Response of Arabidopsis Clones to Toxic Compounds Released by Various Rhizoctonia Species

Response of 3 Arabidopsis clones to 41 strains of eight Rhizoctonia species was studied in model experiments. The seed germination was decelerated in most of the cases, although the inhibitory effect varied within large limits. The pre-emergence damping off and root neck rot leading to damping off were the most frequent symptoms of disease syndrome caused by toxic metabolites. The clone transformed with cDNA clone overexpressing gstf4 gene exhibited significantly improved tolerance as compared to parental one, meanwhile the sensitivity of Dmannose pyrophosphorylase/mannose-1-pyrophosphatase deficient clone dramatically increased. Strains of R. solani of AG-2, AG-4 and AG-7 and Athelia rolfsii produced the most toxic metabolites, however, no strict relationships were revealed between taxonomic position of Rhizoctonia strains and toxicity of their metabolites.


Introduction
Damping off caused by various soil-borne pathogenic microbes is a well-known phenomenon in plant cultivation.The traditional control measures (seed dressing, soil fumigation) cannot be applied in large scale due to high risk of irreversible environmental pollution and possible toxic residuals in food and forage.Moreover, the soilborne fungi (mainly Fusarium spp, Pythium, Rhizoctonia) can attack several hundred cultivated plant species during all vegetation period that makes impossible the long lasting protection of plants with selectively acting synthetic compounds or antibiotic preparations.Recently, none of the marketed fungicides is translocated basipetally, which counteracts their use after development of foliage against root parasiting fungi.Although, Rhizoctonia species are not among the top ten pathogens [1], economic importance of their control is increasing from severe to catastrophic yield losses reported from main wheat cultivating areas [2][3][4].Changes in agricultural practices of cereals with special regard to minimum or no tillage cultivation [5,6] led to increased importance of Rhizoctonia infections [7,8].The most plausible method is the breeding of tolerant varieties.
The Rhizoctonia species are abundant in soils as mutualistic members of microbial consortium associated with plants.Their relationship may change from symbiosis (as in various orchids) to parasitism.The stunted growth can be most frequently observed, which might be related to the effect of Rhizoctonia toxins although few evident experimental proof has been demonstrated yet [9].Presence of parasitic hyphae can also be commonly seen even in symptomless tissues, and in normal conditions does not cause visible disease symptoms either in phyllosphere or in roots.However, when environmental stress factors overwhelm the homeostatic regulation of plant, the disease syndrome may develop at various degrees.Both biotic and abiotic stress manifests as an oxidative stress [10], thus by scavenging free radicals an active antioxidative defense system comprising enzymatic and non-enzymatic antioxidants reduces the level of oxidative stress in plant cells and counteracts evolution of disease.Regulation of substrate pools such as glutathione or ascorbic acid is the key element of cell protection [11], and the capacity of the biochemical path sustaining the oxido-reductive potential of cells has crucial importance in elimination of harmful free radicals [12] and confers in resistance to pathogens [13,14].The high level of ascorbic acid reduces the disease severity [15], while the deficiency increases the sensitivity to environmental stress factors [12].Glutathione S-transferases play an important role in adaptation of various organisms to adverse factors [16][17][18], and in detoxification of electrophilic compounds either exogenic or of natural origin [19,20].The formation of glutathione adducts catalyzed by GST is usually the first step of deterioration [21].The level of GST increased in okra [22] and ricinus [23] seedlings damaged by soil-borne Rhizoctonia, suggesting activation of glutathione conjugation system in defense mechanisms counteracting invasion and eliminating grave consequences of the infection.The deteriorative capacity of plant and associated microbes is related to the tolerance of host plant to the pathogen.Thus, the selection of tolerant plants to these pathogenic fungi is important for quality food production as well.
There is an urgent need in sustainable management strategy to combat damages induced by soil habiting Rhizoctonia complex.No major resistance genes to this pathogen have been identified so far in spite of increasing efforts in studies of physiology and genetics of Rhizoctonia/host interaction [8].Currently, the genetic engineering is widely used in breeding technologies providing genetically modified (GM) plants with a very high resistance against abiotic and biotic factors.Some plants have been objected for increasing tolerance to unfavorable environmental conditions [24] and remediative capacity [25] by gene engineering that can be a determinative factor for producing healthy food [26].
The genomes and physiology of Arabidopsis thaliana are broadly studied, and numerous mutans or transgenic clones are described in literature.The Arabidopsis model system greatly help us to understand specific genetic and biochemical processes.The aim of this work was to get information on the effect of toxic metabolites of Rhizoctonia species complex on seed germination of Arabidopsis.
The scoring of disease incidence and severity meet difficulties in the case of Rhizoctonias as some symptoms of disease syndrome can be induced by toxic metabolites of Rhizoctonia alone [9].In agar cultures, the above two effects can be separated during first days of germination, however, later the damage caused by parasiting hyphae secondary infections of germlings caused by various microbes of spermosphere can mask the visual effect of Rhizoctonia toxins even in agar cultures.To avoid the altering effects, the observations were limited up to five days, as the aim of this work was the evaluation of sensitivity of Arabidopsis to toxic metabolites of Rhizoctonia released to the medium.

Materials and Methods
Response of three A. thaliana clones to metabolites of 41 Rhizoctonia strains five genera (Athelia, Ceratobasidium, Ceratorhiza, Thanatephorus and Waitea) was compared in model experiment applying vertical agar diffusion technique.The seeds were surface sterilized for 2 min in 70% alcohol followed by 5 min in commercial bleach (0.5% hypochlorite) then washed five times in sterile water and diluted in sterile water slightly solidified with 0.1% agar before stratification for 48 hours at 4˚C in the dark.
vtc1 ecotype is deficient in the function of GDP-mannose pyrophosphorylase/mannose-1-pyrophosphatase.This enzyme provides GDP-mannose, which is used for cell wall carbohydrate biosynthesis, protein glycosylation and for ascorbate (vitamin C) biosynthesis.Total acorbic acid content in vtc1 clone is about 40% compared to wild type.Earlier work revealed that vtc1 mutant has increased resistance against Pseudomonas syringae or Peronospora parasitica [30] but more sensitive to Alternaria brassicicola [15].

Test Fungi
Rhizoctonia strains were originated of different locations and various hosts:  The strains were maintained on potato dextrose agar (Merck, Darmstadt, Germany) amended with 2 g soya peptone L44, 0.5 g Trypton T L43 (Oxoid, Basingstoke, sUK) and 1 g yeast extract L21 (Oxoid).

Data Analysis
Fisher's test was applied to evaluate significance of differences between variant at p = 0.05 level.The results of counting of germinated/dormant and healthy/diseased individuals were calculated as percentages.The half time of germination (hours requested for germination of the 50% of seeds) was determined by linear regression analysis, where the percent values were transformed into probits.Similarity of Arabidopsis clones and relationships between parameters of assessment were investigated by Canonic Correlation Analysis (CCA).

Germination of Seeds
The number of dropped seeds varied between 25 ± 6 and they disseminated in a spot with diameter between 10 -14 mm on agar plate.On control plates 96% -100% of them have germinated indicating the good quality of seed material.Seeds of each clone germinated synchronously within 48 hours.The half time of germination approached by linear regression varied within −12 and 18 hours, where the 48 hours of stratification should be taken into consideration for negative value.Predominant majority of Rhizoctonia strains did not break through the agar layer in first five days of incubation, thus most of seeds in each variant could germinate and develop cotyledons until meeting with hyphae.
The hyphae of strains started to colonize the surface after six days of incubation, but their attack cannot be accurately evaluated due to airborne contaminations and bacteria arising of spermosphere.The first visual symptom of toxicose was the formation of brown spot on root-neck of the germlings even before full opening of cotyledons that was followed with damping off within 24 hours.The killed seeds became dark brown.Seedlings that survived the effect of toxic metabolites were subsequently destroyed by fungal attack rapidly in the case of most aggressive strains (B-151, B-245, B-420, B-427 and B-557), while in the case of other strains the variation in number of survivors within series was too high for correct assessment of pathogenicity, thus the data analysis was limited to 5 days of incubation.In some cases germination started after 24 -72 hours lag phase (Table 1).

Toxic Effect of Metabolites
Response of germinating seeds was evaluated in two aspect.First the lethal effect, i.e., the ratio of non germinated seeds was calculated of recorded data at 5th days of incubation for each strain (Table 1).The reaction to metabolites of Rhizoctonias varied within large limit and strain dependents manner.The toxicity of AG-3 (B-446 and B-433) and AG-4 (B-417 and B-430) strain pairs proved to be significantly different.Similarly, great differences were revealed between strains isolated of the same host, the toxicity of potato originated ones varied between 4% and 100%.The genetic manipulation altered significantly the response Arabidospsis clones to Rhizoctonia metabolites (Table 2).The clone A-193 with improved capacity of glutathione conjugation system (GCS) tolerated at higher level the toxins than both parental (A-195) and vitamin C deficient mutant (vtc1), while the latter slightly got behind of A-195.
The other aspect of evaluation of toxic effects was the delay of germination.Toxins of ten strains, except A. rolfsii (B-442) all of them Thanatephorus anamorphs, killed the seeds before seed coat rupture.The introduction of gstf4 gene (A-193) counteracted to this effect in three cases (Table 2), that means the overexpression of GST increased the vitality of Arabidopsis.In some cases the germination started after 24 -72 hours of lag phase (Table 1).In this case, the overexpression of GST unlocked the inhibitory effect as well.The lag phase of germination poorly correlated with lethal effect (Canonic R 2 = 0.389, χ 2 = 13.75, p = 0.13), that means the retardation of emergence is not strictly connected to response of seedlings to toxic effect in subsequent stages of germination (Table 3).The half time of germination (Table 4) varied within large limits (13 -150 hr), nevertheless, this parameter could be calculated surprisingly high precision (the regression could be fitted in p < 0.05 in all cases) that underlines again the good quality of seed material as well as indicates the reproducibility of toxin production following the method applied in our experiments.
The presence gstf4 gene (A-193) accelerated the germination at about 20 hr as related to parental and vitamin C deficient clones, although, this protective effect varied in Rhizoctonia strain dependent manner.
The CCA revealed that the effect on half time of germination correlated slightly better to lethal effect on seedlings than to lag phase (Table 3).Plotting the strains as canonic scores (plots are not shown) no grouping was revealed in the case of HT vs LE and HT vs LP.However, two groups was formed on plot LE vs LP (Table 4).The major one (Group A, 22 strains) did not show any correlation, but in the other one (Group B, 19 strains) the correlation between lag phase and lethal effect was significant (R 2 = 0.75).The difference between this two  The sub-matrix up to diagonal (A) shows χ 2 values, while the sub-matrix down the diagonal (B) Canonic R 2 of the first root.
groups is in the selective effect of their toxins to Arabidopsis clones.The parental clone (A-195) exhibited slightly higher sensitivity to B group than to A (57% and 50%, resp.), while the genetically altered A-193 and vtc1 clones responded exhibited higher sensitivity to A group (48% and 59%, resp.)than to B group (41% and 54%, resp.).No significant relationships is between selective toxicity of taxonomic position, source and origin of strains could be revealed.
The seedlings of A-193 clone differed at higher extent than vtc1 one of parental A-195 in tolerance to Rhizoctonia toxins, but the response of germinating seeds of vtc1 clone altered at higher extent of parental A-195 that those of A-193 (Table 5).The Person's coefficient between responses of A-193 and vtc1 as evaluated by half time of germination was lower than in scoring of lethal effect (0.553 < 0.873) that indicates changes in sensitivity spectrum of clones during the seed germination.

Discussion
Rhizoctonia species are well known soil borne pathogens, which are habiting mainly in rhizosphere, however, they may survive as saprobionts in the upper layer of the soil  The sub-matrix up to diagonal (A) shows similarity (Pearson's coeffients) of clones as evaluated on the base of incidence of diseased individuals (Table 1), while the sub-matrix down the diagonal (B) on the base of delay of germination (Table 3).
forming a mycelial web, thus the undisturbed soil enhance the risk of the infection of young roots.The disease syndrome may evolve rapidly to a fatal consequence in formerly symptomless host (damping off and wilting).
Our data support the assumption of crucial role of mycotoxins in evolution of disease syndrome [9,22].Like to series of host plants [8,10,31] the strain dependent response of A. thaliana clones to Rhizoctonia was demonstrated in our experiments as well.The strains examined highly diverged in toxic properties.The poor correlation between reaction of Arabidopsis seedlings in subsequent steps indicates the qualitative differences in composition of toxic substances released into the medium.Further research requested for both analysis of this complex and elucidation of toxic properties of each component.The fact of increased tolerance of transgenic clone bearing overexpressed alien GST and increased sensitivity of ascorbic acid deficient clone evidently support the importance of elimination of free radicals in response of plants to pathogen attack.

Conclusions
Rhizoctonia strains of various taxonomic position release medium metabolites toxic to Arabidopsis thaliana.Neither taxonomic position nor origin of strains was related to inhibitory effect of their toxins.
The genetic manipulation of A. thaliana significantly altered the response of germinating seeds to Rhizoctonia toxins.Contrary to ascorbic acid deficiency the overexpression of alien glutathione-S-transferase significantly improved the tolerance of seeds.

Table 2 . Response of germinating seeds of Arabidopsis to toxic metabolites of Rhizoctonia strains.
Time requested to outcrop the first seed; 2 Control like germination;3All seeds became dark brown up to 6th day of incubation = 100 percent of inhibition;4Average inhibition of germination recorded at 6th day.