Persian Gulf Crop Protection Available online on: www.cropprotection.ir ISSN: 2251-9343 (online) Volume 3 Issue 1, March 2014 Pages 79-86 First Record Of Pythium oligandrum From Libya Soli N. A. Mohamed1*, Z. I. El-Gali2and A. A. Akila3 Dept. of Plant Protection, Fac. Of Agriculture, Omer Al-Mukhtar Univ. El-Beida,Libya. (*Corresponding author e-mail: [email protected]). Abstract : Pythium oligandrum Drechsler was isolated from surface soil of agriculture field cultivated with vegetable and fruits trees in Ras-Trab, AlGabal Al-Khdar region in the north eastern of Libya. Identification of this fungal was confirmed by both of vegetative (mycelium) and productive structures produced on Corne meal agar media (CMA) incubated at constant temperature (25ºC) for one week period, fast grown, very thin comprises, hyphae due to the evanescent, hardly visible, the optimal growth of P. oligandrum on potato carrot agar (PCA) 63.5mm/day, Glucose Asparagin agar (GAM) 47.6 mm/day. Oogonia production after one week incubation, oogonia terminal or intercalary, subglobose, 27.42 ±5.10 µm conical, pointed projections 3-7µm long; oospores somewhat aplerotic 14.48±2.13µm diam, was observed under light microscopic and measured by with digital camera type Olympus cX21fs1. Key Words: Pythium oligandrum, temperature, soil. Persian Gulf Crop Protection, 3(1): 79-86 79 Introduction 1930). The fungus is distributed globally Several studies explored the role of P. in many countries around the world nd oligandrum as a biocontrol agent against was isolated from soil was new records some plant pathogenic fungi (Vesely, for Argentina(Frezzi, 1956), Italy, Britain, 1979). P. oligandrum is a non pathogenic Cyprus, Australia(Vaartaja & Bumbieris, fungus. It’s recorded on 12 plant species 1964),Hawaii (Klemmer et al., 1964), belonging 6 different families (Brozova, Germany (Domsch et al., 1968), 2002). P.oligandrum dose not attack the Netherlands (Plaats-Niterink, tissue of the crops but occurs on the root 1975),Central Africa (Kobayashi et al., surface (Mohamed et al., 2007) It can be 1977),Iran (Ershad, 1977),as associated of used Biological control, reduces seedling sugarcane cultivars in the field of Rio death by its competition ability on the dejaneiro Pickia (Valdebenito-sanhureca place, Its ability to inhibit the growth of et al 1984), Chain (Duan, 1985),Franc pathogenic fungi according to its (Paul, 1994),Hokkaido (sato et al., 1985), antagonistic action. This antagonistic Japan(Kinoshita et al., 1994), Egypt ability depends upon the type of (Abdelzaher et al., 1997), Pakistan (Sultan pathogenic fungus, antagonistic density et al., 1997), Palestinia Autonomous and the host plant, and It cannot be able to Territory (Ali-Shtayeh et al., 1999). produce antibiotic (Benhamou, Objective of this study isolation Pythium 1997).oospores can be survived in the oligandrum depending on Microscopic form of for a long time (up to 10 years) and Macroscopic characters. without affecting its ability to germinate Materials and Methods (Walther, 1987). the genus Pythium Sample collection: Soil samples were Pringsheim, established by Pringsheim in collected during April 2012 from 1858 (Long et al., 2011) belongs to agriculture fields throughout Ras trab area Pythiaceae, Pythiales, Oomycetes, located in the North East of Libya (Fig 1). Oomycota. Plaats-(Niterink, 1981) used Three samples were collected each field that important morphological each one from diversity of habitats, fruit characteristics for P. oligandrum trees and vegetable. after removing of identification based on morphological surface debris clean hand shovel was used characteristics of vegetative and to collect 500 gram of top soil. Each soil productions structures, beside sample was placed individually in a clean physiological characteristics the influence plastic bag and labelled properly. They of environmental factors such as were brought back to the laboratory as temperature (Waterhouse, 1967) and soon as possible for immediate isolation. (Plaats-Niterink, 1981),revised that their All of the ten individual sample for each hyphae are hyaline and aseptate (not agriculture field were mixed. The pH, Ec, containing cross walls) (Levesque et al., moisture content of the soil sample was 2004) reported that Pythium culture were determined by the assistance of the soil and identified and classified with the aid of water. keys of plaats-Niternik, the taxonomy of Isolation, Purification of P. oligandrum Pythium was mainly based on the from soil: Isolation of the fungus was done morphological characteristics, such as the by modification baiting technique (Harvey, size and shape of oogonia, antheridia and 1925). The mixed soil samples was placed sporangia. However, some important in three sterilized pots, and bromus grass morphological structures are highly plants (Bromus sp) were planted in the variable, overlap considerably and are mixed sample and moisture with 10-15 ml absent in many species. P.oligandrum sterilized water to each pot. After 8 days isolated for the first time in the United The baits (bromus grass plants) were States from the pea roots (Drechsler, rinsed in sterilized water to remove soil Persian Gulf Crop Protection, 3(1): 79-86 80 particles. The leaves cut (1cm2) was placed Determination of physical factors: The in Petri dish contain sterile water and results tabulated in (Table 1) showed that incubated at 25°C; After 2 days, the leaves the charactes of Ras tarb soil as a loamy pieces were transferred to water agar clay type the soil Sample was pH 7.49, medium. after 3 days a faint halo of fungal which electric conduction (EC) 0.4, (RH%) threads was observed on the dish. 17.04%. The results obtained from this For purification P. oligandrum hyphal tip study indicated that the soil content P. technique (Mukundi et al., 2009) was used. oligandrum identified the fungus depended Fungi was transferred to medium consisted on ( colony character, microscopic and of corn-meal agar (CMA :extract of 60 g growth rate). ground maize, 15g agar distilled water 1l). Colony of the P.oligandrum: (Fig 2) 20mg chloramphenicol antibiotic and 10 indicated that the colony of P.oligandrum mg Benomyl and incubated at 25°C for 3 was colorless , meyclium was submerged days. The morphological characteristics, on both media Corn meal agar (CMA) and key suggested by ( Plaat-Niterink ,1981). potato carrot agar (PCA), while on Glucose Were used for identification microscopic asparagin agar medium (GAM) was no and macroscopic characteristics were special pattern and appeared some aerial estimated using the fungus colony features, mycelium, but on GAM medium the such as colony color of the fungus, the mycelium growth was more than PCA nature of the growth. Growth rate was medium. measured on corn meal agar (CMA), Growth rate of P. oligandrum: The Potato carrot agar (PCA) and Gloucose growth rate was differents between the asparagines agar (GAM) after 24 h media. The optimum growth was potato incubation. For Oospores production P. carrot agar (PCA) 63.5±1.96 mm/day after oligandrum was cultured to produces 24 h incubation, while the growth was oospores by grown in 10 ml of V.8 juice recorded 47.6±6.46mm/day and 24.75 ± medium/Petri dish, after 7 days, the 3.07 mm/day on Glucose Asparagin agar mycelium mats was dried by between two (GAM) and Corn meal agar (CMA) spatulas (mycelium without medium) then respectively (Fig 3). also should that the transferred to Erlenmeyer flasks containing mycelium was thin cottony, aerial on 100 ml GAM medium for 21 days. GAM, while on PCA and CAM medium Oospores were obtained, Fifty oospores were embedded and non aerial. For sexual were diameter measured under the structures, the P. oligandrum was produced microscope with camera digital type the same number of oogoina for all media Olympus cX21fs1 (Mohamed, 2006). tested, but the oospores was observed on Morphological characters of asexual GAM media only. organs were studied under the microscope Microscopic characteristics: Concerning with digital camera (Olympus cX21fs1), Microscopic characteristics, results were mycelium diameter, number of antheridia illustrated in (Fig 4)was examined under a and diameter of oogonia was recorded. The light microscope, the mycelium of isolate of fungus was identified according P.oligandrum was non septet, the sexual to the methods of (Waterhouse, 1967), reproduction as oogonia and antheridia was (Abdzaher et al., 1997), (Shtayeh and observed. Oogonia mostly intercalary Saleh 1999), (Lodi et al., 2005), occasionally terminal, ornamented, spiny, (Mstowfizadeh et al., 2005), (Yudiarti et one antheridium clavate are always present al., ,2006) and (Mukundi et al ,2009) and with oogonium. Under the light kept on slant Malt agar in the refrigerator microscopic the structures of P.oligandrum and used for further studies. were measured, the resultswere shown in Results and Discussion (Fig 5) and (Table 2) The main hyphae tested up to 2.49 μm wide, and sexual Persian Gulf Crop Protection, 3(1): 79-86 81 reproduction of P.oligandrum takes place that population levels lowest during by means of oogonia and antheridia, the midsummer, the soil analysis chemical and size of oogonia varied between 15.52 And physioical described as a loamy clay type, - 31.61 (av. 27.52± µm diameter.), Spines pH 7.49 and water content by weighing 6.02 μm long and 1.9 μm wide. The (RH%) 17.04%. The hyphae of this fungus antheridia were 27.3 µm long and5.7 µm were hyaline and the mycelium non septet wide, The oospore 11.67-26.68 µm had and sexual reproduction of oogonia and diameter (mean 19.1 µm), and thick wall antheridia was observed, the size of was 1.34 µm. The results were recorded oogonia varied between (av. 27.52± µm that the fungus P. oligandrum was diameter.), the antheridia were mostly occurrecen in soil of Al-Gabal Al-khtar intercalary 27.3 long and5.7 wide, The area،this results was harmony with oospore (mean 19.1 µm diameter), these (Mohamed, 2010), this fungus re-isolated data agreed with (Abdzaher et al., form collected soil in April 2012, 1997;2004), (Lodi et al., 2005), (Plaats- population levels of P.oligandrum was Niterink, 1981) (Levesque et al., 2004) highest (Ali-Shtayeh 1986), who explained Table 1.