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Ultrastructural studies of the inhibition effect against Phytophthora capsici of rootexudates collected from two garlic cultivars along with their qualitative analysis

Muhammad Azam Khan, Cheng Zhihui*, Xiao Xuemei, Abdul Rehman Khan, Shahjahan Shabbir AhmedCollege of Horticulture, Northwest A&F University Yangling, Shaanxi 712100, PR China

a r t i c l e i n f o

Article history:Received 15 November 2010Received in revised form19 April 2011Accepted 24 April 2011

Keywords:Phytophthora capsiciPhytophthora blightGarlic root exudatesHPLCElectron microscope

a b s t r a c t

The overreliance on and overuse of fungicides is not only a health hazard but also induces naturalresistance in plant pathogens, resulting in an economic burden on agricultural producers and a potentialthreat to natural systems across the globe. It is therefore necessary to identify natural substitutes offungicides. This study was designed to evaluate the inhibitory effect of the root exudates of two differentgarlic cultivars, cv. Gailiang (G064) and Cangshan (G025), against Phytophthora capsici, a pepper fungus.All treatments (T1 to T4, i.e., 25%, 50%, 75% and 100% root exudate concentrations) of garlic cultivar G025showed lower inhibition effects than cultivar G064. An intervarietal inhibition effect comparison at 100%concentration (T4) exhibited a 69.24% decline in hyphal growth for G064 compared with 49.06% forG025. The mycelial growth measured in the control was found to be significantly greater compared withthe garlic treatments. The results of high performance liquid chromatography (HPLC) revealed that G064possessed a large amount of allicin compared with G025. Scanning and transmission electron microscopyshowed that, compared with the controls and hyphae treated with G025, the fungal hyphae treated withG064 exudates were misshaped, fragmented and had a smaller diameter, as well as empty cytoplasmiccontents in the cell wall. Thus, the root exudates of the G064 cultivar had a significant fungicidal effect onP. capsici.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

During the last few years, Phytophthora blight induced byPhytophthora capsici has been recorded as an increasingly severedisease in a wide range of vegetable crops, including cucurbits andpeppers (Babadoost, 2004; Hausbeck and Lamour, 2004; Babadoostand Zitter, 2009). Phytophthora blight can cause up to 100% croploss, which could bankrupt farmers (Babadoost, 2004). P. capsici isa soil-borne oomycete pathogen that causes disease at variousgrowth stages of host plants, including the pepper crop. Thispathogen causes root rot, stem canker, leaf blight and fruit rot inolder plants. Affected plants exhibit sudden wilting and death(Sherf and Mac Nab, 1986). Over-irrigation, heavy rainfall, warmenvironment, a wide host range, and the ability of the pathogen tosurvive in soil and infested seeds are the main reasons for itsspread. Previous studies have failed to identify any cultivar resistantto this pathogen; therefore, different combinations of chemical,cultural and biological control along with sanitary measures areused to manage this problem. Cultural practices such as the

elimination of host weeds and rotation to a non-host crop are oflimited values because of the survival of oospores of P. Capsici fora span of up to 10 years in soil (French-Monar et al., 2003; Hausbeckand Lamour, 2004).

The use of copper fungicides is the most effective treatment, butit is uneconomical (Li, 2002). In some crops of cucumber andpepper, resistance to a common fungicide (Mefenoxam) has beenreported (Lamour and Hausbeck, 2000). Metalaxyl also has excel-lent fungicidal activity, but the development of induced resistanceto these chemicals by some members of peronosporales has causedconsiderable difficulties in its continuous use. In particular, thefailure of Metalaxyl to control naturally occurring Metalaxyl-resistant isolates of P. capsici has been reported. Therefore,mixtures of Metalaxyl with copper oxychloride are being usedextensively to prevent the development of resistance to metalaxylin pepper-growing areas (Oh and Kim, 1992). However, thecontinuous use of fungicides has had several long-term sidenegative effects on human health and the environment (Song et al.,2004; Binod and Bhupendra, 2009), including immune suppres-sion, hormonal disruption, reproductive abnormalities, cancer andthe induction of plant resistance (Ramamoorthy et al., 2001 andGupta, 2004). Soil and ground water pollution are the main envi-ronmental effects of chemicals. Several side effects have been

* Corresponding author. Tel.: þ86 15129183300.E-mail address: chengzh2004@163.com (C. Zhihui).

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reported in many species of insects, mammals and birds by Binodand Bhupendra (2009). The use of non-selective pesticides insoils becomes toxic to beneficial soil microorganisms (fungi,bacteria and protozoa), which dominate both the structure andfunction of natural ecosystems (Pimentel, 1992). Consequently, theuse of copper fungicides in organic agriculture has been restrictedby the European Commission (European Commission, 2002).

With the increasing awareness of the harmful effects of chem-ical control, scientists struggle to explore alternative methods forthe management of crop damage with minimum ecologicalhazards. Among these, plant products that are safe, easily biode-gradable and eco-friendly reflect great potential compared withothers. The efficacy of different plant extracts in terms of their anti-microbial properties has been identified (Afolayan, 2003; Bowersand Locke, 2000; Eksteen et al., 2001; Gulluce et al., 2003; Holand Van-veen, 2002; Magama et al., 2003; Neuhoff et al., 2002;Rohner et al., 2004). Pretorius et al. (2002) identified crude extractsfrom 39 plant species and their anti-fungal potential against seveneconomically important plant pathogenic fungi, and considerablemycelial growth inhibition was obtained with extracts from Aristeaecklonii. Similarly, Demirci and Dollar, 2006 identified cabbage andgarlic plant materials that are effective for the reduction of mycelialgrowth and disease severity in P. capsici of pepper.

The use of biological substances extracted from garlic (Alliumsativum) is found to be an important anti-microbial and anti-lipidemic agent. This agent exhibits anti-bacterial, anti-fungal andanti-viral properties. Thus far, researchers have largely focussed onthe aerial parts of plants (though it may not be practical to add thesame to the soil). In contrast, research on the inhibition effects ofroot exudates is still limited because of the complexity of themethods involved in the collection of root exudates.

The present study was planned to evaluate the efficacy of rootexudates collected from two different garlic cultivars for the inhi-bition of the P. capsici pathogen. Electron microscopy was used toobserve the cytomorphological alterations induced by the rootexudates. High performance liquid chromatography (HPLC) wasused to quantify allicin, an anti-fungal substance found in the garlicroot exudates.

2. Materials and method

2.1. Fungal material

The P. capsici culture was provided by Prof. Dr. Ma Qing, Collegeof Plant Protection, Northwest A & F University, Yangling, China. Theculture was isolated from diseased pepper plants from local fieldsin the Shaanxi province of China. The fungus was maintained ona potato dextrose agar (PDA) medium at 25 � 2 �C.

2.2. Extraction and preparation of garlic root exudates

Two hundred fresh and healthy bulbs of two garlic cultivars (cv.Gailiang（G064）and Cangshan （G025）) were grown in a perlitemedium at 20 �C for 10 days. During culture, the garlic plants wereirrigatedwith distilled water to keep the growingmediumwet. After10 days, all the garlic plants were dug up, washed with tap water toremove perlite and grown hydroponically for 30 days in a growthchamber. Each hydroponic systemwasmainly composed of a cultureplastic pot and an air pump with a time switch. The air was peri-odically recycled by the pump (20 min per h). The exudates werecollected by a continuous root exudate trapping system (Yu andMatsui, 1994; Yu et al., 2000) in which collected water was passeddrop-by-drop through active carbon to trap the exudates. Carbonwashed with acetone was placed for a few minutes in an ultrasoniccleaner to submerge the active ingredient in the acetone from the

carbon. The solution was evaporated with a vacuum evaporator at40 �C and then titrated three timeswith ethyl acetate (CH3COOC2H5),using 50ml each time. The upper transparent portion of the solutionmixed with sodium sulphate (Na2SO4) to absorb the remainingwater. Evaporation was again conducted until the solution was dry.The residues thus obtained were true root exudates (Tang andYoung, 1982) and were stored at �20 �C until further use.

From the 200 bulbs of each cultivar, 0.128 g of garlic root exudatedry matter was collected from G064, and 0.09 g was collected fromG25. We then took an equal amount (0.09 g), measured using ananalytical balance, from each cultivar and dissolved them inacetone; these solutions were mixed with 15 ml of autoclaveddistilled water, obtaining stock solutions (i.e., T4-100%). Threeconcentrations (T1-25%, T2-50% and T3-75%) were prepared fromthe stock solution of each cultivar by adding the calculated amountof autoclaved distilled water. However, the stock solution was usedas treatment T4-100%.

2.3. Effect of root exudates on P. capsici

2.3.1. In vitro effect of root exudates on mycelial growthof P. capsici

The inhibitory effect of different concentrations of root exudatescollected from the two garlic cultivars were examined by themycelial growth rate method. One millilitre from each concentra-tion of root exudates was added in 15ml of PDA in a 9-cm Petri dishand allowed to solidify. A 5-mm mycelial plug of P. capsici froma 7-day old culturewas placed in the centre of each PDA plate underaseptic conditions. All the dishes were completely closed withplastic film bands and incubated for one week in the dark at25 � 2 �C. The inoculated Petri dishes were inspected after 3, 5 and7 days to measure the colony growth of the fungus along twodiameter lines with a vernier calliper. Fungal sensitivity towardsthe concentration of garlic root exudate was expressed in terms ofthe inhibition percentage of mycelial growth and was calculatedaccording to the following formula outlined by Pandey et al. (1982):

Inhibition�%�

¼ Growth in control � Growth in treatmentGrowth in control

� 100

2.3.2. Effect of root exudates on spore germination of P. capsiciThe 10-day-old cultured fungus was used for the spore germi-

nation test. The Petri dishes were kept at 10 �C for 60 min to inducezoospore release. After the cool treatment, the dishes werereturned to 25 �C for 45 min. The inoculum was adjusted to 10�6

zoospores per millilitre with a haemocytometer. Three replicatesper treatment were used. Zoospore germinationwas recorded after2e4 h of culturing under a compound microscope, and a compar-ison was made between the control and both cultivars when thegermination rate of the control’s resting spores exceeded 60%.

2.4. Electron microscopy

Mycelial plugs (1 �1 � 3 mm) of P. capsici of the same age weretaken from the control and both garlic cultivar root exudate treat-ments (T4). These mycelial plugs were prepared for both scanningelectron microscopy (SEM) and transmission electron microscopy(TEM).

In the SEM preparation, the samples were fixed 5 times inphosphate-buffered 3% glutaraldehyde dehydrate with a pH of 6.8for 5, 10, 15, 20, 25 and 30 min, consecutively. Later, the sampleswere placed in increasing concentrations of alcohol (30%, 50% and

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70%) for 15 min and for 20 min (80%, 90%, 95% and 100%) each. Thegraded aqueous series of acetone (25%, 50%, 75%, and 100%) wascritical point dried with CO2 using acetone as an intermediate fluid.The pieces of agar were sputter-coated with plutonium using a JFC-1600 auto fine coater. The fungal hyphae of both the control andtreatments were examined under a JSM-6360LV SEM (JEOL Ltd.,made in Japan) and placed in theMain Hi-Tech Research LaboratoryBuilding of Northwest A & F University, China.

For TEM preparation, the samples were fixed in phosphate-buffered 3% glutaraldehyde, post-fixed in phosphate-buffered 1%osmium tetroxide, and dehydrated in a graded series of ethanolfollowing the procedure described by Bianchi et al. (1997). Plugswere embedded in a plastic fix coating and were dried witha Hitachi Critical Point Dryer. Ultra-thin sections were cut with anLKB ultra tome, stained with uranyl acetate and lead citrate andexamined under a JEOL model JEM-1230 electron microscope.

2.5. HPLC analysis

The quantification of the anti-fungal substance in the garlic rootexudates was conducted using the methods described by Krest andKeusgen (2002) and Zhang et al. (2009). The garlic root exudatesdissolved inmethyl alcohol were filtered through a polyethersulfonmembrane (size 0.45 mm sieves), and 10 ml were then injected intothe HPLC system with a diode array detector. The HPLC softwarewas run with a mixed gradient solution (Solvent A: methanol 85%;B: acetonitrile 12.75%; C: water 2.25%). A Diamonsil C18 (2) 5 m250 � 4.6-mm column was used on a Water 600 controller HPLC,Japan. The spectra were recorded between 200 and 600 nm, andmaximum detection was made at 214 nm for the allicin. Forexternal standardisation, allicin bought from Sigma Internationalwas used with different dilutions.

2.6. Statistical analysis

The experiment was planned in a complete randomised designwith three replicates. The validity of the experiment was obtainedthrough analysis of variance and means separated by Duncan’smultiple range test at a ¼ 0.05. The statistical analysis was per-formed with SPSS software version 16.0.

3. Results

3.1. Efficacy of garlic root exudates on hyphael growth of fungus

Measurementsmade on the thirddayof the culture revealed thatthe garlic root exudates had pronounced inhibitory effects onfungus growth compared with the control (Table 1). However, theintercultivars’ inhibitory effect was significantly different, that is,

the exudates collected fromG064 showedhigher efficacy than thosefromG025. The inhibitory effect induced byG064 (T4)was recordedas 69.24% compared with 49.06% for G025 (T4). Similar but variedtrends pertaining to efficacy levels were observed on the fifth dayof culturing. During the interregnum period, the T4 exhibited63.90% and 45% inhibition for G064 and G025, respectively.

The inhibitory effect of the exudates for both garlic cultivars onfungal growth were persistent till the third round of measurementsmade on the seventh day in comparison with the control (i.e., onlyPDA) (Table 1). The overall results reveal that the garlic rootexudates collected from G064 had a stronger inhibitory effect thanG025. The T4 showed a highly significant inhibition effect, followedby the treatments with 75% exudates (T3) of both cultivars. Thetreatment with 100% root exudates collected from G064 showed70.83% inhibition in comparison with 44.12% in case of G025. Theanalysis of variance (a ¼ 0.05) showed significant differences in allthe treatments of both garlic cultivars compared with the control,as shown in Table 1.

3.2. Effect of garlic root exudates on germinationof P. capsici zoospores

The garlic root exudates also showed inhibitory effects on thegermination of fungal spores. Zoospore germinationwas completelyinhibited by the 100% concentration of G025 (T4). However, thesame observationswere recorded by the 75% concentration of G064(T3) (Table 2). This inhibitory effect is directly linked to theconcentration of garlic exudates. A negligible inhibition wasobserved in the 25% (T1) treatment of G064 and a zero effect inG025at the same concentration. The analysis of variance (a ¼ 0.05)showed significant differences in all the garlic treatments comparedwith the control, except T3-25% of G025,whichwas not significantlydifferent, as shown in Table 2.

3.3. Electron microscopy

3.3.1. Scanning electron microscopyThe mycelial growth of P. capsici under the scanning electron

microscope revealed clear differences in the shapes and sizes of thehyphae among all treatments. The mycelial plugs in the 100%exudates of both cultivars and the control were examined. The rootexudates collected from G064 induced strong morphological alter-ations (mostly collapsed) in the hyphae of P. capsici. Abnormalhyphal swelling, curling, short branching and the accumulation ofprotoplasm in the mycelia were observed. However, the garlic rootexudates collected from G025 showed little change in the size andshape of the hyphae compared G064 and the control. Hyphae of thefungus treatedwith the root exudates of G064were fragmented andsmaller in diameter along with the emptying of the cytoplasmic

Table 1Effect of root exudates on mycelial growth of P. capsici.

Garlic cultivar Concentration 3rd day of culture 5rd day of culture 7th day of culture

Mean of colonydiameter

Inhibition (%) Mean of colonydiameter

Inhibition (%) Mean of colonydiameter

Inhibition (%)

Control 000 27.5 e 0.00 44.9 f 0.00 60.53 g 0.00Gialiang (G 064) 25% 20.2 c 26.66 35.86 d 20.11 48.90 e 19.21

50% 15.1 b 45.22 25.34 c 43.54 39.54 d 34.6875% 9.70 a 64.76 18.82 b 58.08 26.81 b 55.70

100% 8.50 a 69.24 16.20 a 63.90 17.65 a 70.83Cangshan (G 025) 25% 23.60 d 14.41 41.97 e 6.51 54.98 f 9.16

50% 19.4 c 29.37 34.68 d 22.75 48.46 e 19.9475% 14.9 b 45.72 26.07 c 41.92 37.73 d 37.66

100% 14.0 b 49.06 24.55 c 45.32 33.82 c 44.12

Means within a column followed by the same letter are not significantly different.

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contents in the hyphal cell wall comparedwith the other treatments(Fig. 1). The number of hyphae developed on the root exudates ofG064 were smaller than the treatment of G025, followed by thosedeveloped in the control.

3.3.2. Transmission electron microscopyCytomorphological alterations were prominent in both treat-

ments of garlic root exudates compared with the control (Fig. 2).Furthermore, the effectiveness of G064 in terms of the size and

number of vacuoles formed in the hyphal cells remained substan-tially higher than G025. The hyphae developed on the garlic rootexudates of G064 showed strong, thick cell walls compared withthe control and G025. The structures of the tips of the hyphae weredifferent in all the treatments regarding vacuole formation.

3.4. HPLC results

The standard allicin peak was recorded at 10.96 min, and thepeaks of both G064 and G025 were also detected at the same time.It was observed that both garlic root exudates showed differentsizes (heights) of allicin peaks at the HPLC screen, as depicted inFig. 3. From each garlic root exudate, 10 ml aliquot was used toascertain the amount of allicin. The results show that G064 con-tained 0.097 mg/ml allicin, but G025 contained 0.068 mg/ml.

4. Discussion

Although allelopathy is an important ecological phenomenon,recent research has largely focussed on its detrimental effectsrather than its beneficial aspects with reference to agriculture.Allelochemicals are the metabolites released by plants into theenvironment, and they can have deleterious or beneficial effects on

Fig. 1. Scanning electron microscopy: A is control, B belongs to G064 cultivar, C is from G 025 cultivar (same 10 um). D is control; B is G064 and C from G025 with 5 um.

Table 2Effect of garlic root exudates on the germination of spore.

Garlic cultivar Concentration Average germinationof spores

Inhibition (%)

Control 000 62.83 f 00.00Gialiang (G 064) 25% 51.67 d 17.76

50% 19.43 b 69.0775% 000 a 100%

100% 000 a 100%Cangshan (G 025) 25% 57.46 e 8.54

50% 41.09 c 34.6075% 19.00 b 69.75

100% 000 a 100%

Means within a column followed by the same letter are not significantly different.

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other plants. These root exudates contain low and high molecularweight compounds, such as amino acids, organic acids, sugars, anarray of secondary metabolites and high molecular weightcompounds such as mucilage and proteins (Badri and Vivanco,2009).

The present study demonstrates that garlic root exudates can beused as an anti-growth substance for P. capsici causing Phytoph-thora blight in pepper. The results of this study are in agreementwith studies of other plant materials and microorganisms for thecontrol of various pathogens (Daayf et al., 2003; El-Sheikh et al.,2002; Khan and Cheng, 2010). The behaviour of the root exudates ofboth garlic cultivars remained invariable at different growth stages.This variant response of different garlic cultivars may be attributedto the different genetic makeup and the size of the bulbs. It has alsobeen reported that intercropping marigold with tomato candecrease the occurrence of blight through the allelopathic effect ofmarigold on A. solani conidia germination (Gomez et al., 2003).

According to Ren et al. (2008), rice root exudates can suppresspathogenic Fusarium growth, such as spore germination andproduction in watermelon, by intercropping.

Whenwe examine the explanatory power of each cultivar’s rootexudates with reference to the time of application, the mostpronounced inhibition was observed on the third day with G064,followed by a gradual diminishing trend for both cultivars insubsequent days. The results thus obtained can be correlated withthe stability, concentration and volatility of active components inthe garlic root exudates. It is clear from the results that the garliccultivar with a higher amount of allicin performed better atinhibiting fungus growth.

Electron microscopy showed that the hyphae the developed onthe root exudates of G064 had more cytomorphological alterations,particularly the accumulation of lipid bodies and the thickening ofthe cell wall. These effects are similar to those induced by somesynthetic fungicides and garlic extracts (Bianchi et al., 1997; Hippe,

Fig. 2. Transmission electron microscopy: A is treated with garlic root exudates of cultivar G064, B is G025 and C is control, however D belongs to G064, E is G025 and F is of control.

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1991). An increase in the size and number of vacuoles along withother alterations might modify the activity of the membraneenzymes involved in the formation of the cell wall, causinganomalous development. The garlic root exudates of G064demonstrated higher anti-fungal activity against P. capsici. The

garlic root exudates were also effective at reducing mycelial growthand modification in the cell structure of hyphae of the pathogen.

The active anti-fungal substance found in the garlic rootexudates was allicin (diallylthiosulphinate). Allicin crosses the cellmembrane easily and undergoes thiol-disulphide exchange

a

b

M v

0.00

200.00

400.00

600.00

800.00

1000.00

1200.00

1400.00

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1800.00

2000.00

2200.00

R e t e n t i o n T i m e 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.0 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00

10.967

17.805

M v

0.00

200.00

400.00

600.00

800.00

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2200.00

R e t e n t i o n T i m e 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

10.971 17.823

Fig. 3. Allicin peaks. A belongs to garlic root exudates G 064 and B from G 025 cultivar showing clear difference in peak sizes. In both the figures (A&B) first peak appeared at thetime of 10.97 & 10.96 min (retention time) is the peaks of allicin because the peak of allicin standard was also recorded at 10.96 min.

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reactions in the free thiol groups in protein. Because of theseattributes, allicin has several potential targets within the cell,making it difficult for organisms to mutate and develop resistance.Allicin is readily membrane-permeable (Miron et al., 2000;Slusarenko et al., 2008) and can therefore enter the symplast inthe roots. In the present study, HPLCwas used to quantify the allicinfrom garlic root exudates. It is difficult to quantify allicin in the gasphase because the temperature of the injection port in the GC is toohigh and leads to modifications that produce other polysulphides(Block, 1992). Furthermore, it is important to study the chemicalmodification of allicin to enhance its relatively desirable properties.Such studies can also lead to new multi-target plant protectioncompounds to be utilised in conventional agriculture in general andhorticulture in particular.

This study provides a basis for the development of non-chemicaltreatment against the Phytophthora blight of pepper. The overuseof agrochemicals for the control of soil-borne diseases results inecological imbalance and can induce multiple multi-resistantpathogenic microorganisms (Levy, 1997). The use of fungicide hasbeen restricted in some European countries that aim to promoteother controls. The dried plant material of garlic, alfalfa andcabbage has been shown to inhibit the mycelial growth of somefungi (Demirci and Dollar, 2006). Garlic has wide range ofcompounds that have anti-microbial effects. The present studyidentified a stronger inhibition effect from garlic cultivar G064 thanG025. It may not be possible to apply garlic root exudates in soil;however, the inclusion of G064 in rotation or intercropping systemscan be an effective way to control the Phytophthora blight ofpepper. Further research could focus on intercropping G064 withpepper in anticipation of more effective results.

Acknowledgements

This research was supported by the National Key TechnologiesR&D Program of China during the 11th five-year plan period(No.2006BAD07B02) and the project of Commonwealth (Agricul-ture) Scientific Research (No.200903018).

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