Myco-Suppression Analysis of Soybean (Glycine max) Damping-Off Caused by Pythium aphanidermatum

The role of Pythium oligandrum as a biocontrol agent against Pythium aphanidermatum was investigated to avoid the harmful impacts of fungicides. Three isolates of P. oligandrum (MS15, MS19, and MS31) were assessed facing the plant pathogenic P. aphanidermatum the causal agent of Glycine max damping-off. The tested Pythium species were recognized according to their cultural and microscopic characterizations. The identification was confirmed through sequencing of rDNA-ITS regions including the 5.8 S rDNA. The biocontrol agent, P. oligandrum, isolates decreased the mycelial growth of the pathogenic P. aphanidermatum with 71.3%, 67.1%, and 68.7% through mycoparasitism on CMA plates. While the half-strength millipore sterilized filtrates of P. oligandrum isolates degrade the pathogenic mycelial linear growth by 34.1%, 32.5%, and 31.7%, and reduce the mycelial dry weight of the pathogenic P. aphanidermatum by 40.1%, 37.4%, and 36.8%, respectively. Scanning electron microscopy (SEM) of the most effective antagonistic P. oligandrum isolate (MS15) interaction showed coiling, haustorial parts of P. oligandrum to P. aphanidermatum hyphae. Furthermore, P. oligandrum isolates were proven to enhance the germination of Glycine max seedling to 93.3% in damping-off infection using agar pots and promote germination of up to 80% during soil pot assay. On the other hand, P. oligandrum isolates increase the shoot, root lengths, and the number of lateral roots.


Introduction
Soybean (Glycine max L.) is one of the most common widely cultivated food species in the world [1]. Root rot diseases of G. max are widespread in the world and are usually recognized as the main constraint to decrease both yield and quality [2,3]. Pythium as a causal agent for Glycine max root rots has been mentioned as being the main reason leading to soybean yield losses in numerous countries [4,5]. Pythium aphanidermatum has been reported as the species most frequently causing root rot diseases for the greatest variety of crops [4]. The use of chemical fungicides controlling such diseases could lead to degenerating human health, environmental pollution, and the development of pathogen resistance to fungicides. Biological methods are needed for plant protection which are less dependent on chemicals and are more environmentally favorable [5,6].
Pythium is a complex genus, spread worldwide including over 200 species with a wide host range and maintaining in a variety of soil and water environments [7,8]. Sev-eral researchers have explained that Pythium oligandrum efficiency in controlling plant disease, some of them reported that the antagonistic of P. oligandrum enhance plant immune systems through secretion of an elicitin-like protein that induces plant resistances against different phytopathogenic fungi, avoiding their infection. The biocontrol activity of P. oligandrum attributed to directly attack a group of soil-borne fungal pathogens through; (1) mycoparasitism, (2) colonizing rhizosphere of crop plants and competing for space and nutrients, (3) production of an auxin like tryptophan and indolacetaldhyde, which convert to tryptamine (TNH2) that promote plant growth, and (4) secretion of oligandrin and elicitin-like protein activate plant immune-system that increase crop protection against phytopathogenic microbes [4,[9][10][11][12]. A similar mode of action observed by other biocontrol agents like Trichoderma harzianum induces systematic resistance in G. max plants and enhances plant growth through elicitation of bioactive metabolites [13][14][15].
Horner et al. [16] explained the mycoparasitic ability of P. oligandrum through coiling P. oligandrum hyphae around phytophthora hyphae and by secretion of glucanases, cellulose, chitinase and, protease enzymes which degraded the pathogen cell wall. P. oligandrum used in biocontrol of black scurf in potato as the hyphal of biocontrol agent had colonized the sclerotia and established close contact by coiling around the R. solani [17]. Phytium oligandrum has taken serious attention as a biocontrol agent against damping-off diseases, especially those caused by P. aphanidermatum [8,16,18,19].
No attempts have been made to evaluate P. oligandrum as a biocontrol agent against P. aphanidermatum, the causal factor of G. max seedlings damping-off in Egypt and Saudi Arabia. The present work was, therefore, aimed at evaluating the efficiency of using P. oligandrum in the biocontrol of soybean seedling damping-off caused by P. aphanidermatum.

Results
Isolation and identification of biocontrol Pythium isolates showed the presence of three strains of P. oligandrum (MS15, MS19, and MS31) obtained from the rhizosphere soil of Raphanus sativus, Eruca sativa, and Allium cepa, respectively, during the survey of Pythium species in El-Minia Governorate, Egypt. These three isolates were identified morphologically as P. oligandrum Drechsler (Figure 1a). In addition, the sequencing of rDNA-ITS for these isolates was closely related to P. oligandrum with 99.9% similarity to Genbank accession number (AY986954.1).
Isolation of pathogenic strains from the infected roots and seedlings of G. max indicated that P. aphanidermatum was the only fungus present in infected tissues. The morphological identification of pathogenic P. aphanidermatum was illustrated in Figure 1b and the genetic criteria of this isolate were closely related to P. aphanidermatum with 99.9% similarity to Genbank accession number (AB274404).
Antagonistic activity of P. oligandrum isolates (MS15, MS19, and MS31) against the pathogenic P. aphanidermatum using dual culture technique was reported in Table 1. The bioactivity assay showed that P. oligandrum isolates decreased the mycelial growth of P. aphanidermatum and the MS15 was the highest one after 3 days of incubation and reduced the growth by 67.2%. Moreover, the reduction in the growth of pathogenic P. aphanidermatum was increased by increasing the incubation time, where the inhibition reached 71.3% for MS15 after 6 days of incubation, while the results of the isolates (MS19 and MS31) were less effective in all treatments (Table 1).   (3) replicates ± the standard error of the three isolates; values followed by an asterisk (*) are significantly compared with control at p ≤ 0.05. The effect of half-strength culture filtrates of bioagent P. oligandrum isolates on mycelial growth of pathogenic P. aphanidermatum using solid media revealed that the millipore sterilized half-strength filtrates of P. oligandrum isolates were more effective in reducing the mycelial growth of P. aphanidermatum than that of autoclaved cultured filtrate recording inhibition of mycelial growth of P. aphanidermatum by 34.1%, 32.5%, and 31.7%, respectively ( Table 2).  Control of P. aphanidermatum 90.00 * ± 0.02 -Data are means of (3) replicates ± the standard error of the three isolates; values followed by an asterisk (*) are significantly compared with control at p ≤ 0.05.
The effect of culture filtrates of P. oligandrum isolates on the mycelial dry weight of P. aphanidermatum using liquid medium reported in Table 3. The millipore sterilized half-strength filtrates of P. oligandrum isolates reduce the mycelial dry weight of P. aphanidermatum more effectively than autoclaved filtrate recording 40.1%, 37.4%, and 36.8%, respectively, and the height reduction was reported by P. oligandrum (MS 15 ). Control P. aphanidermatum 774.00 *± 0.14 -Data are means of (3) replicates ± the standard error of the three isolates; values followed by an asterisk (*) are significantly compared with control at p ≤ 0.05.
Interaction between the highly effective isolate of P. oligandrum (MS15) and P. aphanidermatum using a light microscope and scanning electron microscope (SEM) were provided in Figures 2 and 3. The characterization of coiling, haustorial branches, infection pegs, and penetration of P. oligandrum to P. aphanidermatum hyphae was shown in the contact area between the two Pythia. Pythium oligandrum attacked P. aphanidermatum by entangling its hyphae with either thin haustorial arms or infection pegs reaching finally to its total destruction. These characters of mycoparasitism were showed clearly at the early stages of the contact region between the two colonies of P. oligandrum and P. aphanidermatum on the surface of Corn Meal Agar CMA plates.  1) and (2) showing infection pegs of P. oligandrum; (3) and (4) showing hyphae of P. oligandrum grow inside hyphae of P. aphanidermatum; (5) and (6) arrows pointed to P. oligandrum twisted on hyphae of P. aphanidermatum.
Pathogenicity of P. aphanidermatum and its possible control measure by P. oligandrum isolates (MS15, MS19, and MS31) in both agar bottles and soil pots assay were illustrated in Table 4 and Figure 4. It was found that the pathogenic isolate of P. aphanidermatum was highly pathogenic causing 100% and 86.7% seedling damping-off when the pathogen cultured with seedlings alone in both 2% WA and soil pots respectively. In contrast, the three isolates of the biocontrol P. oligandrum isolates were detected to be non-pathogenic to G. max seedlings and also improve the seedling growth more than the control treatment. On the other hand, the application of the biocontrol P. oligandrum isolates inhibited the seedling damping-off by 93.3%, 87.2%, and 86.7% in 2% WA assay and 80%, 75.1%, and 76.7%, respectively in soil pots compared with the control. Moreover, P. oligandrum isolate (MS15) was the most effective isolate in both treatments.
Pathogenicity of P. aphanidermatum and its possible control measure by P. oligandrum isolates (MS15, MS 19,and MS 31) in both agar bottles and soil pots assay were illustrated in Table 4 and Figure 4. It was found that the pathogenic isolate of P. aphanidermatum was highly pathogenic causing 100% and 86.7% seedling damping-off when the pathogen  1) and (2) showing infection pegs of P. oligandrum; (3) and (4) showing torsion of P. oligandrum hyphae P. aphanidermatum and the arrow pointed to hyphal collapse and turgor loss of P. aphanidermatum hyphae. Table 4. Effect of P. oligandrum isolates on G. max pre-emergence damping-off disease grown in 2% water agar and in soil pots. The effect of P. oligandrum on the growth parameters of the G. max seedling was recorded in Table 5 and illustrated in Figures 5 and 6. The results showed that the growth parameters of the treated plants with P. oligandrum isolates were enhanced by increasing the shoot and root lengths and increasing the number of lateral roots in all treatments of bioagent isolates. The phytopathogenic P. aphanidermatum affected seedling growth of G. max seeds and reduced their shoot and root length by 43.3% and 34.9%, respectively, as shown in Figure 6 when compared with the healthy plants (no Pythium) in Table 5   1, seeds cultivated alone as a control; 2, seeds cultivated with P. oligandrum alone; 3, seeds cultivated with P. aphanidermatum alone; 4, seeds cultivated with both of P. oligandrum and P. aphanidermatum. Data are means of (3) replicates ± the standard error of the three isolates; values followed by an asterisk (*) are significantly compared with control at p ≤ 0.05.

Discussion
Root damping-off diseases are widely disturbed all over the world and are recognized as a serious problem to soybean cultivators, as they significantly decrease the bean yield. Several control measures of the disease are available, including fungicidal control,

Discussion
Root damping-off diseases are widely disturbed all over the world and are recognized as a serious problem to soybean cultivators, as they significantly decrease the bean yield. Several control measures of the disease are available, including fungicidal control, Figure 6. Growth of G. max seedling parameters: (1), uninfected seedlings (negative control); (2), seedlings cultivated with P. oligandrum (positive control); (3), seedling infested with P. aphanidermatum alone and (4), seedling infested with both P. oligandrum and P. aphanidermatum.

Discussion
Root damping-off diseases are widely disturbed all over the world and are recognized as a serious problem to soybean cultivators, as they significantly decrease the bean yield. Several control measures of the disease are available, including fungicidal control, enhancement of genetic resistance, which may lead to deteriorating human health [5,20]. In Egypt, there is no active control method to reduce damping-off of many crops and vegetable plants caused by P. aphanidermatum although, that pathogenic is widely distributed throughout the country [17,18,21,22]. However, fungicides are regularly used by farmers to manage such diseases without obvious results [6,23]. Pythium aphanidermatum was isolated in this study as the causal agent of G. max damping-off and the pathogenicity test concerned with the pathogenic isolate of P. aphanidermatum was highly virulent to G. max seedlings causing 100% damping off. In contrast, the three isolates of P. oligandrum (MS15, MS19, and MS31) were found to be non-pathogenic and promote seed germination [24,25]. This result was in agreement with the result of Nzungize et al. [20,26].
Biological control of damping-off diseases is a complicated process as a result of the pathogens occurs surroundings the rhizosphere interface. The rhizosphere is indicated by intense microbial action including, a high population of microorganisms, salt concentrations, and osmotic and water potential [27]. The efficient microorganisms for biological control of phytopathogenic fungi include some Pythium spp. as P. oligandrum. The biocontrol agent can preserve the plant from fungal infection through competition with the pathogen for nutrients, the production of antifungal metabolites, parasitism (lysis of the pathogen), or through enhancement of the plant resistance mechanisms [11,27]. The growth of P. aphanidermatum was successfully inhibited by P. oligandrum isolates with growth reductions reached 71.3% with P. oligandrum MS15. The millipore sterilized half-strength filtrate of the bioactive P. oligandrum MS15 showed more activity (34.1%) compared with autoclaved filtrates in the linear growth reduction of pathogenic P. aphanidermatum, which reach 29.4%. These results were confirmed through the reduction of the mycelial dry weight of pathogenic P. aphanidermatum with half-strength millipore sterilized filtrates of the bioactive P. oligandrum MS15 to 40.11% compared with autoclaved filtrate (35.7%). That explains the low activity of autoclaved P. oligandrum filtrates, which may affect and destroy the active metabolites [28][29][30][31].
P. oligandrum isolates (MS15, MS19, MS31) parasitized hyphae of P. aphanidermatum by entangling the hyphae of P. aphanidermatum with thin haustorial branches or infection pegs, eventually leading to destruction, revealing the mode of action of antagonism between P. oligandrum and P. aphanidermatum [20,29] as appears in our results of screening for hyphal interaction under light and scanning microscopy. There were at least three known modes of P. oligandrum action on plant pathogens: mycoparasitism mediated by intimate hyphae interactions, antibiosis and enhancement of plant resistance due to protein metabolite (oligandrin) production [8]. The coiling and penetration of P. oligandrum hyphae to P. aphanidermatum hyphae observed in this study had frequently been reported [17,20]. Additionally, the isolate MS15 of P. oligandrum was the most effective antagonistic one and reduced the disease severity of P. aphanidermatum infected G. max to 93.3% in agar bottles and 80% in soil pots assay. These results confirm the ability of P. oligandrum to significantly reduce the infection by P. aphanidermatum [14,21]. Moreover, the biocontrol agent P. oligandrum was proved to enhance the growth of G. max seedling. These growth promotions of P. oligandrum may be attributed to the elicitation of bioactive fungal metabolites [14,15]. It can be concluded that P. oligandrum isolated in this study worked as mycoparasite and antifungal metabolites producer, which suppresses the plant-parasitic P. aphanidermatum.
The present study confirmed the efficiency and importance of P. oligandrum as a biological control against root diseases avoiding biohazards of fungicides usages.

Materials and Methods
The method of zoospores baiting technique (ZBT) was used for isolation of the biocontrol agent P. oligandrum during the survey of Pythium species associated with the rhizosphere soil of Raphanus sativus, Eruca sativa, and Allium cepa in El-Minia governorate, Egypt during the spring of 2016. The isolation was done using Nystatin Ampicillin Rifampcin Miconazole (NARM) selective medium which was described by Senda et al. [32].
Rotten roots and basal parts of diseased seedling of G. max were collected in March of 2016 from a field in Kidwan, El-Minia governorate, Egypt ( Figure 1) and used for isolation of pathogenic P. aphanidermatum. The deceased tissues were cleaned with 50% ethanol (v/v) for 30 s accompanied by washing with sterile distilled water, then dried with sterile filter paper and transported to NARM medium [32]. All seeded plats were incubated at 20 • C and examined daily. Obtained colonies were then purified and subjected to identification.

Identification of Pythium Species
NARM selective medium was found to repress the bacterial growth in Pythium cultures whilst not affecting Pythium itself. The required reproductive fungal structure including the shape, size, and position of sporangia, antheridia, oogonia, oospores, and zoospores formation was ascertained in grass blade culture [14,16]. The morphological identification of isolated Pythium species was carried out using the keys of Dick and Plaats-Niterink [7,33]. In addition, Pythium species identification was confirmed through the DNA extraction and amplification using polymerase chain reaction (PCR) technique according to [32,34], then the sequencing of rDNA-ITS regions including the 5.8 SrDNA, were analyzed according to the method developed by [21,35] using the basic local alignment search tool (BLAST).

4.2.
Antagonistic Activity of P. oligandrum Isolates against the Pathogenic P. aphanidermatum Using Dual Culture Technique Three isolates of P. oligandrum (MS15, MS19, and MS31) were identified as biocontrol agents. Sterilized PDA plates were inoculated by 5 mm discs cutting from the margins of each of the pathogenic P. aphanidermatum isolate and the three isolates of the biocontrol P. oligandrum at a distance of 4 cm from each other. Three replicates were managed per each treatment and the plates were incubated at 25 • C for 3-6 days. When the growth of control P. aphanidermatum mycelia covers all plates surface, the data were recorded. Percentages of reduction in mycelial growth of the pathogenic P. aphanidermatum were calculated and recorded in relation to the control.

Effect of Half-Strength Culture Filtrate of P. oligandrum on the Linear Growth of Pathogenic P. aphanidermatum
Sterilized filtrate of 5-day old cultures of P. oligandrum isolates grown on potato dextrose broth (PDB) was tested against the growth of pathogenic P. aphanidermatum. The tested filtrates were sterilized by either autoclave or using a 0.22 µm millipore filter. Equal volumes of both sterilized filtrates of P. oligandrum isolates and sterilized double strength PDA medium were mixed (1:1, v/v) before pouring in sterilized Petri dishes. Discs of the 3-day old culture of the pathogenic P. aphanidermatum were put in the middle of these plates and incubated at 25 • C. Data were recorded when the mycelial growth of the control P. aphanidermatum covers all surfaces of plates and of comparing with control, the reduction mycelial growth percentages were recorded [11]. An equal volume of sterilized filtrates (ether by Autoclave or 0.22 Mm millipore) at a rate of 50 mL was mixed with 50 mL sterilized double strength of potato dextrose broth (PDB) in 250 mL Erlenmeyer flasks. Three control flasks containing 50 mL double-strength PDB medium mixed with 50 mL of sterile distilled water. Mycelial discs of 5 days old culture of the phytopathogenic P. aphanidermatum were inoculated and the flasks were incubated at 25 • C for 8 days. Mycelium from each flask was filtered using Whatman's No. 2 filter paper, washed and dried in the oven at 80 • C for 72 h, and finally weighed. All the cultures were maintained in three replicates. The percent inhibition in mycelial dry weight was recorded to indicate the effect of the antagonist's filtrate on the pathogenic fungal growth.

Mycoparasitism between P. oligandrum and P. aphanidermatum Using SEM
Mycelial strips were obtained from the contact area between the two colonies of P. oligandrum and P. aphanidermatum grown on CMA for three days at 25 • C and then fixed in 2.5% gluteraldehyde-sodium phosphate buffer (0.13 M, Ph 7.2) overnight. The specimens were washed three times with buffer and then postfixed in osmium tetraoxide for 2 h, washed again with buffer, and dehydrated using ascending series of ethanol level (30-100%) for 15 min at each step. The specimens were dried at the critical point and coated with carbon to be examined using the scanning electron microscope (JEOL JSM-6380 LA) operating at 20 kV [30]. 4.6. Pathogenicity of P. aphanidermatum and Its Possible Control Measure 4.6.1. In Agar Bottles Assay Pathogenicity and possible control of P. aphanidermatum using P. oligandrum were investigated according to the method described by Abdelzaher and Kageyam [23]. G. max seeds were surface sterilized and germinated to form radicals and plumules for 3 days at 25 • C, then three viable seeds were grown onto 2% WA in a 250 mL Erlenmeyer flask. Three seedling flasks were inoculated by three 5-mm discs cutting from a 5-days old culture of pathogenic P. aphanidermatum to evaluate their pathogenicity as a positive control. Other seedling flasks were inoculated by three 5-mm discs of isolates of biocontrol agent P. oligandrum isolates act as a negative control. While the other seedling flasks were inoculated by three discs of each of P. oligandrum isolates and P. aphanidermatum to evaluate the bioactivity of P. oligandrum isolates (MS15, MS19, and MS31). The inoculated flasks were incubated in a growth cabinet at 25 • C with a 12 h photoperiod (91 µmol m-2S-1). Dampingoff was restricted as the difference between seedlings emergence in non-inoculated controls and inoculated one [21]. The treated seedlings flasks were observed to detect damping-off diseases by recording seedling emergence.

In Soil Pots Assay
To prepare P. aphanidermatum inoculum, five grams of grass blade leaf fragments (0.5 cm × 1 cm) and 2 gm glucose were saturated with 10 mL distilled water in a 250 mL Erlenmeyer flask. After autoclaving, each flask was inoculated with three discs of P. aphanidermatum, then incubated at 25 • C for 7 days. Inoculum concentration of 2.5%, was taken by mixing 1 gm of colonized grass leaf fragments in the Erlenmeyer flask with 50 gm of oven-dried (70-80 • C for 2 days) loam sandy soil (LSS) using a sterilized mortar. Two and a half gm of this mixture was added to 97.5 gm of sterilized soil (LSS). Inoculated soil was placed in plastic pots and exterior sterilized seeds of G. max were sowed after viability test as three seeds in each pot. The pots were acted as a positive control of pathogenicity and incubated in a growth cabinet. Damping-off disease severity was determined as mentioned above.
To evaluate the efficacy of P. oligandrum against the damping-off disease of G. max in artificially infested soil with P. aphanidermatum, mycelial mats of P. oligandrum isolates were got from cultures developed in V-8 juice broth incubated for 10 days in darkness at 25 • C, then the mycelial mats were cleaned with sterile saline water and fragmented by tissue homogenizer. The preparation was utilized to coat G. max seeds using 3% carboxy methyl cellulose solution (CMC). Mycelial mats of P. oligandrum isolates (MS15, MS19, and MS31) at concentrations of 1000 prop gules per ml were mixed with exterior sterilized seeds of G. max. The seeds were spread in the sterile opened Petri dish to dry overnight in a refrigerator at 5 • C. Three coated seeds of G. max were sown into P. aphanidermatum infested soil at 5 mm depth from the soil surface as treated pots.
Some non-coated G. max seeds have been sown into infested soil with P. aphanidermatum and used as a positive control. Another group of coated seeds with the biocontrol P. oligandrum was sown in sterilized soil as a negative control. All pots were put in a growth cabinet as reported previously. Numbers of G. max seeds, which damped-off were counted after 14 days from sowing. Experiments were done with ten replicate pots per treatment.

Effects of G. max Seeds Treatment with P. aphanidermatum and Three Isolates of Bioagent P. oligandrum on Growth Seed Parameters
The growth promotion percentage for the shoot length, root length, and the number of lateral roots as growth parameters of the treated plants with the phytopathogenic P. aphanidermatum and P. oligandrum isolates were measured and calculated according to the formula: The growth promotion percentage = Plant growth of treatment − Plant growth of control Plant growth of control × 100 (1) Statistical analyses software, GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA), was used to statistically analyze the data of antagonistic activity using one-way analysis of variance and Tukey's test. The mean of triplicates was used to tabulate the data (p < 0.05).

Conclusions
The results attained by the present study conclude that P. oligandrum exhibited highly potential bioactivity against P. aphanidermatum mainly through mycoparasitism. Bioassay of P. oligandrum as a control agent showed that bioactivity of P. oligandrum using the dual culture technique was more effective than that of fungal filtrate. The confirmed efficiency of P. oligandrum as a biological control against G. max root diseases avoid the biohazards of fungicides application. This efficiency of P. oligandrum as a biological control agent increases the ability of its usage as an effective natural alternative to chemical fungicides used to control G. max damping-off disease.