W. Kaewduangta1*, W. Sutthisa2, M. Nakornriab3
and P. Rermkratog1

1Department of Agricultural Technology, Faculty of Technology, Mahasarakham University, Mahasarakham 44150, Thailand.
2 Department of Biology, Faculty of Science, Mahasarakham University, Mahasarakham 44150, Thailand.
3Department of Chemistry, Faculty of Science, Mahasarakham University, Mahasarakham 44150, Thailand.

Abstract

The effect of crude extracted and essential oils was comparedfor controlling anthracnose disease in Dendrobium ‘Earsakul’.Dendrobium ‘Earsakul’ was tested to determine the efficiency of five Thai medicinal herbs: galangal, garlic, lemongrass, turmeric and ginger on the growth inhibition of Colletotrichumsp. as the pathogens ofanthracnoseor leaf blight disease.Each medicinalplant was blended and macerated in 95% ethyl alcohol and sterile distilled water at ratio 200 g: 400 mL for seven days. Crude extracts and essential oil were tested for inhibition of their antifungal potentials against Colletotrichum sp. by food poison technique.Results in the laboratory and under greenhouse condition showed that 8,000 ppm essentialoilof ginger inhibited the growth of Colletotrichumsp.both before and after disease infection at 100% similarwith the chemical compound Mancozeb.

Keywords: Dendrobium ‘Earsakul’,plant extract,essential oil,Thai medicinal herb,Colletotrichum sp.

Introduction

Anthracnose or leaf blight disease is caused by fungi in the genus Colletotrichum, a common group of pathogens that are responsible for diseases on many plant species. Anthracnose infects the aerial portion of the orchidandleaves are most often attacked. Leaf tips turn brown, beginning at the apex and proceeding toward the base. Dark brown or light gray patches develop, sometimes as concentric rings or as numerous dark bands across the leaf. The affected area is usually sharply defined and somewhat sunken, while the remainder of the leaf appears normal.  Sporing bodies develop in the infected area. Flowers develop watery, black or brown pustules which are usually raised and occur on the underside of older sepals and petals. The spots may merge and cover the entire flower1,2. The control of this pathogen remains a challenge and is still based upon multiple applicationsof fungicides. Chemical control is effective and efficient but can lead to the development of pathogen resistance, chemical residues in fruit,phytotoxicity to other organisms or environmental and public health problems, as well as the occurrence of fungicide resistant pathogen strains.This has stimulated research on alternative methods to control diseaseswhich are needed because of the negative effects of synthetic chemicals which increase the risk of high levels of toxic residues3,4,5,6.Natural plant extracts are important sources of new agrochemicals with large antimicrobial spectrumproperties for the control of plant diseases7,8. However, the potential toxic effect of applying pesticides toplants on soil beneficial organisms needs to be addressed.Another method is to use plant pathogen control substances which are environmentally friendly. Both these methods leavetoxic residues in the environment butdo not harm the ecosystem.Disease management usingplant essential oils has been applied as an eco-friendly control method9,10. Many plant essential oils showed different levels of antimicrobial efficacies to various ranges of plant fungal and bacterial pathogens, and efficiently reduced the major diseases in crops. Colletotrichum sp. which infectdiverse economically important crops have been successfully managed by plant essential oils and their individual components11.The present work therefore aimed to evaluate plant crude extracts and essential oil for their antifungal activities against Colletotrichum sp. that could possibly lead to their use to control anthracnose disease in vitro and in the greenhouse

Materials and methods

Disease protection activity of the crude plant extracts and essential oils were tested ondetached pseudobulbs or leaves of orchid.Experimentswereconducted in the science laboratory at the Department of Agricultural Technology, Faculty of Technology, Mahasarakham University, Thailand.

Plant material
Topreparetheexperimental Dendrobium ‘Earsakul’ orchidplants,the first step involved inducingprotocorm-like bodies of orchid to seedlingson MSmedia12with 0.5 mg/L benzylaminopurine(BAP) and 0.5 mg/L dichlorophenoxy acetic acid (2,4-D)  for 8 weeks.Seedling growth was then inducedonVWmedia13with 15 mg/L chitosan for 8 weeks withsubculture once a month. Finally, the seedlings were transplantedin thegreenhouseafter one year.

Isolation of target pathogen
Colletotrichumsp. was isolated from pseudobulbs or leaves of orchid showing anthracnose lesions. An isolate of the pathogen grown as a pure culture was maintained in PDA (potato dextrose agar) medium as a stock culture.

Inoculum disc: Seven days old culture of the test fungus was used for the preparation of inoculum discs 5 mm in diameter.

Preparation of plant crude extract and essential oils
Sample collection
Potential plant crude extractsand essential oilswere selected by screening the efficiency of five Thai medicinal herbs (galangal, garlic, lemongrass, turmeric and ginger) on the growth inhibition of Colletotrichumsp.

The method for the preparation of plant crude extracts and essential oils from five Thai medicinal herbs (galangal, garlic, lemongrass, turmeric and ginger) was as follows.  For plant crude extracts,firstly, fresh plant bulbs or rhizomeswere selected and washed thoroughly 2-3 times with running tap water followed bydistilled water.They werethenartificially heated by air dryingin a hot air oven at 50°C for 72 hrs or until stable dry weight. Secondly, each plant sample was mixed in a blender and the powder was soakedin ethanol.An aqueous extract was prepared by blending200 g of eachplant bulb or rhizome in 400mL95% ethanol for sevendays. The macerate wasfiltered through double-layered muslin cloth and centrifuged at 8,000 rpm at 10°C for 30 minutes. The supernatant was filtered throughWhatman No. 1 filter paper followed by evaporation using an R-205 Buchi rotary evaporatorto remove the ethanol to obtain concentrates. The crude extracts were kept at 4°C in sterile universal bottles until required foruse.Essential oils were separated from the crude extracts by the water distillation process.

The inhibitory effects of plant extractsandtheir antifungal potentials against Colletotrichumsp. which causeanthracnosedisease in Dendrobium ‘Earsakul’ orchidwere tested by food poison technique14. Each of the plant crude extracts were dissolved in 1%DMSOand a volume of 5.5 mL of eachconcentrate was aseptically poured into aPetri dish followed by the addition of 9.5 mL of melted PDA andthen agitated gently to achieve a thorough mixing of the contents. Forthe control set, no extract was used. After solidification of the media, one inoculum disc of the test fungus was aseptically inoculated upside down at thecenter of the Petridishand incubated at25°C. Average radial growths of the fungal colonies were measuredon the seventh day of incubation. The treatments were as follows:

Experiment 1
To screen the efficiency of plant crude extracts and essential oils for the control of Colletotrichumsp. in vitroby PDA standard medium with plant crude extracts and essential oils for sevendays.Thisexperiment was conducted in a CRD (completely randomized block design) with four replications.Four discs were prepared for repeated experiments per eachreplication. Four individual experiments wereperformed in vitro.

T1
Control (distilled water)
T2
1,500 ppm Mancozeb
T3
10,000 ppm DMSO (1% DMSO)
T4
10,000 ppm Galangal extract
T5
80,000 ppm Garlic extract
T6
7,500 ppm Lemongrass extract
T7
20,000 ppm Turmeric extract
T8
8,530 ppm Ginger extract
T9
3,000 ppm Galangal essential oil
T10
100 ppm Garlic essential oil
T11
500 ppm Lemongrass essential oil
T12
2,500 ppm Turmeric essential oil
T13
8,000 ppm Ginger essential oil

Following observations, the percentage inhibition of diameter growth (PIDG) values was determined according to the equation below:

Inhibition percentage =100-[(Diameter of sample)/(Diameter of control)×100]

Experiment 2
To study the efficiency of plant crude extracts and essential oils for the control of Colletotrichumsp. by the inoculation modified detached leaf technique in vitro.This experiment was conducted at 7×2 factorials in CRD for 14 treatments.Sevenfactor Asubstances were selected from Experiment 1 (Control, Mancozeb, DMSO, 10,000 ppm Galangal extract, 80,000 ppm Garlic extract, 500 ppm Lemongrass essential oil and 8,000 ppm Ginger essential oil),and the twofactor Btime periods were the time ofusage (before and after pathogen infection) with four replications and10 leaves per replication. Following observations, the PIDG values were determined according to the equation below:

Inhibition percentage =100-[(Diameter of sample)/(Diameter of control)×100]

Experiment 3
To study the efficiency of plant crude extracts and essential oils for the control of Colletotrichumsp. by the inoculation modified detached leaf technique in vivo (greenhouse).This experiment was conducted at 6×2 factorials in CRD for 14 treatments.Sixfactor A substances wereselected from Experiment 2 (Control, Mancozeb, DMSO, 10,000 ppm Galangal extract, 80,000 ppm Garlic extract, and 8,000 ppm Ginger essential oil),  and the twofactor B time periods were the time ofusage (before and after pathogen infection) with four replications and 10plants per replication.

Statistical analyses
ANOVA (analysis of variance) was used to determine the effects of anthracnose treatments at both the laboratoryand greenhouse. Means were compared using Duncan’s multiple range tests. Statistical analyses wereperformed using SPSS version 22 (IBM SPSS Statistics 22.Ink).

Results

The efficiency of plant crude extracts and essential oils for the control of Colletotrichum  sp.  in vitro by PDA standard medium with plant crude extracts and essential oilsfor seven days.Laboratoryresults showed that plant crude extracts of galangal (10,000 ppm), garlic (80,000 ppm), and essential oils from lemongrass (500 ppm) and ginger (8,000ppm) inhibited the growth of Colletotrichumsp. at100% compared with the control andthe chemical compound Mancozeb(Table 1). Other treatmentswere noteffective in inhibiting the growth of Colletotrichumsp. Under a compound microscope,the mycelium showed growth and abnormalities such as bent, knotted, twisted and kinkedhypha (Figure1).

Table 1. Efficiency of plant crude extracts and essential oils for control of Colletotrichumsp.  in vitro by PDA medium for seven days

Treatments Inhibition percentage (%) for 7 days
1 2 3 4 5 6 7
Control (distilled water) 0.00b 0.00b 0.00f 0.00f 0.00d 0.00d 0.00e
Mancozeb 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a
DMSO 100.00a 100.00a 65.02d 39.15d 17.06c 0.00d 0.00e
10,000 ppm Galangal extract 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a
80,000 ppm Garlic extract 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a
7,500 ppm Lemongrass extract 100.00a 100.00a 68.65c 45.33c 18.48c 2.19d 0.00e
20,000 ppm Turmeric extract 100.00a 100.00a 71.21b 50.05b 31.98b 10.77c 0.00e
8,530 ppm Ginger extract 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a 85.67b
3,000 ppm Galangal essential oil 100.00a 100.00a 100.00a 100.00a 100.00a 83.64b 69.12d
100 ppm Garlic essential oil 100.00a 100.00a 62.64e 24.46e 0.00d 0.00d 0.00e
500 ppm Lemongrass essential oil 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a
2,500 ppm Turmeric essential oil 100.00a 100.00a 100.00a 100.00a 100.00a 84.72b 71.17c
8,000 ppm Ginger essential oil 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a
F-test ** ** ** ** ** ** **
CV (%)  8.62 28.26 4.06 1.81 1.34 1.86 1.76

** =  significant difference at p =0.01. Means within a column followed by the same letter do not differ significantly according to DMRT.

Fig. 1. Characteristics of Colletotrichumsp. mycelium causing anthracnose disease tested with different treatment for sevendays.  (a) Control (PDA),(b) DMSO,(c) 7,500 ppm  Lemongrass extract,(d) 20,000 ppmTurmeric extract,  (e) 8,530 ppm Ginger extract,(f) 100 ppm Garlic essential oil, (g) 3,000 ppm Galangal essential oil,(h) 2,500 ppm Turmeric essential oil

The efficiency of plant crude extracts and essential oils for the control of Colletotrichum sp. by the inoculation modified detached leaf technique in vitro. Laboratory results for the main study, factor Asubstances and time factor interaction B were significant at 99%. Ginger essential oil (8,000ppm) inhibited the growth of Colletotrichumsp. at100% similar with the chemical compound Mancozebboth before and after disease infection (Table2).

Table 2. Efficiency of plant crude extracts and essential oils for control of Colletotrichumsp. by inoculation modified detached leaf technique in vitro

Treatment F-test inhibition percentage (%) for seven days
1 2 3 4 5 6 7
Factor A (type of substance) ** ** ** ** ** ** **
Factor B (before-after infection) ns ns ns ns ns ns ns
A×B ** ** ** ** ** ** **
Control (A1) 0.00e 0.00e 0.00d 0.00d 0.00e 0.00d 0.00d
Mancozeb (A2) 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a
DMSO (A3) 0.00e 0.00e 0.00d 0.00d 0.00e 0.00d 0.00d
10,000 ppm Galangal extract (A4) 73.50b 69.75b 50.00b 49.75b 47.75b 24.13b 24.13b
80,000 ppm Garlic extract (A5) 50.00c 49.75c 49.75b 24.69c 24.44c 24.00b 24.13b
500 ppm Lemongrass (A6) 24.75d 24.63d 24.38c 24.25c 19.63d 17.25c 13.63c
  8,000 ppm Ginger essential oil (A7) 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a
before pathogen infection (B1) 50.000 50.000 46.429 42.857 41.857 38.286 37.714
after pathogen infection (B2) 49.500 48.321 46.179 42.482 41.518 37.536 37.107
A1B1 0.00e 0.00e 0.00 0.00d 0.00 0.00d 0.00d
A2B1 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a
A3B1 0.00e 0.00e 0.00d 0.00d 0.00e 0.00d 0.00d
A4B1 75.00b 75.00b 50.00b 50.00b 48.00b 25.00b 25.00b
A5B1 50.00c 50.00c 50.00b 25.00c 25.00c 25.00b 25.00b
A6B1 25.00d 25.00d 25.00c 25.00c 20.00d 18.00c 14.00
A7B1 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a
A1B2 0.00e 0.00e 0.00d 0.00d 0.00e 0.00d 0.00d
A2B2 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a
A3B2 0.00e 0.00e 0.00d 0.00d 0.00e 0.00d 0.00d
A4B2 72.00b 64.50b 50.00b 49.50b 47.50b 23.25b 23.25b
A5B2 50.00c 49.50c 49.50b 24.38c 23.88c 23.00b 23.25b
A6B2 24.50d 24.25d 23.75c 23.50c 19.25d 16.50c 13.25c
A7B2 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a 100.00a
CV (%) 14.70 9.50 10.80 8.80 12.04 6.02 8.40

ns not significant, ** =  significant difference at p = 0.01. Means within a column
followed by the same letter do not differ significantly according to DMRT.

Table 3. The efficiency of plant crude extracts and essential oils for control anthracnose disease of Dendrobium ‘Earsakul’  in vitro

Treatment F-test of disease index for 21 days
7 14   21
Factor A (type of substance) ** **
Factor B (before-after infection) ns ns
A×B ** **
Control (A1) 0.00 2.25a 3.00a
Mancozeb (A2) 0.00 0.00b 0.00d
DMSO (A3) 0.00 2.13a 2.38b
10,000 ppm Galangal extract (A4) 0.00 0.13b 1.00c
80,000 ppm Garlic extract (A5) 0.00 0.25b 1.25c
8,000 ppm Ginger essential oil (A7) 0.00 0.00b 0.00d
before pathogen infection (B1) 0.00 0.67 1.17
after pathogen infection (B2) 0.00 0.92 1.38
A1B1 0.00 2.00a 3.00a
A1B2 0.00 2.50a 3.00a
A2B1 0.00 0.00b 0.00d
A2B2 0.00 0.00b 0.00d
A3B1 0.00 2.00a 2.00b
A3B2 0.00 2.25a 2.75b
A4B1 0.00 0.00b 1.00c
A4B2 0.00 0.25b 1.00c
A5B1 0.00 0.00b 1.00c
A5B2 0.00 0.50b 1.50c
A6B1 0.00 0.00b 0.00d
A6B2 0.00 0.00b 0.00d
CV (%) 10.08 12.70

ns not significant,  ** =  significant difference at p < 0.01. Means within a column followed by the same letter do not differ significantly according to DMRT.

Discussion

This experiment used the DMSO solvent to compare treatments.Resultsindicated that DMSO had no effect on anthracnoseinhibition compared with the control. This findingwas consistent with Pothikhawet et al. (2013)15who tested alfalfa and radish seeds inoculated with Aspergillusparasiticus or A. niger and then soaked in distilled water, 0.5% DMSO, 1,000 ppm Chi-der and1.2 ppm Nano-Pt for 6 hrsbefore washing by distilled water. The 1,000 ppm Chi-der and1.2 ppm Nano-Pt significantly reduced seed infection but enhanced seed germination compared to distilled water and 0.5% DMSO.Prasoetsang and Subtang(2012)16 studied the effect of solvent on the antimicrobial activity of medicinal plant extraction using 1% DMSO as the negative control.The result ofbroth microdilution assay showed that 1% DMSO did notinhibit thebacteria. Medicinal plant extraction and essential oil treatment forColletotrichumsp.  tocontrol anthracnose disease of Dendrobium ‘Earsakul’  before or after disease infection showed that essential oil of ginger (8,000 ppm) inhibited the growth of Colletotrichumsp. by 100% similar to thechemical Mancozeb. Medicinal constituents in ginger essential oil are zingiberene, zingiberol, bisabolene and camphene in high quantities, and extract of ginger also contains phenolic compoundswith antibacterial,rancid and preservative properties17,18,19.  Jamkratokeet at. (1996)20 studied the effect of Boesenbergia rotunda, Curcuma longa and Zingiberofficinalisextracts on postharvest disease fungi. Results demonstrated that the inhibition characteristics of the tested herbs weresignificant at 10,000 ppm for crude extracts and 1,000 ppm for volatile extracts composed in PDA. Radial growth of almost all the tested fungi wasinhibited by the tested herb extracts with various sensitivity. An identical inhibition (83.67 – 87.60%) was determined on fungal colonies formed by Colletotrichumspp. isolated on PDA supplemented with Z. officinalis crude extract. Shovanet al. (2008)21studied the effect of plant extracts on the growth of Colletotrichumdematiumwhich causes anthracnose insoybean by fungicides, plant extracts and Trichodermasp. in the laboratory. Results showed that the most effective material wasgarlic followed by onion, ginger and neem.

Conclusion

Results in the laboratory and under greenhouse conditions showed that essential oil of ginger at 8,000 ppm inhibited the growth of Colletotrichumsp.  both before and after disease infection at100% similarwith the chemical compound Mancozeb.

Acknowledgements

This research was financially supported by Mahasarakham University 2016.

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