Susceptibility of Wheat Varieties to Soil-Borne Rhizoctonia Infection

Response of 19 wheat varieties cultivated in Hungary varied within large limits to soil borne Rhizoctonia infection. The most frequent symptom, usually leading to damping off was the root neck necrosis. Four significant factors influencing the susceptibility of wheat comprised 71% of total variation but none of them was dominant. The inhibition of development of survivors in Rhizoctonia infested soil correlated with overall susceptibility of variety concerned. The varieties Emese, Kikelet and Palotás are proved to be less susceptible, but none of the varieties could be certified as tolerant. No relationships were revealed between pathogenicity of 26 Rhizoctonia strains studied and their taxonomic position or origin. The anamorph strains of Athelia, Ceratobasidium, Ceratorhiza and Waitea similar to Thanatephorus anamorphs selectively infected the wheat varieties, but the syndromatic pictures were undistinguishable with unarmed eye. R. solani was proved to be more aggressive against germinating wheat than R. cerealis. Nine significant factors influencing the virulence of Rhizoctonia strains comprised 82% of total variation, and six of them influenced exclusively Thanatephorus anamorphs.


Introduction
In August 2002, brown patches were observed on turf grasses in parks at four locations in Budapest.The symptoms observed were necrotic lesions on the roots and stems, as well as brown lesions on the leaves.Two types of sclerotia, nearly globose, pinkish to orange and irregularly shaped, dark brown were found on roots.On potato dextrose agar fast growing, colourless colonies with small reddish/coloured scelrotia uprose of the first type.This fungus was identified as Rhizoctonia zeae Voorhees (teleomorph Waitea circinata Warcup and P.H.B. Talbot) [1].Buff-coloured, fast growing colonies of Rhizoctonia solani Kühn (teleomorph Thanatephorus cucumeris (A.B.Frank) Donk) arose of the second type.The study of more than 150 plant species cultivated in Hungary [2] revealed that R. zeae attacked monocotyledonous species more aggressively than dicotyledonous ones, contrarily to R. solani.This latter species was formerly reported as pathogen of winter wheat [3] and oat [4] in Hungary.
Traditionally, farmers paid little attention to field damage caused by soilborne Rhizoctonia infection in wheat, because either seedborne or airborne fungi (rust, mildew, smut etc.) infecting stem, leaves and spikelets had been the main constrains of yield.Due to success in breading and arousal of new synthetic fungicides, these fungi presently do not cause catastrophic yield losses.However, in the last two decades increasing number of papers was published on yield losses (30% to 50%) caused by Rhizoctonia species [2,5] in main wheat cultivating areas [6][7][8].In Europe and North America winter wheat suffered mainly of R. solani AG-8 strains [7] with the R. cerealis [6][7][8], while in Australia AG-1 and AG-8 and in Turkey five different anastomosis groups of R. solani [9,10] were revealed.In South Eastern Hungary damage by the R. cerealis and R. solani has been observed in spring wheat [3].
Rhizoctonia species are well known soil borne pathogens frequently causing damping off prevalently in moist and cool conditions that are the main stress factors re-quested to induce disposition to increased susceptibility of potential host plants [11].These species have no vegetative bodies for spreading, but infected seeds and propagating material can distribute infecting propagula.The disease is more severe in sandy soils, as the fungus can grow more rapidly [12], and the hyphae tend to colonize rhizoplane reducing the vitality of plant even without penetrating into tissues.Infection cushions are formed on the surface before individual hyphae penetrate with minor morphological modifications [13].The ability to produce discrete appressoria is highly variable and regulated seemingly by numerous factors.Hyphae may penetrate without an appressorium through stomata or wounds [14][15][16].All Rhizoctonia species are obligate aerobic fungi, which are habiting mainly in rhizosphere, however, they may survive as saprobionts in the upper layer of the soil forming a mycelial web, thus the undisturbed soil enhance the risk of the infection of young roots [17].Rhizoctonia root rot is more frequent and severe under reduced or conservation tillage [18,19] as the conventional tillage disrupts the mycelial web [20].This underlines the importance of the elaboration of new root health management practices, because formerly minor pest problems, such as Rhizoctonia root rot of wheat became major problems.In last two decades new research has been started and several hundred scientific papers reported data on etiology of root rot and sharp eyespot caused by Rhizoctonia species, on genetic background of susceptibility of wheat as well as on Rhizoctonia virulence.Actually, we can conclude that environment dependent mechanisms regulate the progress of this disease with complex etiology that is not under gene for gene control [21], and further research is requested to understand the multicomponent syndromatic picture.
The disease caused by R. zeae was described for the first time in Florida, by Voorhees in 1934 [22], as sclerotial rot of corn.Since that this fungus distributed in temperate regions, in Europe firstly reported in 2004 [1].In a host-range study, the isolate proved to be highly pathogenic to germlings of several ornamental and cultivated plants, including Beta vulgaris ssp.vulgaris, Callistephus sinensis, Dahlia variabilis, Daucus carota, Lupinus polyphyllus, Papaver somniferum, Pennisetum glaucum, Phaseolus vulgaris, Sesamum indicum, Solanum melongena, S. tuberosum, Sorghum bicolor and Triticum aestivum [1,2,23].The estimation of risk on wheat production caused by this pathogen was the aim of our work.
Actually, both seed and soil borne infections might be well controlled in early stage of germination with seed dressing [24], however, we have no effective methods with reliable cost/benefit ratio for protection of wheat against Rhizoctonia during the vegetation.Although mineral nutrients can manipulate the reaction of plants [25], such treatments can not combat serious yield loss. Sev-eral attempts were made to explore antifungal potential of eubiotic preparations and to desing suppressive soil [26][27][28][29][30][31][32], however, in order to prevent the harm, these developments have had not satisfactory results.In the case of soil-borne infections correlative influences among members of microbial consortia associated to potential host plant may influence both the invading pathogen and disposition of potential host to adverse factors of environment thus change both the virulence of pathogen and the susceptibility of host to pathogen at any time depending on the genetic potential of these partners.Due to complexity of interactions there is difficult to predict the success of biocontrol measures.Currently the effective protective method is the appropriate crop rotation, and the breeding of wheat cultivars for improved tolerance to factors inducing disposition to increased susceptibility to the presence of Rhizoctonia in the soil can be considered.
Our objectives of this study were the comparative evaluation of responses of germinating wheat seeds to Rhizoctonia strains of various origin and taxonomic position as well as to reveal patterns in factors influencing the wheat/Rhizoctonia interaction.

Materials and Methods
Greenhouse experiment was undertaken to compare the infective potential of R. zeae strain with 25 Rhizoctonia strains of various taxonomic position.Susceptibility of two sortiments of Triticum aestivum L., moreover, T. monococcum L., T. turgidum L. and four small seed grains were involved into the tests.No seed dressing or any other manners to depress the microbiota of spermosphere were applied.The potting medium was made by mixing forest soil with peat before autoclaving (1.15 atm per 20 min), at the ratio of 3:1.

Test for Pathogenicity
The potting medium was made by mixing forest soil with peat before autoclaving (1.15 atm per 20 min), at the ratio of 3:1.
The soil was inoculated with Rhizoctonia by the following manner: the sterile soil prepared as above was admixed with chickpea seeds previously infected with the pathogen (10 seeds per 250 g pot), than incubated 96 hours at 26˚C -28˚C for evolving the mycelial net.The seeds were put on the surface of infested soil (1 × 1 cm), than covered with 5 mm layer of sterile soil.Sterile distilled water was used to moist the surface (15 mL per pot), and covered with plastic wrap layer to avoid dessiccation.Subsequently, the pots were evaluated each day counting the emerged germlings, and observing the occurrence of disease symptoms (damping off and leaf spots).The height of seedlings was regularly measured to nearest millimetre to follow the dinamics of growth.The control plants were grown up in Rhizoctonia free soil.The growth inhibition was calculated as a ratio between control and treated plants.
When the coleptyles of control plants had been fully developed (8 days after emergence of first germling) the pathological status of all seedlings was evaluated, their height and mass of measured to nearest millimetre and milligramm, respectively.Inhibition rates were calculated as related to control.The percentages were transformed into probit values, and this transformed data were analyzed according to Sváb [33].The state of roots was assessed as well, and tissue sections were examined under microscope in cases where no visual symptoms were observed.The method was discussed in detail previously [23].The following six fold scale was used to assess the tolerance of test plants at the 8th days: 0 = all seedlings were destroyed; 1 = the majority of seedlings was dead, but at least one survivor was presented either symptomless or bearing severe symptoms (the coleoptyle and the roots damaged, the root neck scoring), 2 = less than half of seedlings survived, the survivor were either symptomless or bearing severe symptoms (the coleoptyle and the roots damaged, the root neck scoring), 3 = more than half of seedlings survived, the symtoms of disease syndrome largely varied, 4 = most of seedlings were similar to control, but as minimum as one diseased, 5 = none of seedlings had any symptoms visible to the naked eye.The results of observations were compiled into data matrix ((19 wheat varieties + 4 small grains) × 26 Rhizoctonia strains).
Surviving specimens were grown up to 21 days and their development and evolution of disease syndrome were observed.

Data Analysis
Box plot analysis was applied to demonstrate alterations both in tolerance of test plants and and virulence of Rhizoctonia strains as well as variations in growth parameters of wheat varieties as influenced by the presence of Rhizoctonia in the soil.The relationships between host (wheat varieties and small grains) and Rhizoctonia strains (potential soil borne pathogens) have been analyzed by multivariate methods: Non-linear Mapping (NLM) [34], Cluster Analysis (CA), Principal Component Analysis (PCA), Regression Analysis combined with Canonical Correlation Analysis (CCA) and Potency Mapping (PM) technique [35] following a previously described scheme [36].PCA was carried out on the correlation matrix [37] and only the components having an eigenvalue greater than one were included in the evaluation of data to demonstrate potential number of factors influencing on host parasite system, and the results were not delineated in details.The protocol of experiments is shown in Figure 1.
Statistical functions of Microsoft Office Excel 2003 (Microsoft, Redmondton, USA) and Statistica5 program (StatSoft 5.0., Tusla, USA) were used for analysis of data.The graphical presentation of result of data analysis was edited uniformly in MS Office Power Point 2003.

Dynamics of Germination
The dynamics of germination of wheat was altered by Rhizoctonia strains in cultivar dependent manner (Figure 2).In majority of cases first germlings emerged within 2 days after sowing in Rhizoctonia free soil, moreover, the process was finished rapidly.The seeds of Pannon Premium sortiment germinated more uniformly, than the other varieties.In the presence of Rhizoctonia strains emergence had been delayed with exception of most tol-erant varieties (Petrence, Emese, Alkor, Hegyes).The last day means the limit after that no further germlings outcropped.Several seeds having been destroyed, seemingly, the susceptible individual were killed either before emergence or suffered damping off within 2 -3 days after outcropping.This ratio strongly varied, and it was not possible to carry out the statistical analysis within frames of the experimental model applied.

Syndromatic Picture of Disease
The growth of infected or diseased seedlings of wheat was conspiciously retarded than the control, however, not all symptoms of disease syndrome turned up.Even seedlings without any visible symptoms suffered damping off before full development of coleoptyle, the roots of such individuals were symptomless in many cases, although brown spots could be frequently observed on roots and root neck, even in the cases of robust survivors too.In some cases small black spots (<1 mm) were found on root necks, but these were not spread later.Leaf spots with dark brown edge were randomly observed after 6 days.The development of seedlings which survived the infec-    tion without root neck symptoms was retarded at various degree even two weeks later.The incidence of robust survivors varied greatly within series, the root system of such individuals was not retarded with exception of most susceptible varieties (Bodri, Lona, Lucilla, Menüett).The reaction of germlings to the presence of Rhizoctionia in the soil was extremely heterogenic, the coefficient of variation was over 30 percent within pot that made impossible the reliable statistical analysis.This was the reason of use of the six fold scale for assessment of plant response to Rhizoctonia infection.In several cases half of seedlings survived while the other half was killed independently of host/pathogen pair, most probably due to environmental effects.
The behaviour of small grains was similar to that of wheat including symptoms and development of disease syndrome.The leaf spots on Eleusine appeared rarely, but this was not in direct contact with virulence of Rhizoctonia strains.The survivors of small grains were not analyzed in details.

Susceptibility of Wheat Varieties
The test plants tolerated the Rhizoctonia in strain dependent manner (Table 1).Among T. aestivum cultivars Lona, Menüett and Bodri proved to be less tolerant, while Emese, Palotás and Petrence exhibited low susceptibility.The response of T. monococcum and T. turgidum was similar to more tolerant T. aestivum cultivars.The Eleusine was less susceptible than other small grains.Unfortunately, none of the test plants tolerated the majority of Rhizoctonia strains at high degree.
The response of wheat cultivars to R. zeae, the new pathogen in Europe, altered significantly of that of R. solani strains.This difference manifested clearly, when variety dependent virulence of R. zeae and associated R. solani strain (B-433) was compared (R 2 = 0.317, n = 19), although their average pathogenicity (AP) was similar (2.9 and 3.1, respectively).R. cerealis proved to be moderately aggressive against germinating cereals (AP = 3.6), and the similarity between activity spectrum of R. solani strains and R. cerealis was low (r 2 < 0.23).
The susceptibility of wheat varieties to Ceratorhiza (B-438) isolated of strawberry and Athelia (B-442) was low, however, the C. ramicola (B-427) isolated of orchid heavily injured five T. aesticum cultivars (Toborzó, Bodri, Lucilla, Toldi and Lona).The orchid symbiont R. stahlii (B-441) exhibited low pathogenicity.Test plants were related applying Nonlinear Mapping based on data comprised in Table 1 (Figure 3).Plants similarly responding to soil borne Rhizoctonia infection formed a loose group, but no clear patterns were revealed.
The taxonomic position did not influence the grouping.This type of plotting did not gave information on the structure of relationships among wheat cultivars, so another nonlinear method, the Cluster Analysis was carried out (Figure 4).
Wheat cultivars clustered into two well separated groups on the dendrogram.Triticum aestivum cultivars were distributed between groups, however T. monococum and T. turgidum were in different clusters.The average tolerance level of groups was similar (A = 3.1 and B = 2.7), but alterations were revealed in the spectrum of suscepti- bility.The difference was particularly spectacular in the case of AG-1 strain of R. solani isolated in Australia, which proved to be especially virulent against T. turgidum and cultivars of Pannon Standard sortiment.

Analysis of Survivors
The inhibition of growth and mass accumulation of survivors were proportional to the potential susceptibility and inhibition of emergence (Figure 5).The rates of inhibition of growth and mass accumulation well correlated to each other: (p < 0.001): Length = 0.8149Mass + 0.8876 (FG = 18, r 2 = 0.8066).Consequently, the response of survivors can be characterized with inhibition of growth.This has importance when survivors have to be grown up for selection or further studies.

Factors Influencing the Plant Response
Four substantial factors could be revealed by PCA (Table 2) that explained 71% of total variation of data matrix edited of the Table 1.None of principal components (PCs) comprised dominant part of variation, and the cultivars clustered into four well defined groups (A = 6, B = 5, C = 3 and D = 4 varieties, respectively).The performance of majority of cultivars was determined by one dominant factor except three (Toborzó, Suba and Karizma) where two factors influenced the response (Table 2).All this indicates, that tolerance of wheat cultivars to soil borne Rhizoctonia infection was regulated by different genetic factors.

Virulence of Rhizoctonia Strains
Majority of Rhizoctonia strains holded back the germination (Figure 6) in the case as minimum as one cultivar.However, this effect was not strictly related to response of seedling in posterior stages of evolution of wheat/ Rhizoctonia association.For example, the AG-1 strain of R. solani proved to be later more aggressive than R. cerealis, although these two strains altered the germination by similar manner.Seemingly, the genetic background regulating the formation of anastomosis between hyphae is not connected directly to expression of pathogenicity against wheat, the host spectrum of two AG-4 strains was different, and these strains inhibited the emergence at various degree.The AG-8 strain of R. solani that causes severe yield losses in China and Australia altered the dynamics of emergence insignificantly, contrary to isolates of imported propagating material of China and Netherlands (B-245, B-521).
Based on results shown in the Figure 4 two matrices were edited of the data of Table 1.The Canonical Correlation Analysis resulted three significant canonic functions (R 2 = 0.886, 0.847, 0.  The PC loadings influencing the response of cultivars significantly were underlined.Varieties 1-6 and 7-13 are of Pannon Standard and Pannon Premium sortiments, respectively.G = Varieties influenced by the same factor were marked with the same letter.PS = potential susceptibility of variety to Rhizoctonia calculated in percents of the Table 1.morphs, indicating that these properties have no decisive role in expression of pathogenicity.The physiological characters, which take place in anastomose contact have also minor importance, for example two R. solani strains of AG-4 anastomosis group (B-417 and B-430) are in notably different position.Similarly, the source of strain had no influence on clustering.For example, the strains isolated of potato tubers were located in different clusters, while anamorphs (B-441 and B-427) of Ceratorrhiza and Thanatephorus of orchid and potato, respectively, were closely related.The taxonomic position of source (host plant) also had no importance, the strains of mono-and dicot plants were placed into the same cluster (see B-417 of Citrus and B-433 of Festuca).Unfortunately, we have few data on exact geographic origin, but surprisingly, all isolates of Hungarian origin were separated into the same cluster.The strains B-413 and B-521 were of imported propagating material.This might be related to role of environmental (biogeographic and bioclimatic) factors.

Emergence (days)
Being typical soil habitants and forming mycelial web in soil [38,39], the soil biota plays a crucial role in the microevolution of Rhizoctonia species, while the assemblage of soil biota significantly depends on both structure and composition of mineral matrix and climatic conditions [40].
respectively).Plotting the strains as canonic scores by first two roots strict linear relationship was revealed, where only few strains deviated (AG-1, AG-5).However, the strains on plot of third canonic function (Figure 7) clustered into two well separated groups (p < 0.001).The members of each group differ by their virulence, and the strains of the same anastomosis group split within two clusters, indicating, that the properties responsible for separation into anastomosis groups have minor importance in formation of these clusters.

Relationships among Rhizoctonia Strains
The Rhizoctonia strains were clustered on the base of their pathogenicity against wheat cultivars (Table 1) based on correlation matrix applying Unweighted Pair Group Average method (Figure 8).

Factors Influencing Virulence of Rhizoctonia Strains
The Principal Component Analysis resulted in nine notable components that explain 82% of total variance (Table 3).Similarly to factors influencing the response of wheat cultivars none of them was superior, the major PC has about twice more weight than the minimally significant one.The groups were sharply separated and formed by strains of various taxonomic positions.Only in two cases was the performance of strains (B-419 and B-410) affected by two factors.The quantitative aspect of virulence was not connected per se to groupping, for example the most virulent AG-1 strain of R. solani (B-415) was linked to significantly less virulent AG-10 strain (B-423).The findings support the concept of multilocal character of interaction between wheat and attacking Rhizoctonia.

Discussion
We focused on soil borne Rhizoctonia infection that has the greatest effect on the growth and yield of wheat among soil borne pathogens [41], and the brown patch disease became devastating in last two decades.This might be related to the changes in both pest management practices and cultivation techniques that resulted the increased frequency of the specialized pathogen genotype in some geographic areas [42,43].The metalloorganics of broad antimicrobial spectrum of activity applied formerly for seed dressing have been banned, and the monosite inhibitors either do not inhibit Rhizoctonia like fungi or they loose activity rapidly due to acquired resistance.Moreover, the formerly dominant tillage based wheat Strains 1-13 refer to anastomosis groups AG-1-AG-11 and AG-E of R. solani, respectively, of CBS collection, strains 14-20 are Hungarian isolates of R. solani and R. stahlii (21), all are Thanatephorus anamorphs.Rhizoctonia strains 22 -26 are anamorphs of Waitea (22), Cerathorrhiza (23,24), Ceratobasidium (25) and Athelia (26).PV% = potential virulence of Rhizoctonia strains against wheat cultivars calculated of the Table 1 applying Potency Mapping.The PC loadings influencing the virulence of strains significantly, were marked with asterisks.The small grains were omitted of calculations.
The efforts to control soil borne infections applying various eubiotic preparations were not successful yet in large scale, mainly due to the low reproducibility of the effect in field conditions which makes the calculation of cost/benefit ratio unreliable.Actually, seed dressing results only sufficient control of pathogens threatening the cultivated plant in early stage of development.Thus the importance of selection of resistant wheat cultivars increased.

Performance of Wheat/Rhizoctonia Pathosystem
Rhizoctonia species are abundant in soils as mutualistic members of microbial consortium associated with plants.
Their relationship with host plants may change from symbiosis (as in various orchids) to destructive parasitism, and these fungi usually do not cause visible disease symptoms.The infection may remain latent for a long period and can rapidly generalize, when environmental stress factors overhelm the homeostatic regulation of host plant, i.e., the disposition of host favours to pathogen either in phyllosphere of in roots.The amount of thallus varies within large limits (0.09 -6 ng per g tissue) in symptomless plants [48].The symptomatic picture is usually variable to a high extent as stunting growth, decrease in mass accumulation, leaf spots of various size, deformations of various organs and rooting tissues can be observed on infected plant alone or in combinations together, the disease syndrome may evolve rapidly to a fatal consequence in formerly symptomless host (damping off and wilting).
In our experiments all the above mentioned variations were observed, and the overall susceptibility of one respective variety influenced only the frequency of various symptoms of disease syndrome caused by soil borne Rhizoctonia infection independently on taxonomic position or origin of the strains.This indicates that some traits used for taxonomic classification can not be tightly associated with the properties determining the character of wheat/Rhizoctonia interaction.The small black spots that were occasionally observed on scutellum and mesocotyl of surviving individuals may be result of hypersensitive reaction (HR) suggesting the defense mechanism against Rhizoctonia attack rapidly activates.Further studies requested to connect this property with wheat response to soil borne Rhizoctonia infection as the involvement or manipulation of HR into breeding programs may open door towards to the control of brown patch disease [49].Furthermore, the role of Rhizoctonia toxins in pathogenesis has to be elucidated as well as factors regulating development of survivors in presence of Rhizoctonia should be identified.
The genetic background of tolerance to Rhizoctonias is not elucidated yet.Discoveries show that both anatomic and physiological features are involved into manifestation.Thus, the structure and composition of cell wall might have importance; the resistance to fungal xylanase is a tolerance factor [50].The multivariate analysis of our experimental results supports the multigenic character of wheat response, although no dominant factor was revealed.Unfortunately, the screening of tolerance to Rhizoctonia in microscale provocative experiments can give data for only preliminary selection of wheat lines, and the survivors should be evaluated in field conditions as well [51].Nevertheless, the data obtained are encouraging and on our opinion the screening of gene banks applying the method demonstrated here can result germlings useful for further manipulations (for example cultivars Emese, Petrence and Toldi).The high variation observed in response of wheat to soil borne Rhizoctonia might be caused by lack of preliminary selection of tested plants.
Members of the genus Rhizoctonia are considered as a complex mixture of filamentous fungi, having in common the possession of a non-spored imperfect state, usually referred to as the anamorphs of five genera: Athelia, Ceratobasidium, Ceratocystis, Thanathephorus and Waitea.
Here we included data on virulence of strains of the above five teleomorph genera, all of them attacked germinating seeds of cereals tested.Our results approve presumtion of Tomaso-Peterson and Trevathan [52] that the new for Europe pathogen, R. zeae, is considered as a hazard for wheat cultivation, with special regard to warm climate areas.
The multivariate analysis of experimental data re-vealed nine significant factors influencing the aggressivity of these strains; moreover, these factors are clearly not related to traits used for taxonomic purposes.The hyphal anastomosis interactions has been widely used for clustering of Rhizoctonia anamorphs within the complex, since other types of diagnostic features are usually scarce in these fungi [53].It was considered, that the anastomosis groups are specialized to defined host plants [54].Our experiments do not support this presumption in the case of wheat.Seemingly, the host range of single strains might be different, and reversely, strains clustered into different taxons, may have similar host range.By this reason, on our opinion, -based on presented results here, -for primary screening of wheat cultivars as minimum as six different strains should be used, including A. rolfsii, C. cerealis and W. circinata.

Future Prospects
The plant rhizosphere is a dynamic environment in which many parameters may influence the population structure, diversity and activity of the microbial community.The soil C:N ratio has critical role in disease incidence caused by Rhizoctonia as it was demonstrated by Kuhn et al. [55].The roles of mycorrhiza in facilitating the acquisition and transfer of carbon (C) and nitrogen (N) is well known.A considerable amount of bidirectional transfer of C between host plant and its fungal symbiont, and a fungus-dependent pathway for organic N can be realized rapidly, thus influencing positively the stress tolerance of plant [56].The micro-and mesofauna also can alter the disease incidence either wounding the roots [57] or decreasing the size of inoculum [58].Induction of suppressive soil by using mixed cropping or applying eubiotic preparations is contradictory, as the iron deficiency may harm the wheat although this can be overcame by leaf nutrition.Nevertheless, we can expect new, usable knowledge of the environmental research on microbial communities and plant microbe interaction studies, which can help to design and sustain suppressive soil that would be the most convenient and economic method for comatting yield losses caused by soil-borne diseases [59].
In our experiments individual resistant to some Rhizoctonia, which attack the majority of its fellows, occurred in each variety, likely to observations of other authors [64].Such survivors can be objects of further breeding or genetic engineering.Unfortunately, the mechanistic approach on concern surrounding the genetically modified maize with Bacillus thuringiensis toxin or the glyphosate tolerant crops borne overall social resistance to gene technology.We should clarify, most of these concerns have no scientific base.First of all, the usefulness of genetically modified (GM) crops can be sustained with careful management [65].
Although, Rhizoctonia species, which cause bare-patch disease and sharp eyespot in wheat are not among the top ten pathogens [66], economic improtance of their control is increasing from severe to catasrophic yield losses reported from main wheat cultivating areas [67][68][69].There is an urgent need in sustainable management strategy to combat damages induced by soil habiting Rhizoctonia complex, first of all, new tolerant wheat cultivars.No major resistance genes to this pathogen have been identified so far inspite of increasing efforts in studies of physiology and genetic background of wheat/ Rhizoctonia interaction.Nevertheless, candidates for transgenic manipulation can be selected (Table 4).The possible improvement of tolerance demonstrated on rice [70] might serve as example for wheat.Some terpenoid phytoanticipins of Pelargonium gravolens [90], Artemisia arborescens [91], Helianthus tuberosus [92] exhibited good antifungal effect, but on our opinion the use of antifungal polypeptides seems to be more promising for transgenic manipulation.For example, a basic oligopeptide of Bacillus subtilis exhibited excellent and broad spectrum antimicrobial activity in our experiments [93] and would fit for control of all four important soil borne pathogens of wheat.The incorporation of Rhizoctonia specific mycovirus into genome of cereals also is a promising possibility [94], with special regard to root border cells.These, detached living cells form the "front line" in the soil, the special part of rhizosphere, described as a system first by Hawes [95], where the plant controls the microenvironment with these specially progammed cells [96].The border cells have indisputably key function in plant defense controlling the dynamics of adjacent microbial populations in the soil to foster beneficial associations and inhibit pathogenic invasion [97], thus these cells are plausible objects of genetic engineering to desing wheat plant with optimum characteristics for root health management.
PCR based molecular methods enable the comparative analysis of genes involved in plant defense [98].The task is complex because the design of PCR based molecular markers linked to the Rhizoctonia resistance genes of wheat cultivars seems to be complicated due to high number of factors influencing on the type of plant response.Synergic joint action of several minor factor may result more stabile repel than a single major one as one gene mutation can not eliminate this type of defense [90].However, we understand little about how genes interact because very few possible genetic interactions have been explored experimentally [99], thus depending on the insertion event, a particular transgene can have large effects on the entire phenotype of a plant and that these effects can sometimes be reversed when plants are moved from the glasshouse to the field [100].On our opinion the more intense research of complex repertoire of small RNAs (microRNAs [miRNAs] and siRNAs) used as guides for post-transcriptional and epigenetic regulation would help to design new, either intraspecific or intrageneric wheat varieties.To promote research in this field it is necessary to make public the data on cereal crop genomes and discuss the contribution: what proteins, and their genome sequence organisation, play in plant defence.Although these data are extremely valuable tool for detailed analysis, and the emergence of informatic market makes difficulties as patent applications back out of scientific disputation [101], nevertheless, such free disputation in large scale of scientific community would significantly accelerate the progress in breeding of wheat tolerant to soil borne diseases.

Conclusions
No relationship was found between taxonomic position and origin of Rhizoctonia strains, indicating that traits used for their classification are not closely related to expression of their pathogenicity against wheat cultivars.Nine factors were revealed that significantly affect their virulence in wheat/Rhizoctonia system.
We have got empirical evidence from plant/pathogen system on the possibility of selection; the wheat phenoltype resistant to soil-borne Rhizoctonia, that verifies our approach of using simplified scale for disease assessment.

Figure 1 .Emergence
Figure 1.Flow diagram of the experimental protocol.The labels F and T in ellipses mark figures and tables in the body text where the results of computations were used for demonstration, while V means verbal interpretation of result.The zero hypothesis was: on the base of screening large number of varieties there is possible to select candidates for breeding wheat cultivar tolerant to soil borne Rhizoctonia infection.

Figure 2 .
Figure 2. Susceptibility of wheat varieties to soil borne Rhizoctonia infection.The gray and black strips mark varieties grown up in Rhizoctonia infected (+R) and non-inculated (control) pots, respectively.Wheat varieties are listed on the x-axis, grouped as: other wheat varieties, Pannon Premium group of varieties, Pannon Standard group of varieties.F and L are upper and lover values of the limits of emergence of first and last seeds, respectively.

Figure 3 .Figure 4 .
Figure 3. Nonlinear map of test plants.The size of balls is proportional to potential tolerance of test plant to Rhizoctonias.Triticum aestivum cultivars are marked with light grey, those correlated by their response to Rhizoctonia strains (r > 0.5) are linked with lines.The clusters P and S comprise varieties of Pannon Standard and Pannon Premium sortiments.The curve A marks possible pattern.

Figure 6 .
Figure 6.Influence of Rhizoctonia strains on germination of wheat seeds.The gray strips mark strains that significantly delayed the germination.F and L are the limits of emergence of first and last seeds calculated as average for 19 wheat cultivars.The skew lined area mark the interval (p = 0.05) of germination of seeds in Rhizoctonia free soil.

Figure 7 .HFigure 8 .
Figure 7. Separation of Rhizoctonia strains with Canonical Correlation Analysis.According to groups shown in Figure 3 two submatrices were edited of the data comprised in Table 1 and were related by means of CCA.Strains B-409 and B-430 (AG-4) were omitted of calculations.The size of black (A) and gray (B) balls is proportional to potential aggressivity of strains to wheat, respectively.The fitness of regression was over r = 0.97 (p < 0.001) for both function.