sporulation.  At the same time, several volatile chemicals in essential oils are considered to inhibit sporangia development.

Reduction of symptoms on taro corms inoculated with fungus. The mycelial discs inserted into taro corms began infestation 7  days after inoculation, whereas negative control treatments and certain EO treated corms i.e., 1250 ppm or lower concentrations presented characteristic deterioration symptoms. Dark brown spots appeared around the inoculation sites and the surface of corm was apparently impaired. In contrary Metalaxyl treatment and higher concentration of essential oil treatments i.e., 2500 ppm and above did not show any visible symptoms on inoculated corms. Inhibition of infection in taro corms probably because of volatility of EOs that enabled quick absorbance in fibrous tissues of corms. In addition to blight symptoms on leaf, (TLB), disease is also a major cause of severe deterioration in corms after harvest and storage. In contrast, oospores hibernate on the surface of corms for a long time and act as the main inoculant, causing primary infection and leading to epidemics.

Conclusion

In the context of returning to nature, with the innovation of science and technology for improved living standards, people have begun to seek solutions for food hygiene without chemical additives. Therefore, biological pesticides and plant-oriented chemicals have received special attention from researchers because they are environmentally friendly and nonhazardous, sustainable, and effective alternatives against many noxious phytopathogens. Considering the perilous nature and direct/indirect paraphernalia of systemic fungicides on human and environmental health. At the same time, ginger is inexpensive and easily available with high oil yield and possesses extensive biological properties. Conducted this preliminary study using ginger essential oil as an impending alternative for toxic fungicides. Results are derived from this fungal species and a hypothesis that involves further research on other plant pathogens to demonstrate the overall potency of essential oils.

Reference:

Kalhoro, M.T., Zhang, H., Kalhoro, G.M., Wang, F., Chen, T., Faqir, Y. and Nabi, F., 2022. Fungicidal properties of ginger (Zingiber officinale) essential oils against Phytophthora colocasiae. Scientific Reports, 12(1), pp.1-10.