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	�:	Effect of certain plant extracts to control of purple blotch diseases of onion (Allium cepa L.)
Sobhy I. I. Abdel-Hafez1 Kamal A. M. Abo-Elyousr2 and Ismail R. Abdel-Rahim1
1) Plant Botany Department Faculty of Science Assiut University Assiut 71516 Egypt. 2) Plant Pathology Department Faculty of Agriculture Assiut University 71526 Assiut Egypt.
Corresponding author Kamal A. M. Abo-Elyousr Plant Pathology Dept. Faculty of Agriculture Assiut University 71526 Assiut Egypt Tel. +201003821743 Fax.+20882331384 E-mail:  HYPERLINK "mailto:kaaboelyousr@yahoo.com" kaaboelyousr@yahoo.com
Abstract 
The present investigation aimed to isolate the causative agents of onion purple blotch disease and evaluate the efficacy of certain plant extracts against the pathogen in vitro and under greenhouse condition. Antifungal activity of some aqueous plant extracts (Azadirachta indica Cydonia oblonga Datura stramonium Eucalyptus globulus Foeniculum vulgare Ocimum basilicum Rosmarinus officinalis and Salix mucronata) was assayed in vitro by well diffusion technique. The data indicated that there were significant differences between these extracts in their effect on fungal growth of Altenaria porri and the best were A. indica and D. stramonium. Under greenhouse condition application the aqueous extract of A. indica either before or after 48h A. porri inoculation produced the highest reduction in disease severity comprising 70% and 74.7% respectively. According to our results it can be concluded that plant extracts of A. indica and D. stramonium can be used for the bio-control of purple blotch disease instate of applied fungicide used to minimize the risks and hazards of toxic fungicides.
Keywords: Antifungal activity plant extract Azadirachta indica Datura stramonium Alternaria porri.
Introduction
Onion (Allium cepa L.) is one of the main important and oldest vegetable crops grown in Egypt. About 60 onion and garlic diseases were described by Schwartz and Mohan [1] forty of which were caused by fungi fourteen by bacteria one by yeasts six by nematodes three by viruses and one by phytoplasma-like organism. Most of fungal diseases can cause extensive crop losses and are important throughout the onion- and garlic-producing areas of the world in addition to the highest annual production and storage losses.
Purple leaf blotch (PLB) is one of the most serious diseases that widespread in many parts of the world restricted to Allium spp. [2, 3] is more prevalent in warm and humid environments [4].  The pathogen usually associated with the PLB symptom is Alternaria porri (Ellis) Cif. The disease is however more severe on seed crop as compared to bulb crop [5,6] causing sometimes 100% loosing of the seed production [7, 8]. Although chemical control of onion blotch had been practiced its success depends largely on high frequency of spraying [9] but today there are strict regulations on chemical fungicide use and there is political pressure to remove the most hazardous chemicals from the markets due to carcinogenic effects residual toxicity problems environmental pollution [10]. Additionally using of synthetic fungicides can cause development of fungicide-resistant strains as well as the appearance of diseases which are increased in their severity by the use of specific chemical product has stimulated. Therefore there are a large number of studies have been devoted to searching for alternative control strategies to plant pathogens [11,12,  13].
Higher plants were considered one of the main sources of bioactive economically important natural compounds pharmaceuticals and pesticides [14]. Plant extracts of many higher plants have been reported to exhibit antibacterial antifungal and insecticidal properties under laboratory trails [15, 16]. Neem (Azadirachta indica Juss.) the sources of biocontrol agent has already emerged at the top of the list of plants with the highest potential as insecticides and fungicides [17, 18]. The extracts from neem leaves have been used to control several fungal plant pathogens such as Alternaria alternata Alternaria porri Aspergillus flavus Fusarium oxysporum. f. sp. phaseoli Fusarium solani Monilinia fructicola Rhizoctonia solani and Sclerotinia sclerotiorum [19, 20, 21, 22, 23]. Neem contains at least 35 biologically active substances of which triterpenoides e. g. Nimbin azadirachtin and Nimbidine are the most active fungicidal and insecticidal ingredients [24]. 
Leaf Plant extracts of Datura stramonium Eucalyptus globules Rosmarinus officinalis Salix spp. Ocimum spp. Foeniculum vulgare and Cydonia oblonga have been used successful to control several fungal diseases in plants [25, 26] 
The present investigation aims to evaluate the effectiveness of certain plant extracts against the pathogens of purple blotch disease of onion plants Alternaria porri in vitro and under greenhouse condition. 
Material and methods
Isolation and identification of the causal agent
The leaves and floral stalks of onion (Allium cepa L.) plants showing typical symptoms of purple blotch disease were collected from commercial onion farms at Assiut governorate by cutting and packed into clean polyethylene bags and transferred to Mycological laboratory to isolate and identify the causative agents. 
Infected lesions showing typical PLB disease symptom were cut into small pieces equal to 1 cm2 each washed with tap water and surface sterilized by immersing it in 5% aqueous sodium hypochlorite for 5 min and 70% ethanol for 0.5 min rinsed several times in sterile distilled water. Five pieces were deposited on a Petri dish (9 cm diameter) containing 20 ml PDA medium [4]. Three replicates were used to each sample. The plates were incubated in the dark at 25�C for 5-6 days. The fungus was purified by inoculation it in Petri dishes containing PDA medium. Identification of fungal isolates of the pathogens was carried out based on the macro- and microscopic characteristics [27]. The pure identified culture of the fungus was transferred to PDA slants and kept in refrigerator at 4�C to further studies. 
Inoculum preparation, method of inoculation and disease assessment
Inocula of the tested isolates were prepared by growing isolates on PDA medium in Petri plates (9 cm. diameter) at 25�C for 10 days. At the end of incubation period ten ml of sterile distilled water were added to each plate and the mycelial growth were carefully scraped with a sterile needle and the resulting conidial suspension from each isolate was used for infection. Pots (25 cm. in diameter) were filed with sterilized clay sandy soil. Each pot was cultivated on November 2010 by one bulb of onion (Giza 6) and cared to grow for 110 days then applied the pathogenicity testes upon these plants. Three pots replicates were used for each isolate test. After inoculation the plants by spraying the colonial suspension (3x106 conidia/ml) then they were covered with polyethylene bags for 48 h. to maintain high humidity. After this period the bags were removed and plants were kept under normal condition. Disease severity percentage was recorded after 15 days from inoculation date [28]
Disease severity of onion seed-stalk was recorded as average percentage of infected seed-stalk area readings were converted to disease index (0- 4) scale as the following:
0 = No infection 1 = 1 - 25% seed-stalk areas infected 2 = 26 - 50% seed-stalk areas infected 3 = 51 - 75% seed-stalk areas infected 4 = 76 - 100% seed-stalk areas infected.
The disease severity (%) was determined according to the following equation:
Disease severity % = [� (n x V) / 4 x N] x 100
Where: n = Number of seed-stalk within each infection category V = Numerical values of infection categories N = Total number of seed-stalks examined 4 = Constant highest numerical value.
Screening of some plant extracts to control the pathogen of PLB disease in vitro
Plant material
Fresh healthy free leaves of eight plant species were collected from Botanical garden of Faculty of Science Assiut University (Table 1). 
Preparation of aqueous extracts
Healthy and fresh leaves of each plant mentioned above were washed thoroughly in cold running tap water then sterilized water then air-dried and separately micronized with a hammer mill into a fine powder. Known weight of the powder of plant materials was added to distilled water in the ratio of 1:1 (w/v) and then transferred to shaker for 24 h.  The pulverized mass of a plant leaves were squeezed through double layered muslin cloth and then centrifuged at 5000 rpm for 20 min under 4�C. The supernatant was sterilized by passing it through bacterial filter 0.20 �m (Seitz). The plant extract was collected in sterilized brown bottles and kept in refrigerator at 4�C until using [29].
Assay the effect of plant extract against the pathogen PLB in vitro
Assay the effect of plant extract on mycelial growth of the PLB pathogens was determined by well diffusion plate method as the following:
Autoclaved sterilized PDA medium poured into Petri dishes under aseptic conditions, and then these Petri dishes were inoculated with PLB pathogens at the center of the plate. Three wells of with 10, 7 and 5 mm diameter were made using cork borer that was sterilized with alcohol and flame, then these wells were filled with 500, 200, 100 �L of plant extract, respectively. The inoculated plates were incubated at 25�C for five days. Five replicates plates for each treatment were maintained. PDA medium plates with wells containing sterile distilled water served as control. The inhibition zones were measured and recorded [30].
Effect of application of aqueous plant extracts as well as Ridomil gold plus on disease severity of purple blotch under greenhouse condition. 
 Plant extracts that showed highest inhibition to Alternaria porri in vitro under laboratory condition were selected to control the disease under greenhouse condition. All treatments were applied by two methods the first was applied before 48 hours of pathogen�s inoculation while the second was applied after 48 hours of pathogen�s inoculation. Disease assessment of each treatment was recorded after 15 days from inoculation and measuring disease severity percentage was determined as previously described [28]. 
The foliar fungicide Ridomil gold plus (2g/liter) was used for comparison purpose with the biocontrol agents in controlling the disease incidence. The chemical composition of Ridomil gold plus (w/w): Mefenoxam 2.5%; Dicopper chloride trihydroxide 69%; Adhesive components 6%; Carrier 22.5%. Mefenoxam is the common name of Methyl N - (methoxy acetyl) - N - (2.6-xylyl) - DLalaninate; methyl (R) � 2 - [(2 6 -dimethylphenyl) methoxyacetyl] amino-propionate.
Statistical analysis
The data were subjected to one-way ANOVA using the SPSS 10.0 software program. Mean and standard errors were calculated for three replicate values. Means were compared by the Duncan's multiple tests and statistical significance was determined at 5% level. 
Results and Discussion 
Screening on the ability of aqueous plant extracts to suppress Alternaria porri in vitro 
Well diffusion method was used to illustrate the antifungal activity of the eight tested aqueous extracts against Alternaria porri. The mean inhibitory zone of eight aqueous plant extracts against A. porri was summarized in Table 2.
By using 100 �l of aqueous plant extract, Azardiachta indica showed the highest significant inhibition zone value against A. porri contributing 7.2 mm. At 200 �l of aqueous plant extract, the maximum inhibition values against A. porri were detected by A. indica, Datura stramonium and Ocimum basilicum contributing 9.4, 8.6 and 8.6 mm, respectively without significant difference between the three treatments. At 500 �l of crude aqueous extract, both A. indica and D. stramonium showed highest significant inhibition zone value against A. porri (11.2 and 10.8 mm, respectively). The amount of minimal concentration of aqueous extract giving highest inhibition zone values was recorded by A. indica at 100 �l and D. stramonium at 200 �l against the pathogen.
In this respect Meena [23] reported that using 5% aqueous extract of Azadirachta indica and Palmarosa oil showed inhibition on the mycelial growth of A. porri comprising 52.94% and 97.05% respectively. Other study reported that plant extract of Mentha arvensis was the most effective in controlling the growth of A. porri. Usha [31] investigated that Datura stramonium Calotropis gigantean and Azadirachta indica (neem) showed strong antifungal activity against Fusarium mangiferae the pathogen of mango floral malformation. Extract of D. stramonium contains alkaloids such as scopolamine and hyoscyamins that well known for their medicinal properties. 
Other studies were reported that the extract of neem leaves have been used to control several fungal plant pathogens such as Monilinia fructicola Alternaria alternata Aspergillus flavus Fusarium oxysporum. f. sp. phaseoli Rhizoctonia solani and Fusarium solani Sclerotinia sclerotiorum [19, 20, 21, 22, 32].
Several researchers reported that the fungicidal and bactericidal properties of extract from neem leaves attributed to the presence of several antimicrobial active ingredients such as desactylimbin quercetin and sitosterol [33, 34]. Whereas other researchers explained this activity by the presence of active ingredients like triterpenes or the limonoids such as meliantriol azadirachtin desactylimpin quercetin sitosterol nimbin nimbinin nimbidin nimbosterol and margisine and/or to different bitter substances such as alkaloids phenols resins glycocides terpenes and gums [35].
Effect of application of two plant extracts as well as Ridomil gold plus on disease severity of onion purple blotch under greenhouse condition 
The aqueous plant extracts of Neem and Datura as well as foliar fungicide Ridomil gold plus (2g/liter) were selected for greenhouse applications to control the purple blotch disease in vivo (Table 3). 
In case of PLB disease caused by Alternaria porri the treatment with aqueous plant extract of Azadirachta indica showed the highest reduction in disease severity (70%) before 48h A. porri inoculation. Meanwhile the treatment with aqueous extract of A. indica and Ridomil gold plus caused disease reduction (more than 70%) after 48h A. porri inoculation. 
In this respect Mishra et al. [36] reported that Datura leaves extract showed the highest efficacy in reduction onion blight disease caused by Stemphylium vesicarium under green house condition. Iranbakhsh [11] investigated the strong antifungal effects Datura stramonium leaf extract on phytopathogenic fungi. It was concluded that the effective antimicrobial ingredient goes back to atropine alkaloids. In conclusion our results support the uses of aqueous plant extracts of Azadirachta indica and Datura stramonium to control onion purple blotch and Stemphylium blight diseases caused by Alternaria porri 
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Table 1: List of plants tested for antifungal activity against PLB pathogens.Binomial nameCommon nameFamilyAzadirachta indica A. Juss.NeemMeliaceaeCydonia oblonga Mill.QuinceRosaceaeDatura stramonium L.Jimson weed devil's trumpet devil's weed thorn apple locoweed DaturaSolanaceaeEucalyptus globulus Labill.Blue GumMyrtaceaeFoeniculum vulgare Mill.FennelApiaceaeOcimum basilicum L.Basil HYPERLINK "http://en.wikipedia.org/wiki/Lamiaceae" \o "Lamiaceae" LamiaceaeRosmarinus officinalis L.Rosemary HYPERLINK "http://en.wikipedia.org/wiki/Lamiaceae" \o "Lamiaceae" LamiaceaeSalix mucronata AnderssonCape Silver Willow or Safsaf willowSalicaceae

Table 2: Inhibition zone by mm as a parameter to detect the antifungal activity of aqueous plant extracts against Alternaria porri 
Pant extractAlternaria porri 100 �l200 �l500 �lAzardiachta indica7.2 � 0.4a9.4 � 0.5a11.2 � 0.8aCydonia oblonga3.6 � 0.5c,d5.0 � 0.0c6.4 � 0.5dDatura stramonium5.8 � 0.8b8.6 � 0.5a10.8 � 0.8aEucalyptus globulus3.0 � 0.7d6.2 � 0.8b8.6 � 0.5cFoeniculum vulgare0.0 � 0.5e3.8 � 0.8d3.4 � 0.8eOcimum basilicum5.6 � 0.5b8.6 � 0.5a9.6 � 0.5bRosmarinus officinalis4.4 � 0.5c6.2 � 0.4b8.2 � 0.8cSalix safsaf3.2 � 0.8d5.6 � 0.5b,c6.6 � 0.5d
Numbers within each column are means of three replicates. Different letters within column indicate significant differences (Pd"0.05) based on One-way ANOVA analysis.
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