Occurrence, Identification and Pathogenicity of Pythium aphanidermatum, P. diclinum, P. dissotocum and Pythium "Group P" Isolated from Dawmat Al-Jandal Lake, Saudi Arabia
Hani M.A. Abdelzaher
Pythium species are considered to be an aquatic fungus which occurs much abundantly in aquatic media. There are no sufficient efforts in studying the presence of them in water as compared with study in soil. For this reason, these fungi were studied in the waters of Dawmat Al-Jandal Lake which is essentially not yet attained their roles of biological and environmental studies in this country. In this study, Pythium biota has been investigated in Dawmat Al-Jandal lake, Saudi Arabia in order to clarify a diversity of this important fungus. Pythium species were isolated from water by baiting method with grass blades using Pythium selective medium. Sequencing of the internal transcribed spacer regions of ribosomal DNA (rDNA-ITS) including the 5.8 SrDNA of isolated pythia confirmed identification based on morphological characteristics. Fifty-nine isolates of Pythium spp. were obtained, of these, a total of three species and one group could be identified. Species isolated in the water of the lake were P. aphanidermatum, P. diclinum, sterile P. dissotocum and Pythium 'group P'. P. aphanidermatum was highly pathogenic to tomato seeds causing 100% damping-off but P. diclinum and the sterile isolate of P. dissotocum were moderately pathogenic, while Pythium "group P" was non-pathogenic to tomato seeds causing 60 and 0% damping-off, respectively. It is concluded that Pythium species is present in Dawmat Al-Jandal lake and some of these fungi appeared their virulence to tomato seedlings. It is possible that these pythia represent a risk factor in dissimilation of such microorganisms to the surrounded areas.
to cite this article:
Hashem Al-Sheikh and Hani M.A. Abdelzaher, 2012. Occurrence, Identification and Pathogenicity of Pythium aphanidermatum, P. diclinum, P. dissotocum and Pythium "Group P" Isolated from Dawmat Al-Jandal Lake, Saudi Arabia. Research Journal of Environmental Sciences, 6: 196-209.
Received: October 15, 2012;
Accepted: January 09, 2013;
Published: February 16, 2013
Dawmat Al-Jandal Lake is located near Dawmat Al-Jandal city, northern part of Saudi Arabia (29°5156″N, 39°5120″E) and it has been formed during the eighth decade of the last century. The Lake formed due to accumulation of agricultural drainage water and rain water into the region. Formation of Dawmat Al-Jandal Lake has led to a number of changes in the existing microhabitats that a large number of different species of biota, flora and fauna became reestablished in such new ecological setting. The area thus opened several possibilities for various forms of development in tourism and recreational activities, fisheries and limited agricultural or soil reclaimatory activities. Before this study, no studies on the occurrence of Pythium species in the water Dawmat Al-Jandal lake has been done.
One of the most important aquatic fungus is the genus Pythium which
was established by Pringsheim 1858 and placed in the
family Saprolegniaceae. The genus was subjected to several taxonomical alternation
through more than 14 decades (Berlese and De Toni, 1888;
Butler, 1907). Now-a-days, the genus belongs to the Kingdom
Stramenopile, excluded from the traditional true fungi of the Kingdom
Myceteae. Aquatic media is necessary for production of zoospores and thus, Pythium
spp. have been referred to commonly as belonging to aquatic molds. Consequently,
standing water in fields, ditches, ponds, lakes and hydroponic systems are places
that Pythium spp. can exist, multiply to great numbers and spread. Zoospores
can swim for a short time (approximately 1/2 h) to a limited distance of three
inches on the thin film of water around soil particles. Without free water they
die rapidly or sometimes they can encyst by forming a thick wall and may survive
up to 7 days in soil. Zoospores swim towards all directions and may be reached
to new roots during irrigation. The zoospores are then attracted by chemicals
to root tips where infection typically occurs. Natural or artificial movement
of water infested with Pythium spp. provides a method by which rapid
spread of these plant pathogens can occur over greater distances (Kucharek
and Mitchell, 2000).
Keys for identification of Pythium species are based only on morphological
basis (Middleton, 1943; Waterhouse,
1968; Van der Plaats-Niterink, 1981; Dick,
1990). Recently, morphological characteristics of a species are increasingly
supported by molecular characteristics (Matsumoto et
al., 1999; Levesque and de Cock, 2004; Kageyama
et al., 2005; Tsukiboshi et al., 2007).
Amplification of ribosomal genes by Polymerase Chain Reaction (PCR) is used
for the genetic identification of many fungi (Masih and
Paul, 2003). Additionally, The ribosomal nuclear DNA consists of transcribed
and non-transcribed regions [ITS1 (internal transcribed spacer) and ITS2] can
be amplified with the PCR method by using the universal primers ITS1 and ITS4
(Chen et al., 1992). Sequences of these parts
are used to assist in identification of these species within the genus Pythium.
Species of Pythium species are cosmopolitan. They may present as saprophytes,
muturalists and parasites and distributed in aquatic and terrestrial places
(Van der Plaats-Niterink, 1981; Ichitani
and Goto, 1982; Al-Sheikh and Abdelzaher, 2010a,
b). Recently, some species of Pythium caused
diseases to fishes, animals (Kitancharoen and Hatai, 1998;
Helman and Oliver, 1999). Pythium diseases to
human (Pythiosis) occurs in animals and human who contacting aquatic habits
that contain Pythium insidiosum de Cock (Mendoza
et al., 1987; Shenep et al., 1998).
Pythium spp. is commonly occurred in agricultured and non-agricultured
soils of the world (Al-Sheikh and Abdelzaher, 2012).
They present most abundantly in agricultured zones (Van
der Plaats-Niterink, 1975), less commonly in non-agricultured or acid soils
where the genus Trichoderma is the reason for their absence (Barton,
1958). Pythium species have also been isolated from different soil
types of arable, pastures, forests, nurseries, marshes, swamps and water (Senda
and Kageyama, 2006). Deserts, dry forests and salt marshes are generally
poor in this taxon (Moharam, 2010). Species of Pythium
dormans in soil as saprophyte and by the formation of resistant sexual structures
(oospores) (Elnaghy et al., 2009). They germinate
when humidity increase and by any other soil factor that may give it a competitive
advantage against other soil microorganisms. They also become pathogenic and
enter plant tissues; it spreads rapidly but can soon be overcome by competitors.
Sparrow (1968) and Abdelzaher et
al. (1995) postulated that "the ecology of fresh-water Pythium
spp. has not received the same attention reached by that of soil pythia. Studies
have been made on Pythium spp. associated with aquatic basins (Sanchez
and Gallego, 2001). Until recently, studies on the occurrence of aquatic
pythia in Saudi Arabia are scarce.
In Saudi Arabia, researching on this genus has begun recently. Little investigations
were performed concerning some virulent Pythium spp. (Sunboul,
2001, 2006). International Migration Institute (IMI)
(records for geographical unit Arabian Peninsula) has an isolate of P. aphanidermatum
which isolated from Al-Madinah city and was parasitic on tomato plants in 1983
(Herb IMI 291421). Some researchers isolated some species of Pythium
from the diseased and of healthy plants cultivated in Saudi Arabia, others have
also isolated two species of Pythium from accumulated rainfall water
in the Riyadh region, Saudi Arabia (El-Nagdy and Nasser,
2000) but descriptions and illustrations of the pure cultures of the isolated
Pythium spp. were not obtained as well as identification based on morphological
and molecular basis were not also supplied (Sunboul, 2001;
Occurrence of Pythium, especially in soil of near different plants,
have been carried out by several researchers all over the world. In Saudi Arabia,
scare studies has been performed for isolation and identification of this economic
fungus. Except for few studies performed in certain places, no comprehensive
study has been done concerning this line. Therefore, studying this unique fungus
in Saudi Arabia is of importance and more researches should be done. In a series
on study of Pythium in Saudi Arabia, Al-Sheikh and
Abdelzaher (2012) have isolated some species of Pythium from rhizosphere
soil of different cultivated plants. Generally, no data were available on isolation
of Pythium from aquatic habitats in Saudi Arabia.
To receive more information, this study was done to get information about the presence of Pythium species in Dawmat Al-Jandal lake during winter of December of 2012. A complete description of the isolated species based on morphological and molecular criteria was provided. Pathogenicity of the isolated Pythium species using tomato was also tested. This study is one of a series of research on the unique fungus of Pythium in Saudi Arabia.
MATERIALS AND METHODS
Survey points: Collection were made from Dawmat Al-Jandal lake in Al-Jouf governorate, Saudi Arabia (29°5156″N, 39°5120″E), during December of 2012.
Surface water was collected from a depth of 30 cm at a distance of about 100 cm from the bank in 1 L sterilized plastic bottles and hermetically sealed.
Samples of water were collected in clean dry plastic bottles and at the laboratory,
30 mL poured in sterilized glass Petri-dishes and baited with 4 autoclaved wheat
leaf pieces (0.5x1.0 cm). After incubation at 25°C for 5-7 days, the baits
then were removed, washed several times with sterilized distilled water and
blotted dry with sterile tissue paper. Baits were then placed on the colonial
edges of a Petri-dish containing Nystatin Ampicillin Rifampicin Miconazole (NARM)
medium (Gamo et al., 2004) for selective isolating
of Pythium species. Baits were then incubated at 20°C for 3 days
or until colonial appearance.
Morphological identification: NARM selective medium was used for purification
and then identification. Identification using morphological characteristics
was performed with the aid of keys of Middleton (1943),
Waterhouse (1968), Van der Plaats-Niterink
(1981) and Dick (1990).
Temperature-growth relations: Minimum, optimum and maximum temperatures of one each fungus are concluded on cornmeal agar (CMA; 17 gl-1, BD-BBLTM) inoculated with 5 mm dam discs from stock cultures on CMA plates. All plates must be incubated at 25°C for 24 h before starting growth determinations. Cardinal temperatures were evaluated at 5, 10, 15, 20, 25, 30, 35 and 40°C.
DNA extraction, amplification, cloning and sequencing: Adequate mycelia
of the tested pythia were got from the growth in V8 agar medium at 25°C
for 7 days. Mycelia from the edge of Pythium colony grown on the plate
were placed in 200 μL of the Reagent (Prep Man Ultra Sample Preparation;
Applid Biosystems, CN, USA) in a 2.0 mL microcentrifuge tube in order to obtain
extract of total genomic DNA. DNA of each fungus were then whirled for 30 sec
and heated at 100°C for 10 min. DNA samples were then wind for 30 min in
a centrifuge at 15000 g. The remaining supernatants were subjected to PCR. Amplification
of The nuclear rDNA region of the Internal Transcribed Spacer (ITS), including
the 5.8S rDNA, with the universal primers ITS4 (5′TCCTCCGCTTATTGATATGC3′) and
ITS5 (5′GGAAGTAAAAGTCGTAACAAGG3′) was done. Primers of ITS1 (5′TCCGTAGGTGAACCTGCGG3′)
and ITS2 (5′GCTGCGTTCTTCATCGATGC3′) were, sometimes, used according to White
et al. (1990) and Matsumoto et al. (1999).
The amplicons were 700-900 bp long. In some Pythium species, 563 bp of
the cox II gene was amplified with the primer pair FM66 (5′TAGGATTTCAAGATCCTGC3′)
and FM58 (5′CCACAAATTTCACTACATTGA 3′) (Martin, 2000).
Amplification of the target template of sequencing was done using Thermal Cycler
with a profile of pre PCR at 94°C for 5 min, then denaturation at 94°C
for 1 min, another one minute of primer annealing at 55°C for the internal
transcript spacer, after the final cycle, 52°C for gene cox II and elongation
at 72°C, further two min for 40 cycles, with a 7 min extension at 72°C.
Five micro liters of the PCR reaction mixture was loaded in 2% L03 (Takara Bio)
agarose gel, electrophoresed at 100 V, 20-30 min and stained with ethidium bromide
to ensure presence of PCR product. Sequencing templates were purified with GenElute
PCR Clean-up kit following the manufacturers instructions (Sigma Chemical
Co., St Louis, Missouri, USA). Sequencing was performed with BigDye Terminator
v3.1 Cycle Sequencing Reaction kit (Applied Biosystems) using the same primers
in the initial PCR step. After purification of the sequencing reaction mixture
by using ethanol precipitation, it was then run on ABI 3100 DNA Sequencer (Applied
Biosystems). Some PCR products were cloned in the pT7Blue T-vector (Takara Bio)
with the Ligation kit (Takara Bio) according to the manufacturers instruction.
The cloned ITS region was amplified using M13M4 (5′GTTTTCCCAGTCACGAC3′) and
M13Rv (5′CAGGAAACAGCTATGAC3′) primers. The PCR products were purified using
the Gene Elute PCR cleaning kit (Sigma, Ronkonkoma, NY, USA). The BigDyeTM
Terminator Cycle Sequencing Ready Reaction kit *Applied Biosystems) was used
for cycle sequencing with primers M13M4 and M13Rv according to the manufacturers
instructions. The sequencing reaction mixture was purified by ethanol precipitation
and run on an ABI 3100 DNA sequencer (Applied Biosystems). The consensus sequences
were generated based on the forward and reverse primer sequences.
Pathogenicity test: Inocula of tested species were developed to be suitable for the present study. Five gram of grass blade leaf segments and 2 g glucose were moistened by adding distilled water (10 mL) each in 250 mL conical flask. After sterilization, each conical flask containing the above mentioned mixture was supplemented with 3 colonized agar disks (7 mm dam) by Pythium spp. and incubated at 25°C for 10 days. Inoculums concentration of 2.5% was prepared by mixing one gram of colonized grass leaf segments in the conical flask with 50 g of dried clay sandy soil using a sterilized mortar and pestle. The 2.5 g of this mixture were then mixed with 97.5 g of sterilized clay loam soil. Tomato seeds (Solanum lycopersicum L.) were surface sterilized by using sodium hypochlorite 2% for 3 min and then washing well by sterilized H2O followed by 1 min in 70% ethanol and finally three times using sterilized distilled water. Pre germination test was done to select viable seeds for the pathogenicity test. Twenty tomato seeds were planted in each pot to perform the pathogenicity test. The experiments were tested in a growth chamber at 25°C with illumination of 12 h photoperiod (91 μmol m-2 S-1). Damping-off percentage was determined by the difference between tomato seeds emergence of the infested pot and the control one.
Occurrence and distribution of Pythium spp.: All baits produced Fungi. Grass leaf blades baits were very useful for isolating different species of Pythium. Pythium aphanidermatum, P. diclinum, sterile P. dissotocum and Pythium group P were isolated using this bait.
Morphological identification: Fifty-nine isolates of Pythium spp. were obtained from the studied area in Dawmat Al-Jandal lake, Al-Jouf governorate, Saudi Arabia (Table 1). Of these, a total of six species could be identified on the basis of morphological and molecular criteria. The remaining isolates, which lacked sexual structures, have filamentous sporangia and one of them was identified molecularly as P. dissotocum others which lacked sexuality and have proliferated sporangia were identified as Pythium group P.
Pythium aphanidermatum (Edson) Fitzp.(JU00010) (Fig. 1): Mycelial growth of this fungus showed heavy with cottony appearance especially on rich medium such as Potato Dextrose Agar (PDA). Main hyphae were up to 10 μm wide. Zoosporangia consisted of terminal complexes of swollen hyphal branches of varying length and up to 22 μm wide. Zoospores were formed at 15-35°C. Encysted zoospores had a diameter of 12 μm. Oogonia were terminal, globose, smooth and of (20-) 22-25(-26) μm (av. 24 μm) dam. Antheridia were mostly intercalary, sometimes terminal, broadly sac-shaped, 11-15 μm long and 9-15 μm wide, 1(-2) per oogonium and monoclinous or diclinous. Oospores were not filling oogonia (aplerotic) (19-)20-24 μm (av. 22 μm) in dam and their walls are 1-2 μm thick. This fungus was deposited in Department of Biology, College of Science, Al-Jouf University, Saudi Arabia with number JU00010.
Pythium diclinum Tokunaga (JU00020) (Fig. 2):
Mycelial growth of this fungus showed submerged growth on poor medium such as
CMA and with little aerial mycelia on the rich medium. Main hyphae was up to
7 μm wide. Sporangia were filamentous or non-inflated, sometimes branched.
Zoospores formed at 20-25°C; vesicles are varying from very small containing
2 zoospores to big containing many zoospores. Encysted zoospores were about
7 μm dam. Oogonia were globose, to subglobose and sometimes ovoid, smooth,
mostly terminal or subterminal, occasionally intercalary, 18-24 μm (av.
22 μm) dam. Antheridia were typically diclinous, 1-2 per oogonium, about
12x15 μm. Antheridial stalks not branched. Oospores were single, aplerotic;
16-20 μm (av. 19) dam, wall up to 3 μm thick.
|| Linear growth of Pythium spp. isolated from water
of Dawmat Al-Jandal lake at various temperatures
|*Growth is the mean of 5 replicates and no significant difference
in diameter within replicates of each measurement was observed
||Morphology of Pythium aphanidermatum (JU00010), (a)
A toruloid zoosporangium with a vesicle (arrow), (b) A vesicle contains
differentiated zoospores, (c) Young oogonium and young intercalary diclinum
antheridium, (d) Intercalary antheridia (arrows) and (e, f) Aplerotic oospores
(arrows). Bar on photo (f) is equal 20 μm and is applicable to the
This fungus was deposited in Department of Biology, College of Science, Al-Jouf
University, Saudi Arabia with number JU00020.
Pythium dissotocum Drechsler (JU00030) (Fig. 3):
Colonies on corn meal agar were submerged, without a special pattern and with
little aerial mycelia. Main hyphae was up to 6 μm wide.
||Morphology of Pythium diclinum (JU00020), (a) A filamentous
zoosporangium with a vesicle (arrow), (b) Intercalary oogonium and diclinous
antheridium, (c, d) Terminal oogonia and diclinum antheridia (arrows) and
(e) An aplerotic oospore (arrows). Bar on photo (4) is equal 20 μm
and is applicable to photos 1, 2, 3 whereas bar on photo 5 is equal 20 μm
and it is for its photo only
Sporangia were filamentous forming very little inflated dendroid structures.
Zoospores formed at 20°C. Encysted zoospores were up to 8 μm dam. This
isolate failed to produce sexual structures and it was deposited in Department
of Biology, College of Science, Al-Jouf University, Saudi Arabia with number
Pythium group P Plates Niterink (JU00040) (Fig.
4): Colonies on corn meal agar were submerged, with radiate pattern
and with some aerial mycelia. Main hyphae was up to 8 μm wide.
||Morphology of a sterile isolate of Pythium dissotocum
(JU00030), (a) Terminal hyphae, (b) A young vesicle (arrow), (c) Young and
old vesicles contains differentiated zoospores (arrow), (d) Vesicles and
evacuation tube after zoospores liberated from the vesicle (arrow) and (e)
Liberation of zoospores from the vesicle. Bar on photo (a) is equal 20 μm
and is applicable to the rest photos
Sporangia were proliferating internally by forming a new sporangium inside the old one, often 20-30 μm dam, 25 μm on average, mostly forming short evacuation tubes, sometimes germinated by 1 or 2 germ tubes. Zoospores produced at 20-25°C. Encysted zoospores were up to 12 μm dam. No sexual reproduction observed. This fungus was deposited in Department of Biology, College of Science, Al-Jouf University, Saudi Arabia with number JU00040.
Temperature growth relations: P. aphanidermatum showed temperature
maximum for growth higher than that of the other Pythia studied.
||Morphology of Pythium "group P" (JU00040), (a, b) Hyphae,
(c) Proliferated zoosporangium, (d) Young zoosporangium, (e-h): Stages of
vesicle formation and zoospores liberation and (i-l): Internal proliferation
of zoosporangia. Bar on photo (h) is equal 20 μm and is applicable
to rest photos
The optimum temperatures for all the other Pythium spp. investigated
lied between (25 and 30°C) and the minimum temperature supporting mycelial
growth of these spp. was around 5°C except P. aphanidermatum which
started to grow at 10°C (Table 1).
Molecular identification: The sequence of JU00010 was closely related
with that of P. aphanidermatum (AY598622.1) with 100% similarity. The
sequence (with the aid of TA-cloning) of JU00020 was closely related with that
of P. diclinum (EF153676) with 100% similarity.
||Nuclear rDNA region of the internal transcribed spacer (ITS),
including the 5.8S rDNA, of P. aphanidermatum, P. diclinum
and P. dissotocum
The sequence of JU00030 was closely related with that of P. dissotocum
(AY598631) with 99% similarity. Sequencing of Pythium group P
was though to be Pythium helicoides, therefore, sequencing of cox II
gene was done. It was confirmed that this isolate of group P was not P. helicoides.
Pathogenicity test: Pre-emergence damping-off test was performed in
pots using 2.5% inoculum concentration of the tested Pythia added to soil and
incubated for 2-3 weeks before cultivated tomato seeds. P. aphanidermatum
was highly pathogenic to tomato seeds causing 100% damping-off. P. diclinum
and P. dissotocum were moderately pathogenic producing 65% damping-off
while Pythium "group P" was non-pathogenic to tomato seeds (Fig.
This is the first study dealt with aquatic pythia in Dawmat Al-Jandal lake,
Dawmat Al-Jandal city, Al-Jouf governorate, Saudi Arabia. The species obtained
in this study were; P. aphanidermatum, P. diclinum, sterile P.
dissotocum and Pythium group P. P. diclinum and
the sterile P. dissotocum have filamentous sporangia, while P. aphanidermatum
has inflated lobulate sporangia and Pythium group P forms
proliferated sporangia. P. aphanidermatum was isolated several times
from aquatic habitats such as irrigation water (Abdelzaher,
1999; Moustafa et al., 2010). P. diclinum
was originally isolated from a paddy field in Japan (Van
der Plaats-Niterink, 1981). P. dissotocum has also been isolated
from water in the formal USSR and the Netherlands (Van der
Plaats-Niterink, 1981). Pythium group P was isolated
many times from aquatic habitats (Van der Plaats-Niterink,
1981; Abdelzaher, 1999; Abdelzaher
et al., 1995). Therefore, result of isolation of such species from
water here is in harmony with the previous findings.
Results appear that grass leaves segments was very useful as baits for isolation
of Pythium spp. It is worth to mention that, the results (Fig.
7) of the rDNA-ITS and cox II sequence analysis of the Pythium isolates
agreed with the morphological features of each isolate and were of great helpful
in confirming identification and overcome hesitating and doubtfulness.
||Pre-emergence damping-off of tomato seedlings grown in clay
sand soil infested with tested Pythium spp. Bars indicate standard
errors of 100 measurements
||Pre-emergence damping-off of tomato seedlings grown in clay
sandy soil in plastic pots infested with Pythium aphanidermatum (2),
P. diclinum (3), P. dissotocum (4). Control pot (1)
does not contain any Pythium inocula
Results here appeared that three out of four pythia isolated from water of
Dawmat Al-Jandal lake proved ferocity against tomato seedlings (Fig.
5, 6). As shown in results, P. aphanidermatum was
highly pathogenic causing 100% damping-off, P. diclinum and P. dissotocum
caused 65% damping-off to tomato germinating seeds. Therefore, the presence
of such fungi that cause some diseases of crop plants is a source of danger,
especially if the lake water is used for irrigation. Care must be taken of pollution
of the agricultural fields by using water of the lake. In previous studies,
the pathogenicity of the isolated Pythium spp. in this study was proven.
P. aphanidermatum can cause root rot, damping-off, stalk and rhizome
rot, soft rot, fruit rot or cottony blight of many economic plants (Van
der Plaats-Niterink, 1981). P. diclinum caused severe damping-off
of wheat in Egypt (Abdelzaher, 2004). P. dissotocum
was pathogenic to sugar cane, pecan and peach trees (Van
der Plaats-Niterink, 1981). Therefore, above mentioned previous studies
support results of the present investigation.
It is worth mentioning that Dawmat Al-Jandal Lake is an artificial lake created by nearly 30 years in a desert area of continental climate. Explanation of occurrence of Pythium spp. which possess zoospores floating in the lake is of important from the environmental perspective. Where this fungus is not air borne, it could be explained that entry of it to the lake by either droppings of migratory birds or wash of agricultural soil adjacent to the lake.
Experimental scientific explanation for this phenomenon has not carried out in this study and needs further investigation.
This study was partially supported by King Faisal University (small grant No. 131001). We are grateful for the partial support of this project by Eid and Otto Internationale, S.A. P.O. Box 793 Belize City, Belize, C.A.
1: Abdelzaher, H.M.A., 1999. The genus Pythium in Egypt. Proceedings of the 2nd International Conference on Fungi: Hopes and Challenges, September 29-October 1, 1999, Cairo, pp: 1-31.
2: Abdelzaher, H.M.A., 2004. Occurrence of damping-off of wheat caused by Pythium diclinum tokunaga in El-Minia, Egypt and its possible control by Gliocladium roseum and Trichoderma harzianum. Archiv. Phytopathol. Plant Prot., 37: 147-159.
3: Abdelzaher, H.M.A., T. Ichitani, M.A. Elnaghy, S.K.M. Hassan and E.M. Fadl-Alla, 1995. Materials for Pythium flora of Japan. X. Occurrence, identification and seasonality of Pythium spp. in three pond waters and mud soils in Osaka. Mycoscience, 36: 71-85.
Direct Link |
4: Al-Sheikh, H. and H.M.A. Abdelzaher, 2010. Differentiation between two isolates of Pythium ultimum var. ultimum isolated from diseased plants in two different continents. J. Biol. Sci., 10: 306-315.
CrossRef | Direct Link |
5: Al-Sheikh, H. and H.M.A. Abdelzaher, 2012. Materials for Pythium flora of Saudi Arabia (I) Occurrence, pathogenicity and physiology of reproduction of Pythium aphanidermatum (Edson) Fitzp. isolated from north and east regions of Saudi Arabia. Res. J. Microbiol., 7: 82-100.
CrossRef | Direct Link |
6: Al-Sheikh, H. and H.M.A. Abdelzaher, 2010. Isolation of Aspergillus sulphureus, Penicillium islandicum and Paecilomyces variotii from agricultural soil and their biological activity against Pythium spinosum, the damping-off organism of soybean. J. Biol. Sci., 10: 178-189.
CrossRef | Direct Link |
7: Barton, R., 1958. Occurrence and establishment of Pythium in soils. Trans. Br. Mycol. Soc., 41: 207-222.
8: Berlese, A.N. and J.B. De Toni, 1888. Phycomyceteae. In: Saccardo's Sylloge Fungorum, Saccardo, P.A. (Ed.). R. Friedlander and Sohn, Berlin, Germany, pp: 181-322.
9: Butler, E.G., 1907. An Account of the Genus Pythium and Some Chytridiaceae. Mem. Dep. Agri. Indian Bot. Vol. 1, Agricultural Research Institute, Pusa, Pages: 160.
10: Chen, W., R.W. Schneider and J.W. Hoy, 1992. Taxonomic and phylogenetic analyses of ten Pythium species using isozyme polymorphism. Phytopathology, 82: 1234-1244.
11: Dick, M.W., 1990. Keys to Pythium. College of Estate Management, Reading, Pages: 64.
12: El-Nagdy, M.A. and L.A. Nasser, 2000. Occurrence of zoosporic and terrestrial fungi in accumulated rainfall water in the Riyadh region (Saudi Arabia). Fungal Diversity, 5: 175-183.
Direct Link |
13: Elnaghy, M.A., E.M. Fadl-Allah, H.M.A. Abdelzaher and S.A. Moharam, 2009. Pythium longisporangium Paul, A new record from Egyptian soil. Assiut Univ. J. Bot., 38: 81-91.
14: Gamo, Y., M. Tojo and S.T. Ohki, 2004. Evaluation of selective media using fluazinam or miconazole nitrate for quantitative isolation of Pythium spp. from soil. Japn. J. Phytopathol., 70: 215-215.
15: Helman, R.G. and J. Oliver, 1999. Pythiosis of the digestive tract in dogs from Oklahoma. J. Am. Anim. Hosp. Assoc., 35: 111-114.
16: Ichitani, T. and H. Goto, 1982. Distribution of Pythium zingiberum causing rhizome rot in ginger growing and its surrounding uncultivated soils. Ann. Phytopath. Soc. Japan, 48: 647-676.
Direct Link |
17: Kageyama, K., A. Nakashima, Y. Kajihara, H. Suga and E.B. Nelson, 2005. Phylogenetic and morphological analyses of Pythium graminicola and related species. J. Gen. Plant Pathol., 71: 174-182.
CrossRef | Direct Link |
18: Kitancharoen, N. and K. Hatai, 1998. Some biochemical characteristics of fungi isolated from salmonid eggs. Mycoscience, 39: 249-255.
19: Kucharek, T. and D. Mitchell, 2000. Diseases of agronomic and vegetable crops caused by Pythium. Plant Pathology Fact Sheet, Florida Cooperative Extension Service, Institute of Food and Agricutural Service, University of Florida, USA.
20: Levesque, C.A. and W.A. de Cock, 2004. Molecular phylogeny and taxonomy of the genus Pythium. Mycol. Res., 108: 1363-1383.
PubMed | Direct Link |
21: Martin, F.N., 2000. Phylogenetic relationships among some Pythium species inferred from sequence analysis of the mitochondrially encoded cytochrome oxidase II gene. Mycologia, 92: 711-727.
Direct Link |
22: Masih, I. and B. Paul, 2003. Pythium regulare sp. Nov., isolated from the Canary Islands, its taxonomy, its region of rDNA and comparison with related species. Curr. Microbiol., 47: 309-313.
23: Matsumoto, C., K. Kageyama, H. Suga and M. Hyakumachi, 1999. Phylogenetic relationships of Pythium species based on ITS and 5.8S sequences of the ribosomal DNA. Mycoscience, 40: 321-331.
CrossRef | Direct Link |
24: Mendoza, L., L. Kaufman and P. Standard, 1987. Antigenic relationship between the animal and human pathogen Pythium insidiosum and nonpathogenic Pythium species. J. Clin. Microbiol., 25: 2159-2162.
25: Middleton, J.T., 1943. The taxonomy, host range and geographic distribution of the genus Pythium. Memories Torrey Bot. Club, 20: 1-171.
Direct Link |
26: Moharam, S.A., 2010. Occurrence, pathogenicity and physiology of Pythium species associated to some wild plants in Egypt. Ph.D. Thesis, Faculty of Sciene, Minia University, Minia, Egypt.
27: Molan, Y.Y., 2009. Detection of presumptive mycoparasites in soil placed on host-colonized agar plates in Riyadh Region, Saudi Arabia. Asian J. Plant Pathol., 3: 22-26.
CrossRef | Direct Link |
28: Moustafa, S.M.N., H.M.A. Abdelzaher and S.K.M. Hassan, 2010. Occurrence, isolation and identification of aquatic Pythium species from water streams for irrigation in Egypt. Assiut. Univ. J. Bot., 39: 13-49.
29: Van der Plaats-Niterink, A.J., 1975. Species of Pythium in the Netherlands. Netherlands J. Plant Pathol., 81: 22-37.
30: Van der Plaats-Niterink, A.J., 1981. Monograph of the genus Pythium. Stud. Mycol., 21: 1-244.
31: Pringsheim, N., 1858. Contribution to morphology and systematics of algae. 2, The Saprolegniales. Jb Wiss. Bot., 1: 284-306.
32: Senda, M. and K. Kageyama, 2006. Pythium species in cool-temperate forest soil. Proceedings of the 8th International Congress on Mycological, August 20-25 2006, Cairns, Australia -.
33: Shenep, J.L., B.K. English, L. Kaufman, T.A. Pearson and J.W. Thompson et al., 1998. Successful medical therapy for deeply invasive facial infection due to Pythium insidiosum in a child. Clin. Infect. Dis., 27: 1388-1393.
34: Sparrow, Jr. F.K., 1968. The Ecology of Freshwater Fungi. In: The Fungi, Ainsworth, G.C. and A.S. Sussman (Eds.). Vol. 3. Academic Press, London, UK., pp: 41-93.
35: Sunboul, Y.H., 2001. Basal stem rot of vegetables in controlled environment greenhouses I western Saudi Arabia: (A) Studies on the causal agent Pythium aphanidermatum. J. King Abdulaziz Univ. Meteorol. Environ. Arid Land Agric. Sci., 12: 1-7.
36: Sunboul, Y.H., 2006. Solarizaton of commercial peat in transparent polyethylene bags and its effect on survival of some plant pathogenic fungi. JKAU Sci., 18: 1-11.
Direct Link |
37: Tsukiboshi, T., Y. Chikuo, Y. Ito, Y. Matsushita and K. Kageyama, 2007. Root and stem rot of chrysanthemum caused by five Pythium species in Japan. J. Gen. Plant Pathol., 73: 293-296.
38: Waterhouse, G.M., 1968. The genus Pythium pringsheim. Mycological Papers No. 110, pp: 1-50.
39: White, T.J., T.D. Bruns, S.B. Lee and J.W. Taylor, 1990. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. In: PCR Protocols: A Guide to Methods and Applications, Innis, M.A., D.H. Gelfand, J.J. Sninsky and T.J. White (Eds.). Academic Press, San Diego, CA., USA., ISBN-13: 9780123721808, pp: 315-322.
40: Sanchez, J. and E. Gallego, 2001. Pythium spp. present in irrigation water in the Poniente region of Almeria (south-eastern Spain). Mycopathologia, 150: 29-38.
Direct Link |