Behavior of fungus Rhizoctonia solani under faba bean cotyledons when away from host and the effect of its starvation on aggressiveness

Behavior of Rhizoctonia solani on their hosts was carried out-in many studies when the pathogen was in close proximity with its host. The purpose of this study was focused on the behavior of this fungus on faba bean cotyledons when it was away from the host and the effect of nutritional status on its pathogenicity was also taken into consideration. Observations led to speculation that R. solani feels its host even when it was away from it and begins to assemble its strength to grow toward the host then start to form its pathogenic structures when it reach it. Scanning electron microscopic (SEM) observations which was carried out after 24, 48 and 72 h from exposing the pathogen to the host when the fungus was away from it revealed that when the hyphal pathogen feel the host, they swell and some hyphae begin to differentiate and appear very thick, forming likelihood mycelial strands and such hyphae grow vigorously and vertically toward the host. Starvation of the pathogen was found to be vital factor in R. solani pathogencity. Growing of the fungus on water ager led to dramatic increase in the pathogen. This strength was confirmed by visual determination of disease severity and by determination of polyphenol oxidase activity in infected cotyledons. These results exclude the role of host exudates in the attraction of the R. solani toward its host.


INTRODUCTION
Rhizoctonia solani (Kuhn) = Thanetophorus cucumeris (Frank) Donk is one of the most important soil borne pathogen all over the world.It causes very serious diseases of wide varieties of plants ranging from damping-off till stem canker led to dramatic effects on plant nutrition and its physiology (Baker, 1970;Hanounik, 1978;Anderson, 1982;Salt, 1982;Ogoshi, 1987, Wallwork, 1996;Hsiang et al., 2006, Simonetta et al., 2007;Kammerer and Harmon, 2008).Infection structure of this fungus was studied either under light microscopy or by using scanning electron microscopy (SEM).All studies revealed that the fungus produce infection cushions on the host surface and many infection pigs form on the underside of the cushions for penetrating the host (DeSilva and Wood, 1964;Dodman et al., 1968;Bassi et al., 1978;Marshall and Rush, 1980b;Armentrout and Downer, 1986;Kazuho Matsuura, 1986;Demirici and Doken, 1997;Zheng and Wang, 2011).
There is strong evidence which suggests that infection cushion formation by R. solani is induced by host exudates (Wyllie, 1962;Flentje et al., 1963;Martinson, 1965;Dodman and Flentje, 1970).However DeSilva and Wood (1964) were able to produce infection cushions-like structures on washed strips of host cuticle and epidermis without the addition of the exudates.Weinhold and *Corresponding author. E-mail: mostafahelmymostafa@hotmail.com. Bowman (1974) induced repression of virulence of R. solani by glucose and 3-O-methylglucose.Moreover, Marshall and Rush (1980) have found that the exogenous supply of 3-O-methylglucose and glucose resulted in a decided reduction in lesion development on rice seedlings and they postulated that the nutritional status of R. solani hyphae prior to penetration had a decided effect on aggressiveness of the pathogen.
The purpose of this study was to investigate the behavior of R. solani (AG 4) under faba bean cotyledons when it was away from the fungus and test the effect of nutritional status of the fungus on its aggressiveness.

Pathogen isolation and pathogenicity
R. solani isolates were isolated from the diseased plants of faba bean (Vicia faba L.) collected from farm of Faculty of Agriculture, Ain Shams University, Qualiobya Governorate, Egypt.Specimens were rinsed in tap water then cut into small pieces (2-5 mm), washed three times in sterile distilled water and blotted dry on sterile filter paper.Pieces were placed on 2% water agar (WA) and incubated at 25°C for 2 days.Emerging hyphal tips were transferred on potato sucrose agar (PSA: 200 g of potato, 20 g sucrose and 20 g agar) and pure culture was transferred to PSA slants.
Pathogenicity test of R. solani isolates was carried out by putting sterilized germinated faba bean seeds on fungal growth of tested isolates growing on PSA medium.The most aggressive isolate on faba bean cotyledons was chosen for further studies.This isolate was found to belong to AG4.

Scanning electron microscopic observation
The fungus R. solani was grown on PSA medium till the dishes were completed by fungal growth.Faba bean cotyledons were prepared as mentioned later, then the top of a plastic tip of micropippete was immersed in the central part of cotyledons and the base part was immersed in fungal growth.Six millimeter distance was left between fungal growth and cotyledon surface.After 24, 48 and 72 h fungal growth laid beneath cotyledons was taken for observation of SAM.Control fungal pieces were taken from the same dish in a wide distance from the site of cotyledons.
The material to be examined by SEM was fixed for 2-3 h in 5% glutaradehyde in 0.1 M phosphate buffer (pH 7), rinsed twice with the same buffer, and then dehydrated by immersing in increasing concentrations of ethanol (10-100% in 10% increments, 15 min in each concentration).Mycelia were attached with colloid on stubs and immediately immersed again in liquid nitrogen for 20 min before coating with 20 µm (200 A°) thick gold in Separating Coating Unit under vacuum (Matsuura, 1986).The material was then examined in stereo scan electron microscope at 28 KV.SEM was kindly carried out at Electron microscopy unit, Faculty of Science, Ain Shams University using (JEOL) JEM.1200EXII electron microscope.

Effect of nutritional status of R. solani on its aggressiveness
The fungus R. solani was grown in Petri dishes (9 cm in diameter) contained PSA medium at 25°C.When hyphal growth reached the edge of the dish, a piece (5 mm in diameter) was transferred to the center of another Petri dish contained water agar (2%) and incubated at the same temperature in the dark till the growth filled the dish (first transfer), a piece of fungal growth was taken from the edge of the first transfer and transferred to another Petri dish contained water agar and incubated till the growth reached the edge of the plate (second transfer).
Seeds of faba bean (Giza 2 cv.) were rinsed several times in tap water, then in sterilized distilled water.Seeds were dried on filter papers then rinsed in 2% sodium hypochlorite for 5 min for surface sterilization, finally, they were washed again in sterilized distilled water and left to germinate in plastic box in the dark.Apparently healthy germinated seeds have been selected then peeled.To avoid the mass effect of the pathogen on disease incidence, the tip of a plastic tip of micropipette (2 cm long) was immersed in the central part of cotyledons and the base part of the tip was immersed in fungal growth (either on PSA, first transfer or second transfer) leaving 6 mm distance from the surface of fungal growth (Figure 1a, b).
Dishes contained cotyledons were incubated at 25°C for three days in the dark, then they were photographed and disease incidence was estimated on cotyledons using adopted scale ranged from 1-5 where: 1: apparently healthy cotyledons; 2: very weak infection; 3: moderate infection; 4: very severe infection; 5: cotyledons completely covered by fungal mats and died.Three dishes were used as replicates for each treatment and five cotyledons were put in every dish.This experiment was repeated at least three times.Average of disease incidence was calculated and standard deviation(s) was estimated (Ghahramani, 2002).Sticky strips were used to make a finger print of hyphal growth and infection cushion on faba bean cotyledons and to observe them using light microscope (Leica DM2500) connected to computer in Histopathology Unit, Plant pathology Department, Faculty of Agriculture, Ain Shams University.In another experiment, R. solani was grown in PSA medium till the fungus fills the dish.Strips 1 cm width were carried in the growth then transferred to another sterilized Petri dish contained filter paper.Strips of fungal growth contained the medium were pasted on sides of the dish and filter paper was wetted by 1 ml of sterilized distilled water.Tow cm.apart from fungal growth sterilized cotyledons of faba bean were put on wetted filter paper, and tow cotyledons were put in every dish.Control dishes were carried out as mentioned without faba bean cotyledons.Dishes were incubated at 25°C in the dark and observed periodically and photographed after 3 days.

Determination of polyphenol oxidase activity (PPO)
PPO activity was determined in five seeds with the highest degrees of disease incidence for each particular treatment.Infected seeds were grinded in pistil and mortar at high speed in phosphate buffer solution (pH 5.5) (1:2 w/v).Suspension was centrifuged at 3000 g for 10 min at 4°C.Supernatant was taken for determination of PPO activity using catechol as substrate according to the method described by Arnnok et al., 2010.The absorbance at 410 nm was recorded continuously at 25°C for 15 s using Unico UV-2100 ultraviolet-visible spectrophotometer, USA.

Behavior of the fungus R. solani when away from host
As shown in Figure (1a) when the fungus was far from its host either vertically or horizontally (Figure 2a) the fungus try to reach it, where it gathering pace to reach the host.As shown from Figure (2a) fungal hyphae were collected together and grown vigorously toward the host comparing with control plate were the fungus poorly grows regularly toward the moist filter paper.When it reaches its host it begins to produce its infection cushions (Figure 3) in order to penetrate host tissues.
This phenomenon was deeply studied using SEM.Germinated faba bean cotyledons were fixed vertically away from the fungus and a piece of the fungus laid beneath cotyledons was taken for SEM after different intervals that is, 24, 48 and 72 h.Figures (4a, b, c, d and  f) illustrate the response of the fungus when it feels its host.As Figures indicate, after 24 h the fungal hyphae swelled to very great extent comparing to fungal hyphae very far from the host, the deviation from the normal case (control) include hyphal anastomosis, strong hyphal branching, hyphal likelihood strands and swelling.After 48 h, in addition to the previously mentioned observations, fungal hyphae started to differentiate forming strands consist of at least two thick hyphae and started to grow vertically.After 72 h these strands grow vigorously vertically toward the host.Swellings of hyphae began to decline indicating that nutrients were translocated from non invading hyphae to invading ones.

Effect of starvation of R. solani on its aggressiveness
The fungus was starved by successive cultivation from PSA medium to water agar.The transfer on water agar     was carried out twisly.Germinated faba bean cotyledons were put away (6 mm) from fugal growth in order to rule out the effect of fungal mass on disease incidence.This experiment was repeated three times, and data are presented in Table 1, Figures 5 and 6.It is evident that starvation of R. solani resulted in significant increase of disease incidence.More starvation led to the higher aggressiveness.The higher five cotyledons severely injured from the three experiments were chosen for photographing (Figure 5).As shown in the figure, starvation of the fungus led to severe disease incidence.
In order to make sure that starvation of the fungus led to increase in the aggressiveness of the pathogen, PPO activity was determined in severely infected five cotyledons subjected to the pathogen.Data are illustrated in Figure 7. PPO activity significantly increased in cotyledons subjected to the more starved growth compared to cotyledons subjected to feed growth.

DISCUSSION
What is going on when the fungus R. solani was away from its host?Does it feel it and what are the events that will be followed when the pathogen sense the host?These questions were the subject of the present study.It was noticed that the pathogen R. solani sense its host even when it was away from it.This phenomenon was noticed either when the host was found vertically or horizontally for the pathogen.What are the stimuli which moved to recognize the pathogen host?In this regard, many authors -as mentioned before -postulated that infection cushion is induced by host exudates.On the other hand, Weinhold and Bowman (1974) found that glucose and 3-O-methyl glucose repressed virulence of R. solani on rice seedling.It could explain these results on the base that these compounds may down regulate pathogenicity genes of R. solani (Lopez-Berges et al., 2010).All the previous studies were carried on models where R. solani was in close proximity with its host.The obtained results revealed that the fungus sense its host either by moisture or heat emitted from the host or by evaporate substances or by all of them.This means that these stimuli interact with the pathogen through receptor sites located in cytoplasmic membrane of R. solani.The results of this interaction may lead to provoke the fungus to react with it resulting in great effects on its morphology.The interaction between stimuli and receptors during pathogenesis still obscure and need further research.However the interaction between host and pathogen during elicitation of immune system in plants was deeply studied (Chishelm et al., 2006;Spoel and Dong, 2008).The most noticed morphological deviations from the normal state -in this study are swelling the hyphae beneath the host which was observed after 24 h from the diffusion of this stimulus, this phenomenon was followed by differentiation of fungal hyphae, and strengthen of hyphae by anastmosis and by formation of something likelihood mycelial threads which grow toward the host forming infection cushions.This result revealed that not all hyphae able to infect the host but only some newly differentiated ones had the ability to infect the host.Other non invading hyphae lost their thickness after the differentiation of invading hyphae had been occurred, this action may due to the migration of biomass from non invasive hyphae toward invading ones and it is very important to investigate the type of stimuli that provoke fungal differentiation.
In this study, the role of nutritional status of the fungus on its aggressiveness was investigated.In order to reach this goal, the fungus was grown on PSA medium.After the fungal growth has reached the edge of the plate, a piece of fungal growth at the edge was taken and transferred to another dish contained water agar and this technique was repeated again on WA.
Germinated sterilized cotyledons were put on the growth leaving 6 mm distance between fungal growth and cotyledon surface.Three days later at 25°C disease severity was assayed either visually or by determination of PPO activity in the most five injuries cotyledons since PPO activity is increased in Rhizoctonia diseased hosts (Stackwell and Hanchey, 1987;Seo et al., 2012).Data obtained indicated that starvation of R. solani led to great increase of its aggressiveness, and this increasing of aggressiveness was increased more than an increase of starvation.
How the starvation does increase the aggressiveness of R.   have found that nitrogen limitation act as a signal to trigger the in planta expression of virulence genes, They postulated that the mitiogen-activated protein kinase Fmk1 is required for plant infection, but the results obtained in the present study revealed that expression of genes of pathogenecity occurring prior to infection and before it reaches the host pathogen.
The enhancing effects of starvation on fungal aggressiveness may explain how organic amendments, such as animal and green manure, organic wastes, composts and peats, have proposed to control diseases caused by soil borne pathogens (Baker and Cook, 1974;Hoitink and Fahy, 1986).There are many examples of soil borne pathogen controlled effectively by the application of organic amendments among these R. solani (Papavizas and Davey, 1960).The explanation of this phenomenon was not clear until the discovery of the role played starvation in elicitation of pathogenecity genes during infection.

Figure 1 .
Figure 1.Hyphae of Rhizoctonia solani growing up, reached the cotyledons, invade it forming disease symptoms.A) Cotyledons fixed on fungal growth on water agar, b) water agar free from fungal growth.

Figure 2 .
Figure 2. Hyphae of Rhizoctonia solani grow horizontally to reach the cotyledons.A) In the presence of cotyledons, b) in case of absence of cotyledons.

Figure 4 .
Figure 4. Behavior of Rhizoctonia solani hyphae which laid beneath to cotyledons .Where A, C, and D :Hyphae very far from cotyledons, after 24, 48 and 72 h respectively.B, D and F: Hyphae laid beneath cotyledons, after 24, 48 and 72 h respectively.In B stars indicate abnormal hyphal thickness, arrows indicate anastmosis and circle indicates abnormal hyphae.In D arrow indicates mycelium likelihood strand.In F arrow indicates newly differentiated hyphae, growing toward cotyledons.

Figure 5 .
Figure 5. Disease symptoms due to Rhizoctonia solani invasion of faba bean cotyledons.PSA: the fungus was grown on potato sucrose medium; 1 st , the first transfer from PSA to water agar, 2ed second transfer from water agar to water agar.

Figure 6 .
Figure 6.Effect of starvation of Rhizoctonia solani on its aggressiveness on faba bean cotyledons.Results are average of three distinct experiments.

Figure 7 .
Figure 7. Polyphenol oxidase activity in cotyledons infected either by Rhizoctonia solani grown on PSA, 1 st transfer to water ager or 2ed transfer from water agar to water agar.

Table 1 .
Disease severity on faba bean cotyledons infected by Rhizoctonia solani grown either on potato sucrose medium (PSA), water agar (1 st transfer) or another water agar (2ed transfer).Three distinct experiments were carried out.
Disease severity was determined according to the following scale: 1: apparently healthy cotyledons; 2: very weak infection; 3: moderate infection; 4: very severe infection; 5: cotyledons completely covered by fungal mats and collapsed.