EFFICACY EVALUATION OF INHIBITORY ACTIVITY OF SOME BIOLOGICAL PRODUCTS, ESSENTIAL OILS, PLANT EXTRACTS AND INDUCED RESISTANCE AGENTS AGAINST ERYSIPHE HERACLEI DC, THE PATHOGENIC POWDERY MILDEW OF PARSLEY (PETROSELINUM SATIVUM L.)

-

EFFICACY EVALUATION OF INHIBITORY ACTIVITY OF SOME BIOLOGICAL PRODUCTS, ESSENTIAL OILS, PLANT EXTRACTS AND INDUCED RESISTANCE AGENTS AGAINST ERYSIPHE HERACLEI DC, THE PATHOGENIC POWDERY MILDEW OF PARSLEY (PETROSELINUM SATIVUM L.)

Abdel-Radi T. Bakeer ¹; Hoda M. H. Ahmed ¹;

Mohamed A. M. Baiuomy ² and Ekram F. M. Fatoh ²

¹ Botany Dept. (Plant Pathology), Fac. Agric., Fayoum Univ., 36514 Fayoum, Egypt.

² Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt.

Corresponding author: Hoda M. H. Ahmed, hmh03@fayoum.edu.eg

Key Words: Parsley, Erysiphe heraclei, powdery mildew, biocide, essential oil, plant extract, Induced-resistance agents and fungicides.

ABSTRACT

Parsley (Petroselinum sativum L.) is one of the most important medicinal plants which belong to the family of Apiaceae and believed to be natives of the Eastern Mediterranean region. Parsley is infected with many foliar diseases, powdery mildew disease caused by Erysiphe heraclei DC is considered one of the most important of these diseases, which is spread and attack plant in the different cultivation areas, causing severe damage to the vegetative crop. The typical symptoms of powdery mildew were observed on parsley during disease survey monthly at growing season, 2016/17, from January to April at four Fayoum governorate districts. In an attempt to control of this disease, there are several experiments conducted using safe materials in order to preserve human health and avoid environmental pollution but, without ignore the role of chemical control which can be used if need be. In-vitro experiments were conducted to assess the impact of using different concentrations of biocides (Plant-Guard, Bio-ARC 6 % and Biobac 50 %), plant extracts (moringa leaf, garlic cloves and bluegum leaf extracts), essential oils (citronella, thyme and anise) and induced resistance agents (Sodium bicarbonate, Ascorbic acid and Ammonium dihydrogen phosphate) besides two fungicides (Thiovit jet 80 % and Score 25 %) on conidiospore germination.

All the tested treatments achieved an obviously significant reduction to the spore germination of E. heraclei compared with reference to the control treatment. This reduction gradually increased by increasing the tested concentration. Among tested biocides, Biobac 50 % was the most efficient one being 61.95 % reduction to the spore germination followed by Bio-ARC 6 % (49.16 %), then Plant-Guard in descending order (32.88 %). Concerning plant extracts, garlic cloves extract was the superior antifungal activity against spore germination followed by moringa leaf extract, the corresponding percentages of reduction to the spore germination were recorded 47.98 and 36.85 %, respectively. Meanwhile, bluegum leaf extract in descending order (8.28 %).Among three essential oils tested, maximum reduction to the spore germination was recorded 66.06 % by thyme oil followed by anise oil (59.88 %), then citronella oil in descending order (46.62 %). Concerning induced resistance agents, the best antifungal activity against spore germination obtained by Ascorbic acid, is 60.51 % reduction to the spore germination followed by Sodium bicarbonate (53.82 %) then, Ammonium dihydrogen phosphate in descending order (22.85 %). Finally, complete inhibition in spore germination occurred by Score 25 % fungicide followed by Thiovit Jet 80 % (90.02 %).

INTRODUCTION

Parsley (Petroselinum sativum L.) is considered one of the most important medicinal plants cultivated in Egypt, and is believed to be natives to the Eastern Mediterranean region. In Egypt, the statistical studies indicate that the cultivation areas of parsley during 2015 and 2016 seasons reached 1843 and 715 feddan, produced 38085 and 13494 tons, respectively from fresh herb (Anon., 2015 & 2016).

Powdery mildew disease caused by Erysiphe heraclei DC is one of the most important foliar diseases, causing severe damage to the vegetative crop, which in turn reflected negatively on the number of inflorescences and essential oil yield (McGrath, 2013 and Ahmed, 2016). Although the high efficiency of chemical fungicides controlling powdery mildew, a great hazard on the human health due to the residual toxicity in the consume food, environmental pollution and development of resistance in the pathogen populations (Yoon et al., 2013). Therefore, there is a need for developing novel plant protectants that interferes with the fungal pathogenicity factors, so great efforts by scientists are spends to search about safe alternative methods to manage plant diseases. In recent years, several fungicide alternatives including, mineral salts, essential oils, plant extracts, and biological agents have been developed to control plant pathogens (Ryals et al., 1996; Pasini et al., 1997; Istvan, 2002; Kiss, 2003; Rosato et al., 2007 and Pereira et al., 2011).

The objective of this study was to evaluate the efficiency of some safe materials such as some commercial products of bioagents, cold watery plant extracts, essential oils and induced resistance agents against powdery mildew of parsley caused by Erysiphe heraclei DC in vitro to determine the antifungal activity of suggested control units by estimating the reduction percentage of pathogen' spores germination.

MATERIALS AND METHODS

Survey of parsley powdery mildew disease survey at Fayoum province:

The typical symptoms of powdery mildew disease on parsley (Petroselinum sativum L.) were observed and inspected for the most parsley widespread cultivation areas at four Fayoum governorate counties (El-Fayoum, Etsa, Sinnoures and Abshaway; three villages/county) during growing season, 2016/17. Parsley plants were randomly examined in each location. The percentage of disease incidence and disease severity were determined monthly four times/ season started from January to April.

Disease assessment

Percentage of Disease Index (PDI) was determined using the following formula

   (Koitabashi, 2005)

While, disease severity was assessed using 0-4 scale given by Whitney et al. (1983) with slight modification. Plants were evaluated according to their percentage of powdery mildew covered area. Where, 0= (No mildew colonies observed), 1= 1-25 %, 2= 26-50 %, 3= 51-75 % and 4= 76-100 % area of plant covered by mildew.

Whereas, the powdery mildew infection percentage of parsley was calculated as the following equation:

Identification of Powdery Mildew Causal Organism:

Parsley plants showing symptoms of powdery mildew infection were collected from many fields at different Fayoum governorate' counties. Microscopic preparations for identification of the causal fungus were made by placing epidermal strips taken from the infected parts (bearing the fungal conidiophores and conidia) on glass cover then, examined by using the light microscope at 10, 20 and 40 x and identified according to (Baiuomy and Shalaby, 2007; Rosa et al., 2008; Koike and Glawe, 2009; Park et al., 2010; Ziedan, 2010 and Bubici, 2015). The identification was kindly confirmed by the mycology and plant diseases survey dept., Plant Patholo. Res. Inst., ARC, Giza, Egypt.

Evaluation of inhibitory activity of some safe materials against Erysiphe heraclei DC spore germination:

Spore Germination Technique:

Conidia of powdery mildew fungus were obtained from young sporulation lesions to avoid the old unviable conidia (Nair et al., 1962 and Godwin et al., 1987). Collecting freshly conidia applied using sterilized camel brush from the infected parts and were put in each tested concentration on a sterilized slide (2.5 × 7.5 cm), then slides were placed on U-shaped glass rod in a sterilized Petri-dish containing a piece of wetted cotton by sterilized distilled water to provide high relative humidity. The control treatment was carried out by putting conidia in sterile distilled water film. Three replicates were used for each treatment and three dishes for each replicate. Preparations were incubated in an incubator at 25±1°C for 24 hours. One drop from lacto-phenol cotton blue stain was added at the time of slide examination to fix and killing the germinated conidia. The percentage of spore germination (SG) was calculated by the following formula given by Kiraly et al., (1974):

× 100

While, the spore germination reduction percentage for different treatments with reference to the control one were calculated according to Manici et al. (1997) using the following equation:

× 100

Evaluation of inhibitory activity of biocides:

Three commercial products of biocides i.e., Plant-Guard (Trichoderma harzianum, 30×10⁶ spore/ml), Bio-ARC 6 % WP (Bacillus megaterium, 25×10⁶ cell/g) and Biobac 50 % WP (B. subtilis, 1×10⁹ cell/g) were tested for their antifungal activity against the spore germination of E. heraclei under in-vitro conditions. Four concentrations (1000, 2000, 3000 and 4000 ppm) were prepared depending on the active ingredient of each biocides by dissolving known quantity from these bioagents in sterile distilled water. Biocides were obtained from Central Lab for Organic Agriculture, ARC, Giza, Egypt.

Evaluation of inhibitory activity of plant extracts and essential oils:

Three cold watery plant extracts i.e., moringa leaf, garlic cloves and bluegum leaf extracts (Table 1) with three concentrations for each; 25, 50 and 100 % were tested for their antifungal activity against E. heraclei's spore germination under in-vitro conditions. As well as three essential oils of three plant species i.e., citronella, thyme and anise (Table 1) with four concentrations; 1000, 2000, 3000 and 4000 ppm each, were tested, also. Plants were obtained from the Medicinal and Aromatic Plants dept., Horticulture Res. Inst., ARC, Giza, Egypt. Required concentrations were prepared by dissolving known quantity from the previous materials in sterile distilled water. The essential oils were emulsified with 0.05% of tween-80 before application (Reuveni et al., 1996).

Table (1): List of plant extracts and essential oils used for controlling parsley powdery mildew.

Plant extracts preparation:

Samples were collected from apparently healthy plants, then washed thoroughly in tap water and dried in the oven at 45^o C for 2hrs. The dried samples were powdered in the grinding machine. 100 g of powdered material was soaked in excess water for 24 hrs. The extract was filtered through a double cheesecloth fabric and Whatman no.1 filter paper and centrifuged for 10 minutes at 300 rpm to get a clear plant extract. Sterilization was made using 0.45 µm Millipore filter disc. The crude extract was diluted with sterile distilled water to give a series of concentrations of its original volume.

Extraction of essential oils:

Extraction of essential oils from subjected plants was carried out using a modified Clevenger-type apparatus (Negahban et al., 2007) at the following condition: 40 g of air-dried plant material, 500 ml distilled water and 4 hrs distillation. Anhydrous sodium sulfate was used to remove water from extracted essential oils after extraction. The resulting oil placed into sealed dark glass tubes (Negahban et al., 2006) and was stored in a refrigerator at 4 °C.

Evaluation of inhibitory activity of induced resistance agents:

Three induced resistance agents, i.e. Sodium bicarbonate (NaHCO₃), Ascorbic acid (C₆H₉O₆) and Ammonium dihydrogen phosphate (NH₄H₂PO₄) were evaluated for their antifungal activity against the spore germination of E. heraclei under in-vitro conditions. Required concentrations (1000, 2000, 3000 and 4000 ppm) were prepared by dissolving known quantity from these chemical inducers in sterile distilled water. The previous inducers were obtained from El-Nasr Chemical Co., Egypt.

Evaluation of inhibitory activity of fungicides:

Two commercial fungicides i.e., Thiovit jet 80% WG (80 % micronized sulphur) and Score 25% EC [(25 % difenconazole) (1-{2-[4-(4-chlorophenoxy)-2-chlorophenyl- (4-methyl-1, 3-dioxolan-2-yl)-methyl}-1h-1, 2, 4–triazole] were evaluated under in-vitro conditions via spore germination technique against E. heraclei spores. Required concentrations (250, 500, 750 and 1000 ppm) were prepared by dissolving known quantity from these fungicides in sterile distilled water separately under aseptic conditions. The tested fungicides were obtained from Syngenta Co., Egypt.

Statistical analysis:

Data obtained were subjected to the statistical analysis according to the standard methods recommended by (Gomez and Gomez, 1984) using the computer program (Costat). The means differences were compared by least significant difference test (LSD) at 5 % level of significance according to (Fisher, 1984).

RESULTS

Disease symptoms of powdery mildew on parsley (Petroselinum sativum L.):

The symptoms of powdery mildew disease on parsley plants were observed and descripted throughout the period starts from January to April months of growing season, 2016/17 at Fayoum governorate. The symptoms firstly appeared on parsley plant at vegetative growth and pre-flowering stages as a thin white to light gray powdery colonies or irregular patches scattered on the lower parts of the plant near the soil surface, which include stems and leaves. With favorable weather conditions, these spots gradually expand in size and combine to cover large areas of the plant, in the severe case the infection extends and spread over the inflorescences. With the disease progressed, the infected leaves and stems become yellow, then dry and died. In addition, infected inflorescences do not reach their normal size and eventually fail to form the fruits, this leads to an economic loss in the crop yield (Figure 1).

Figure (1): The symptoms of powdery mildew disease on parsley (P. sativum L.) under the natural infection conditions.

Survey the incidence and severity of powdery mildew disease on parsley in Fayoum governorate:

Data presented in table (2) show that among four districts surveyed at Fayoum governorate during January, February, March and April months at the growing seasons,2016/17 and 2017/18, powdery mildew of parsley was more sever in Etsa county followed by El-Fayoum and Abshaway counties, the corresponding values of the average of percentage of disease severity were recorded, 58.25, 53.81 and 52.04 %, respectively. Meanwhile, the lowest disease severity mean percentage was recorded in Sinnoures county with 46.33 %, average of percentage of disease severity. On the other hand, the highest average of disease incidence was recorded in Etsa county (70.33 %) followed by Abshaway (67.25 %) and El-Fayoum (65.11 %). While the lowest values were recorded in Sinnoures (63.18 %).

Table (2): Survey of parsley powdery mildew disease incidence and severity averages at different Fayoum counties during 2016/17 growing season.

Identification of powdery mildew causal organism:

Microscopic examination of prepared slides reveals that based on the observations of the morphology of Anamorph stage, the causal agent of parsley powdery mildew was identified as Erysiphe heraclei DC. The conidiophores were cylindrical to ovate and ranged in length from 28 to 50 µm and width from 8 to 11 µm with an average of 39 × 9.50 µm. Conidia produced singly, oblong-elliptical to oblong and ranged in length from 25 to 48 µm and width from 12 to 18 µm with an average of 36.50 × 15 µm. Cleistothecia were not found during trial period  (Figure 2).

Figure (2): The causal agent of parsley powdery mildew Erysiphe heraclei DC,  (A) conidiophores and (B) conidia.

Evaluation of inhibitory activity of some safe materials against spore germination of Erysiphe heraclei DC:

1- Inhibitory activity of biocides:

Data presented in table (3) and shown in figure (3; A) indicate that all the tested biocides achieved an obviously significant reduction to the spore germination of E. heraclei with reference to the control treatment. This reduction was gradually increased by increasing the tested concentration. In addition, Biobac 50 % WP (Bacillus subtilis) was the most efficient one in this regard, being 61.95 % of  mean reduction to the spore germination followed by Bio-ARC 6 % WP (B. megaterium) (49.16 %). Meanwhile, Plant-Guard (Trichoderma harzianum) was the lowest efficient one being, 32.88 % of mean reduction to the spore germination.

2- Inhibitory activity of essential oils:

Data presented in table (3) and shown in figure (3; B) show that all the tested essential oils resulted in a significant reduction to the spore germination of E. heraclei compared with the control treatment. This reduction was highest at 4000 ppm concentration in comparison with the other tested concentrations from all the tested essential oils. In addition, maximum reduction to the spore germination was recorded 66.06 % mean percentage by thyme oil followed by anise oil (59.88 %). Meanwhile, minimum reduction to spore germination was recorded, 46.62 % by citronella oil.

3- Inhibitory activity of induced resistance agents:

Data presented in table (3) and shown in figure (3; C) show that all the tested plant resistance inducers were significantly reduced the spore germination of E. heraclei in comparison with the control treatment. This reduction was gradually increased by increasing the used concentration. In addition, ascorbic acid (C₆H₉O₆) was the most efficient one in this regard, being 60.51 % reduction to the spore germination followed by sodium bicarbonate (NaHCO₃) (53.82 %). Meanwhile, ammonium dihydrogen phosphate (NH₄H₂PO₄) was the lowest efficient being, 22.85 % reduction to the spore germination.

Table (3): Germination percentage of Erysiphe heraclei DC spore affected by; (a): biocides, (b): essential oils and (c): induced resistance agents under in vitro conditions.

Figure (3): E. heraclei spore germination reduction by: (A) biocides, (B) essential oils, and (C) induced resistance agents under in vitro conditions

4 - Inhibitory activity of plant extracts:

Data presented in table (4) and shown in figure (4; A) show that all the tested cold watery plant extracts achieved an obviously significant reduction to the spore germination of E. heraclei under in vitro conditions compared with the control treatment. This reduction was gradually increased by increasing the used concentration. Besides, garlic cloves extract (Allium sativum) was the superior antifungal activity against spore germination followed by moringa leaf extract (Moringa oleifera), the corresponding values of percent reduction to the spore germination were recorded, 47.98 and 36.85 %, means respectively. Meanwhile, bluegum leaf extract (Eucalyptus globulus) resulted in the lowest reduction as recorded 8.28 %.

Table (4): In-vitro effect of plant extracts on Erysiphe heraclei spore germination.

5 - Inhibitory activity of fungicides:

Data presented in table (5) and shown in figure (4; B) indicate that all the tested fungicides caused a significant reduction to the conidial germination of E. heraclei compared with the control treatment. This reduction was gradually increased by increasing the tested concentration. Also, complete inhibition in spore germination was occurred by Score 25 % EC fungicide followed by Thiovit Jet 80 % WG, the corresponding values of inhibition percentage to the conidial germination were 100 and 90.02 %, respectively.

Table (5): In-vitro effect of fungicides on Erysiphe heraclei spore germination.

Figure (4): In-vitro reduction percentage of E. heraclei spore germination by: (A): watered plant extracts and (B): two different fungicide concentrations.

DISCUSSION

The disease survey trial which was done in the four counties in Fayoum governorate (three villages for every county) throughout January to April months of the growing season 2016/17, indicates that powdery mildew of parsley was more sever in Etsa county followed by El-Fayoum and Abshaway counties, the corresponding values of the average of percentage of disease severity were recorded, 58.25, 53.81 and 52.04 %, respectively. Meanwhile, the lowest percentage of disease severity was recorded in Sinnoures county with 46.33 %, average of percentage of disease severity. On the other hand, the highest average of disease incidence was recorded in Etsa county (70.33 %) followed by Abshaway (67.25 %) and El-Fayoum (65.11 %). While the lowest values were recorded in Sinnoures (63.18 %). These results are in agreement with the results obtained by Baiuomy and Shalaby (2007) who conducted a survey of powdery mildew of parsley in Fayoum, Beni-Suef and Qalyubia governorates and reported that the highest average of disease incidence was recorded in Qalyubia governorate. In general, the severity of the disease depends on several factors, including the variety or cultivar involved, the age and condition of the plant, when the host is infected, and the weather conditions during the growing season. In this respect, powdery mildews flourish when the days are warm to hot, the nights are cool, and dew forms on the foliage. Powdery mildews are most severe on crowded plants growing in the shade where air circulation is poor (Anon., 1987). The variation of disease severity in the various localities may be attributed to the climatic factors like temperature and relative humidity followed by cultural practices (Dinesh, 2009). In the same way, Ashtaputre (2006) reported that the higher severity of powdery mildew of chili was attributed to the temperature and relative humidity prevailed during the crop period which are favorable for development and spread of the disease. Also, Kolte (1985) mentioned that the climatic conditions like cool temperature and low relative humidity prevailed during the crop growth favored the buildup of inoculum level of pathogen, causing severe epidemics of sunflower powdery mildew. Also, Aust and Jurgren (1986) reported that cool nights, dry weather situation and age of the crop are more favorable for the powdery mildew to become severe.

In-vitro, microscopic examination of prepared slides reveal that based on the observations of the morphology of Anamorph stage, the causal agent of powdery mildew of parsley was identified as the fungus Erysiphe heraclei DC. The conidiophores were cylindrical to ovate and ranged in length from 28 to 50 µm and width from 8 to 11 µm with an average of 39 × 9.50 µm. Conidia produced singly, oblong-elliptical to oblong and ranged in length from 25 to 48 µm and width from 12 to 18 µm with an average of 36.50 × 15 µm. Cleistothecia were not found during trial period. These results are in agreement with those obtained by (Koike and Saenz, 1994; Baiuomy and Shalaby, 2007; Rosa et al., 2008; Anon., 2011; McGrath, 2013; and Ahmed, 2016). Powdery mildew fungi belonging to the order Erysiphales of the phylum Ascomycota with only one family, the Erysiphaceae are obligate biotrophic parasites of higher plants (Huckelhoven, 2005). They are further subdivided into 5 tribes (Erysipheae, Golovinomycetinae, Cystotheceae, Phyllactinieae and Blumerieae) and further sub-tribes, making more than 10 genera in total (Braun et al., 2002). Taxonomy and identification of powdery mildews were based primarily on the characteristics of the teleomorphs, such as shape of the appendages on the cleistotheium and number of asci in the cleistothecium (Braun, 1995). Also, anamorphic stage of powdery mildew include more than twelve morphological characters such as conidia, conidiophores, appressoria, houstoria, fibrosin bodies and mycelium (Boesewinkel, 1980).

Under in-vitro conditions, the present results indicate that all the tested treatments resulted in significant reduction to the spore germination of E. heraclei. This reduction was gradually increased by increasing the tested concentration. Among three commercial products of biocides, Biobac 50 % WP (Bacillus subtilis) was the most efficient one, being 61.95 % reduction to spore germination followed by Bio-ARC 6 % WP (B. megaterium) (49.16 %). Meanwhile, Plant-Guard (Trichoderma harzianum) was the lowest efficient one being, 32.88 % reduction to spore germination. These results are in agreement with those obtained by (Ahmed, 2004; Raja, 2010; Ahmed, 2011; Awad et al., 2012; Abada and Ahmed, 2014; Channaveeresh and Kulkarni, 2015 and Bakeer et al., 2016). Some of them reported that Bio-Zeid (T. album) was the most effective in reducing spore germination of Sphaerotheca fuliginea followed by Bio-ARC 6 % (Ahmed, 2011). Also, Awad et al. (2012) reported that Bio-ARC 6 % was the most effective against spore germination of S. fuliginea followed by Bio-Zeid. In addition, Abada and Ahmed (2014) stated that B. thuringiensis, B. subtilis and B. chitinosporus were the most efficient bacteria against the spore germination of Leveillula taurica. Also, Channaveeresh and Kulkarni (2015) reported that B. subtilis at 6 g/L achieved the maximum conidial inhibition of E. polygoni. Similarly, Bakeer et al. (2016) tested the antifungal activity of four commercial products of bioagents against conidial germination of E. heraclei and reported that Bio-Zeid at 2000 ppm was the superior antifungal activity against conidial germination followed by Clean-Root (B. subtilis) and Bio-ARC 6 % at same concentration, while Blight-Stop (Trichoderma spp.) at 250 ppm in descending order. In general, the superior antifungal activity of antagonistic microorganisms, Trichoderma sp. and Bacillus sp. in inhibition of spore germination may be due to the ability to synthesize and production of a wide variety of metabolic compounds such as antibiotics, hydrolytic enzymes and toxic compounds, these chemicals may be cause degradation and lysing to the cell wall of spores or inhibition of enzymes that are essential for completely germination process. Also, competition by bioagents is effective when the conidia need exogenous nutrients for germination and germ tube elongation (Blakeman, 1993). Also, Abd-Alla (2012) reported that deformation and completely damage of fungal hyphae and haustoria of E. pisi was achievement 24 h after application of B. subtilis.

Among three cold watery plant extracts tested against spore germination of Erysiphe heraclei DC, garlic cloves extract (Allium sativum) was the superior antifungal activity against spore germination followed by moringa leaf extract (Moringa oleifera), the corresponding values of percentage reduction to the spore germination were recorded, 47.98 and 36.85 %, respectively. Meanwhile, bluegum leaf extract (Eucalyptus globulus) resulted in the lowest reduction as recorded 8.28 %. These results are in agreement with those obtained by (El-Naggar, 1997; Dhaliwal et al., 2002; Ahmed, 2004; Chovatiya, 2010; Ahmed, 2011; Kalyanji, 2012; Mahalakshmi and Alice, 2013; Al-Surhanee, 2013 and Shalaby et al., 2016). Some of them reported that out of nine plant extracts tested against conidial germination of E. polygoni under in vitro conditions, garlic extract gave the best inhibition to the conidial germination (80 %) followed by ginger (63.75 %) and onion (62 %) (Chovatiya, 2010). Similar finding was also reported by Mahalakshmi and Alice (2013) who revealed that among 31 plant extracts only, garlic extract at 10% gave the superior inhibition to conidial germination of E. polygoni. Also, Shalaby et al. (2016) reported that garlic extract at 75% was the superior antifungal activity against conidial germination of E. heraclei followed by bluegum extract at the same concentration, the corresponding values of percentage of inhibition to the conidial germination were recorded, 75.87 and 72.62 %, respectively. Meanwhile, capsicum extract followed by fennel extract, each at 12.5 % concentration resulted in the lowest inhibition as recorded 0.92 and 2.29%, respectively. In general, the plant extracts show antifungal activity against a wide range of fungi (Davidson and Parish, 1989). The antifungal properties of naturally occurring substances in plant species were capable of inhibiting the spore germination at different degrees (Kalyanji, 2012). The activity of these extracts in inhibition of spore germination may be attributed to their containing phytochemical that exhibits antimicrobial and cytotoxic effects on microorganisms (Feldberg et al., 1988).

In relation to the tested essential oils, the maximum reduction to the spore germination of E. heraclei under in vitro conditions was recorded 66.06 % by thyme oil followed by anise oil (59.88 %). Meanwhile, minimum reduction to spore germination was recorded, 46.62 % by citronella oil. These results are in agreement with the results obtained by many researchers who studied the antifungal activity to essential oils against spore germination of many plant pathogenic fungi (Dhaliwal et al., 2002; Ahmed, 2004; Taxenna and Belattar, 2006; Baiuomy and Shalaby, 2007; Huang and Laksman, 2010 and Shalaby et al., 2016). They concluded that many of these substances able to achievement significant inhibition to the spore germination under in vitro conditions. The antifungal activity of essential oils against spore germination may be due to their contain specific components, antifungal compounds and fungitoxic agents that can inhibit the ability of spore germination and suppress germ tube elongation (Abd-El-Kader et al., 2003 and Sheng et al., 2005).

Out of three induced resistance agents tested against the spore germination of E. heraclei under in-vitro conditions, ascorbic acid (C₆H₉O₆) was the most efficient one in this regard, being 60.51 % reduction to the spore germination followed by sodium bicarbonate (NaHCO₃) (53.82 %). Meanwhile, ammonium dihydrogen phosphate (NH₄H₂PO₄) was the lowest efficient being, 22.85 % reduction to the spore germination. These results are in agreement with those obtained by (Ahmed, 2004; Eisa et al., 2006; Awad et al., 2012; Abada and Ahmed, 2014 and Bakeer et al., 2016). Some of them reported that the highest reduction to the spore germination of Sphaerotheca fuliginea was recorded by copper sulphate (100 %) followed by ascorbic acid (92.9 %) (Eisa et al., 2006). Also, Abada and Ahmed (2014) stated that salicylic acid was the most efficient against conidial germination of Leveillula taurica, being 41.6 % followed by chitosan, being 42.5%. Meanwhile, zinc sulphate (45.5%) followed by humic acid (44.5%) were the lowest effecting ones. Similary, Bakeer et al. (2016) tested the antifungal activity of five chemical resistance inducers against spore germination of E. heraclei under in vitro conditions and reported that maximum inhibition to the conidial germination was recorded 76.03 % when spores treated with ascorbic acid at 2000 ppm followed by potassium silicate (67.96 %) and potassium dihydrogen phosphate (64.11 %) at the same concentration. Meanwhile, the lowest value was recorded 53.13 % by the treatment of ammonium dihydrogen phosphate at the same concentration. In general, the antifungal activity of chemical inducers against spore germination may be due to the ability of these substances to damage or lysing the cell wall of spores as a result to completely contacts among these substances and spore surface.

Among two fungicides tested against spore germination of E. heraclei under in-vitro conditions, complete inhibition to the spore germination was occurred by Score 25 % EC fungicide followed by Thiovit Jet 80 % WG, the corresponding values of inhibition percentage to the spore germination were recorded100 and 90.02 %, respectively. These results are in agreement with those obtained by (Sharma and Gupta, 1994; Thind and Chander, 1995; Ahmed et al., 1995; Malani et al., 1998; Eisa et al., 2006; Shivanna et al., 2006; Zyton, 2008; Chovatiya, 2010; Ahmed, 2011; Kalyanji, 2012; Devi et al., 2013; Gogoi et al., 2013; Bheemaraya, 2014; Channaveeresh and Kulkarni, 2015 and Bakeer et al., 2016). In general, triazole fungicides group such as penconazole, propiconazole, hexaconazole and difenconazole were found to be highly effective in inhibition the spore germination of different powdery mildew pathogens. In this respect, Kalyanji (2012) reported that among systemic fungicides, hexaconazole was the highest inhibition to the conidial germination of E. cruciferarum, the causal agent of mustard powdery mildew with (84.10 %) followed by penconazole and difenoconazole (Score) with 80.05 and 78.09 %, respectively, then azoxystrobin in descending order (53.98 %). Similar finding was also reported by Bheemaraya (2014) who mentioned that propiconazole was found to be significantly superior in the inhibition of conidial germination of E. cichoracearum (94.05%) followed by penconazole (92.08%), hexaconazole (90.93%) and carbendazim (87.89%). Also, Ahmed (2016) reported that propiconazole followed by penconazole gave the best inhibition to the conidial germination of E. heraclei, the corresponding values of the percentage of inhibition to spore germination were, 100 and 96.10 %, respectively. The present results also, show that Thiovit Jet 80 % WG was found to be effective against spore germination of E. heraclei. Similar finding was also reported by Chovatiya (2010) who reported that wettable sulphur recorded maximum inhibition (80 %) to the spore germination of E. polygoni in fenugreek followed by mancozeb (72.25%) and dinocap (70%). Also, Kalyanji (2012) reported that among non-systemic fungicides, dinocap followed by wettable sulphur were the most effective against spore germination of E. cruciferarum, the causal agent of mustard powdery mildew with 79.98 and 77 %, respectively. Similarly, Gogoi et al. (2013) tested the efficacy of four forms of sulphur fungicide at 1000 ppm against spore germination of E. cichoracearum and reported that all sulphur fungicides significantly reduced the germination of conidia compared with the control.

REFERENCES

Abada, K.A. and M.A. Ahmed (2014). Effect of combination between bioagents and antioxidants on management of tomato powdery mildew. American J. of Life Sci., 2 (6-2): 26-32.

Abd-Alla, F.M.F. (2012). Studies on powdery mildew disease affecting pea plants in Egypt. Ph.D. thesis, Plant Pathol. Dept., Agric., Cairo Univ., 152 pp.

Abd-El-Kader, Dawlat, A.; A.A. Hilal; A.Z. Aly and M.G.A. Nada (2003). Effect of essential oils and volatile substances of some medicinal and aromatic plants on squash powdery mildew. Proc. 10th Cong. Phytopathol., Giza, Egypt., 179-192.

Ahmed, G.A. (2004). Using plant extracts to control powdery mildew disease that attack cucumber plants under protected houses. M.Sc. Thesis, Plant Pathol. Dept., Fac. Agric., Moshtohor, Zagazig Univ., 170 pp.

Ahmed, H.F.A. (2011). Management of some methods to control powdery mildew disease of chamomile. M.Sc. Thesis, Plant Pathol. Dept., Fac. Agric., Fayoum Univ., 152 pp.

Ahmed, H.F.A. (2016). Using different measures to control powdery mildew on coriander (Coriandrum sativum L.) and dill (Anethum graveolens L.) in Fayoum Governorate. Ph.D. Thesis, Plant Pathol. Dept., Fac. Agric., Fayoum Univ., 171 pp.

Ahmed, M.; M.A. Khan and M. Iqbal (1995). Evaluation of fungicides against conidial germination of Erysiphe pisi D.C. and powdery mildew disease on pea. Pakistan J. Phytopathol., 7 (2): 154 - 156.

Al-Surhanee, Ameena, M. (2013). Efficiency of the extracts of some plants on squash powdery mildew. Mediterranean J. Soc. Sci., 4 (11): 39-49.

Anonymous (2016). Annual report of agricultural statistics. Agric. Economic statistics Dept., Min. Agric. and land Reclamation, Egypt, 164 pp.

Anonymous (2015). Annual report of agricultural statistics. Agric. Economic statistics Dept., Min. Agric. and land Reclamation, Egypt, 159 pp.

Anonymous (2011). Pathogen of the month -2011, Erysiphe heraclei DC. Andrew Watson Sci. & Res., Primary Industries. Online, Andrew. Watson @ industry.nsw.gov.au.

Anonymous (1987). Powdery mildews of ornamentals. Report on Plant Disease. Dept. Crop Sci., Illinois Univ. At Urbana-Champaign, RPD No. 617.

Ashtaputre, S.A. (2006). Studies on loss assessment, epidemiology and management of powdery mildew of chili caused by Leveillula taurica (Lev.) Arn. Ph.D. Thesis, Univ. Agric. Sci., Dharwad, Karnataka, India, 79-81.

Aust, H.J. and H.H. Jurgen (1986). Microclimate in relation to epidemics of powdery mildew. Ann. Rev. Phytopathol., 29: 144-145.

Awad, M.E.; K.A. Abo-Elyousr and M.F. Abdel-Monaim (2012). Management of cucumber powdery mildew by certain biological control agents (BCAs) and resistance inducing chemicals (RICs). Archives of Phytopathol. and Plant Prot., 45 (6): 652- 659.

Baiuomy, M.A.M. and I.A. Shalaby (2007). Parsley powdery mildew and its management. Egypt . J. Appl. Sci., 22 (1): 45- 67.

Bakeer, A.T.; M.A.M. Baiuomy and H.F.A. Ahmed (2016). Impact of spraying with some biological products and chemical resistance inducers on controlling powdery mildew disease, growth quality and yield components of coriander and dill. Egypt . J. Appl. Sci., 31 (2): 17-39.

Bheemaraya, A. (2014). Sunflower powdery mildew caused by Erysiphe cichoracearum DC-epidemiological considerations. M.Sc. Thesis, Plant Pathol. Dept., Fac. Agric., Univ. Agric. Sci., Dharwad, India, 88 pp.

Blakeman, J.P. (1993). Pathogens in the foliar environment. Plant Pathol., 42: 479-493.

Boesewinkel, H. (1980). The morphology of imperfect states of powdery mildews (Erysiphaceae). Bot. Rev., 46: 167-224.

Braun, U. (1995). The powdery mildew (Erysiphales) of Europe. Jena, Germany: Gustav Fischer-Verlag., 116-117.

Braun, U.; R.T.A. Cook; A.J. Inman and H.D. Shin (2002). The taxonomy of the powdery mildew fungi. In: The powdery mildews. Eds. Belanger, R.R., Bushnell, W.R., Dik, A. J. and Carver, T.L.W., APS Press, St. Paul, Minnesota, U.S.A., 13-55.

Bubici, G. (2015). First formal identification of Erysiphe heraclei causing powdery mildew on dill (Anethum graveolens) in Italy. New Disease Reports, 31: 22.

Channaveeresh, T.S. and S. Kulkarni (2015). In vitro evaluation of fungicides, bioagents and botanicals against Erysiphe polygoni DC in black gram. Int. J. in Physical and Appl. Sci., 2 (3): 41-53.

Chovatiya, A.J. (2010). Management of powdery mildew disease of fenugreek (Trigonella foenum graecum L.). M.Sc. (Agric.) Thesis submitted to Junagadh, Agric. Univ., Junagadh, 57 pp.

Davidson, P.M. and M.E. Parish (1989). Methods for testing the efficacy of food antimicrobials. Food Technol., 43:148 - 155.

Devi, P.; K. Vinothini and V. Prakasam (2013). Effect of azoxystrobin and Mancozeb on powdery and downy mildew pathogens under in vitro conditions. J. Agric. and Veterinary Sci., 6 (4): 50-55.

Dhaliwal, H.S.; T. Thind; C. Mohan and B. Chhabra (2002). Activity of some essential oils against Uncinula necator causing powdery mildew of grapevine. Indian Phytopathol., 55 (4): 529-531.

Dinesh, B.M. (2009). Studies on powdery mildew of sunflower caused by Erysiphe cichoracearum DC. M.Sc. Thesis, Plant Pathol. Dept., Fac. Agric., Univ. Agric. Sci., Dharwad, India, 72 pp.

Eisa, Nawal, A.; A.I. El-Fiki; F.G. Mohamed and M.H. El-Habbak (2006). Biochemical changes in squash leaves sprayed with some chemicals for inducing resistance to powdery mildew. The second conf. on farm IPM, Fayoum Univ., 16-18 January.

EL-Naggar, M.A.A. (1997). Effect of some plant extracts as germicides on controlling powdery mildew disease of pepper under plastic house. 8th cong. Egypt. Phytopathol. Soc., 173 - 184.

Feldberg, K.S.; S.C. Chay; A.N. Kotica; M. Nadler; Z. Neuwirth; D.C. Sunderstroma and N. Thompson (1988). In vitro mechanism of inhibition of bacterial cell growth by allicin. Antimicrob. Agents Chemother., 32: 1763-1768.

Fisher, R.A. (1984). Statistical methods for research workers. Oliver and Boyd, London, U.K.

Godwin, J.R.; J.W. Manstield and P. Darby (1987). Microscopical studies of resistance to powdery mildew disease in hop cultivar wye target. Plant Pathol., 36: 21-32.

Gogoi, R.; P.K. Singh; R. Kumar; K.K. Nair; I. Alam; C. Srivastava; S. Yadav; G. Gopal; S.R. Choudhury and A. Goswami (2013). Suitability of Nano-sulphur for biorational management of powdery mildew of okra caused by Erysiphe cichoracearum. J. Plant Pathol. Microb., 4: 2-4.

Gomez, K.A. and A.A. Gomez (1984). Statistical procedures for agricultural research 2 nod, John Wiley and Sons, Inc., New York.

Huang, Q. and D.K. Laksman (2010). Effect of clove oil on plant pathogenic bacteria and bacterial wilt of tomato and geranium. J. of Plant Pathol., 92: 701-707.

Huckelhoven, R. (2005). Mini Review: powdery mildew susceptibility and biotrophic infection strategies. FEMS. Microbiol. Lett., 245: 9-17.

Istvan, U. (2002). Transforming natural products into natural pesticides-experience and expectations. Phytoparasitica, 30: 439-442.

Kalyanji, K.K. (2012). Management of powdery mildew disease (Erysiphe cruciferarum Opiz ex. Junell) of mustard [Brassica juncea (L.) Czern. & Coss.]. Ph.D. Thesis, Fac. Agric., Junagadh Univ., India, 178 pp.

Kiraly, Z.; S.J. Klement; Vores and K. Solymosy (1974). Methods in plant pathology with special reference to breeding for resistance. Elsevier Scientific Publishing Company, New York, 212 pp.

Kiss, L. (2003). A review of fungal antagonists of powdery mildews and their potential as biocontrol agents. Pest Manag. Sci. 59: 475- 483.

Koike, S.T. and D.A. Glawe (2009). First report of powdery mildew of poison-hemlock caused by Erysiphe heraclei in north America. Online. Plant Health Prog. doi:10.1094/PHP-2009-0312-01-BR.

Koike, S.T. and G.S. Saenz (1994). Occurrence of powdery mildew on parsley in California. Plant Disease, 78: 1219.

Koitabashi, M. (2005). New biological method for parsley powdery mildew by the antifungal volatiles- producing fungus. Kyu-W63. J. Gen. Plant Pathol., 71:280-284.

Kolte, S.J. (1985). Diseases of annual edible oilseed crops III. CRC Press, Florida, 9-96.

Mahalakshmi, P. and D. Alice (2013). Management of powdery mildew (Erisiphe polygoni) disease in green gram by botanicals. Int. J. Appl. Bio-Res., 18 (18): 23-29.

Malani, S.S.; N. Khare; N. Lakpale and R. Kumar (1998). Efficacy of some fungicides against powdery mildew of grass pea (Lathyrus sativus L.). Ann. Plant Prot. Sci., 6 (2): 131-135.

Manici, L.; L. Lazzeri and S. Palmieri (1997). In vitro fungitoxic activity of some glucosinolates and their enzyme-derived products toward plant pathogenic fungi. J. Agric. and Food Chemistry, 45: 2768-2773.

McGrath, M.T. (2013). Powdery mildew of carrot. Cornell Univ., Long Island Horticultural Res. and Extension Center. Vegetable MD Online, mtm3 @ cornell.edu, February, 2013.

Nair, K.R.; S. Sivan and A.H. Ellingboe (1962). A method of controlled inoculations with conidiospores of Erysiphe graminis var. tritici. Phytopathol., 52: 714.

Negahban, M.; S. Moharramipour and F. Sefidkon (2007). Fumigant toxicity of essential oil from Artemisia sieberi Besser against three stored-product insects. J. Stored Prod. Res., 43:123–128.

Negahban, M.; S. Moharramipour and F. Sefidkon (2006). Insecticidal activity and chemical composition of Artemisia sieberi Besser essential oil from Karaj, Iran. J. Asian Pac. Entomol., 9: 61–66.

Park, M.; J. Choi; J. Kim and H. Shin (2010). Outbreak of powdery mildew caused by E. heraclei on Pleurospermum kamtschaticum in Korea. New Disease Reports, 21, 14.

Pasini, C.; F. D’Aquila; P. Curir and M.L. Guliino (1997). Effectiveness of antifungal compounds against rose powdery mildew (Sphaerotheca pannosa var. rosae) in glasshouses. Crop Prot., 16: 251-256.

Pereira, R.B.; G.C. Lucas; F.J. Perina; M.L.V. Resende and E. Alves (2011). Potential of essential oils for the control of brown eye spot in coffee plants. Ciência Agrotecnol., 35: 115-123.

Raja, S.A. (2010). Management of powdery mildew, Phyllactinia corylea of Mulberry (Morus sp.) using chosen biocontrol agents. J. Biopesticides, 3 (2): 483 - 486.

Reuveni, M.; V. Agapov and M. Reuveni (1996). Controlling powdery mildew caused by Sphaerotheca fuliginea in cucumber by foliar sprays of phosphate and potassium salts. Crop Prot., 15: 49-53.

Rosa, D.D.; C.T. Ohto; M.A. Basseto; N.L. De Souza and E.L. Furtado (2008). Brazil, a new location for powdery mildew on parsley and fenchel plants. Plant Pathol., 57: 373.

Rosato, A.; C. Vitali; N. De laurentis; D. Armenise and M. Antonietta Milillo (2007). Antibacterial effect of some essential oils administered alone or in combination with norfloxacin. Phytomedicine,14: 727-732.

Ryals, J.A.; U.H. Neuenschwander; M.G. Willits; A. Molina; H.Y. Steiner and M.D. Hunt (1996). Systemic acquired resistance. Plant Cell, 8: 1809-1819.

Shalaby, O.Y.; M.A.M. Baiuomy and H.F.A. Ahmed (2016). Impact of spraying with some plant extracts and essential oils on controlling powdery mildew, growth quality and yield components of coriander (Coriandrum sativum L.) and dill (Anethum graveolens L.). Egypt . J. Appl. Sci., 31 (9): 194-215.

Sharma, K.K. and V.K. Gupta (1994). Influence of fungicides on spore germination, sporulation and control of apple powdery mildew. Indian J. Mycol. Plant Pathol, 24 (1): 24-28.

Sheng, Y.; F. Pin and T. Shang (2005). Antifungal activities of essential oils and their constituents from indigenous cinnamon leaves against wood decay fungi. Bioresouce Techol., 96: 813-818.

Shivanna, E.; A. Sataraddi; B. Janagoudar and M. Patil (2006). Efficacy of fungicides for the management of powdery mildew, E. cichoracearum of okra. Indian J. Plant Prot., 34 (1): 85-88.

Taxenna, A. and N. Belattar (2006). Effect of oils extracted from Nigella sativa seeds and some terpen compounds on Mauginiella scaettae spore germination. 9^th Arab Cong. of Plant Prot., 19-23 November 2006, Damascus, Syria, E-157.

Thind, T.S. and M. Chander (1995). A laboratory method for evaluating fungicides against Uncinula necator. Indian Phyopathol., 48: 207-209.

Whitney, E.D.; R.T. Lewellen and I.O. Skoyen (1983). Reaction of sugar beet to powdery mildew: Genetic variation, association among, testing procedure and resistance breeding. Phytopathol., 73: 182 - 185.

Yoon, M.Y.; B. Cha and J.C. Kim (2013). Recent trends in studies on botanical fungicides in agriculture. Plant Pathol. J., 29 (1): 1-9.

Ziedan, E.H.E. (2010). First record of dill powdery mildew caused by Erysiphe heraclei DC in Egypt. Archives of Phytopathol. and Plant Prot., 43: 728-735.

Zyton, Marwa A. (2008). Pathological studies on powdery mildew of okra in Egypt. M.Sc. Thesis, Plant Pathol., Dept., Fac. Agric., Cairo Univ., Egypt, 81 pp.

تقییم کفاءة النشاط التثیبطی لبعض مرکبات المقاومة الحیویة والزیوت العطریة والمستخلصات النباتیة وعوامل المقاومة المستحثة على الفطرErysiphe heracle  مسبب مرض البیاض الدقیقی فی البقدونس

عبد الراضی طاهر بکیر¹، هدى محمد حسین أحمد¹، محمد أحمد محمد بیومی²، أکرام فتوح محمد فتوح²

¹ قسم النبات الزراعی (أمراض النبات)– کلیة الزراعة– جامعة الفیوم

²معهد بحوث امراض النبات– مرکز البحوث الزراعیة- الجیزة

یعتبر البقدونس من أهم النباتات الطبیة التی تنتمى الى العائلة الخیمیة وتعتبر منطقة حوض البحر المتوسط هی الموطن الأصلی له. یصاب البقدونس بالعدید من أمراض المجموع الخضری ویعتبر مرض البیاض الدقیقی المتسبب عن الفطر Erysiphe heracleiمن أهم هذه الأمراض والذى ینتشر فی اماکن زراعته المختلفة مسببا بذلک اضراراً بالغة بالمحصول الخضری والذی ینعکس بدوره بالسلب على کمیة المحصول من النورات والزیت العطری. تم مشاهدة اعراض البیاض الدقیقی بوضوح على البقدونس خلال فترة حصر المرض والتی امتدت من ینایر وحتى ابریل خلال موسم نمو 2016/2017 فی العدید من المراکز فی محافظة الفیوم. فی محاولة لمقاومة المرض هناک العدید من التجارب التی اجریت باستخدام مواد آمنة بهدف الحفاظ على صحة الإنسان وعدم تلوث البیئة مع عدم إغفال دور المقاومة الکیمیائیة واللجوء إلیها عند الحاجة. فی المعمل أجریت العدید من التجارب لتقییم تأثیر استخدم ترکیزات مختلفة من المبیدات الحیویة (بلانت جارد، بیوأرک 6 %، بیوباک 50 %)، والزیوت العطریة (السترونیلا، الزعتر، الینسون)، والمستخلصات لنباتیة (مستخلص أوراق المورینجا، مستخلص فصوص الثوم، مستخلص أوراق الکافور)، وعوامل المقاومة المستحثة (بیکربونات الصودیوم، حامض الاسکوربیک، فوسفات الامونیوم ثنائیة الحامضیة) الى جانب اثنین من المبیدات الکیمیائیة (ثیوفیت جیت 80 %، سکور 25 %) على انبات الجراثیم الکونیدیة للفطر المسبب للمرض.

فی اختبار المعمل اوضحت النتائج ان کل المعاملات المختبرة خفضت انبات الجراثیم الکونیدیة للفطر مقارنة بمعاملة الکونترول وکان هذا الانخفاض یزداد بزیادة الترکیزات المستخدمة. وأوضحت النتائج انه من بین ثلاثة من المرکبات الحیویة کان المرکب بیوباک 50 % الافضل فی تثبیط انبات الجراثیم تلاه المرکب بیوأرک 6 % بینما کان المرکب بلانت جارد هو الاقل تأثیرا فی هذا الصدد، وکانت القیم المقابلة لنسبة الخفض فی انبات الجراثیم هی 61.95، 49.16، 32.88 % على الترتیب. ومن بین المستخلصات النباتیة المستخدمة أحدث المستخلص المائی لفصوص الثوم التأثیر الافضل (47.98 %) تلاه المستخلص المائی لأوراق المورینجا (36.85 %) بینما کان مستخلص أوراق الکافور هو الاقل فاعلیة (8.28 %). ومن بین الزیوت العطریة کان الزیت العطری للزعتر هو الافضل فی تثبیط انبات الجراثیم (66.06 %) تلاه الزیت العطری لکل من الینسون (59.88 %) والسترونیلا (46.62 %). ومن بین مستحثات المقاومة المستخدمة أحدث حامض الاسکوربیک التأثیر الافضل (60.51%) تلاه بیکربونات الصودیوم (53.82 %)، بینما کانت فوسفات الامونیوم ثنائیة الحامضیة هی الاقل تأثیرا فی هذا الصدد (22.85 %). وأخیرا، فقد أدى استعمال المبید الکیمیائی سکور 25 % الى احداث تثبیط کلی لإنبات الجراثیم الکونیدیة للفطر تلاه المبید ثیوفیت جیت 80 %.