THE ARTICLE WAS PUBLISHED IN JOURNAL: J. REP. ACAD. SCI. UKR. ( JOURNAL “Dopovidi Akademii nauk Ukrainy”) 11. (2012). P. 185 – 190 (IN UKR.).

GENETIC MECHANISMS OF WHEAT AND CHICKPEA (CICER ARIETINUM L.) INHERITANCE OF RESISTANCE

TO PATHOGENIC MICROMYCETE OF FUSARIUM L. GENUS

                                                  V. А. Tsygankova 

Institute of Bioorganic Chemistry and Petrochemistry, NAS of Ukraine, KyivE-mail: vTsygankova@ukr.net

 In vivo experiments the inheritance of resistance to pathogenic micromycetes (Fusarium graminearum and Fusarium oxysporum f. ciceris) of wheat and Cicer arietinum L. plants of  the next generation, obtaining from the seeds of plants the first generation, that at ontogenesis process were processed by growth regulators Biolan, Regoplant, Stimpo on an infectious background, was investigated.  According to morpho-physiological indexes the heterosis-like effect of resistance to pathogens of experimental Cicer arietinum L. and wheat plants is set. The molecular-genetic analysis with using DOT-blot hybridization method showed the considerable difference in the percent of homology between populations of si/miRNA with immune-protective properties of experimental plants and mRNA of control plants.

 Introduction. Plant-growing of almost all world countries suffers from harmful organisms: insects and mites, pathogenic

and parasitic microorganisms (bacteria, fungi, viruses, nematodes) as well as weeds [1]. Diseases which are caused by

pathogenic micromycetes of Fusarium L. genus, including Fusarium graminearum L. (sexual stage, Gibberella zeae), damage grain production of wheat, barley, oat, corn and other cereals, especially in recent 2 decades. According to Food Agricultural organization at UN (FAO) data, the raise of damage of cereal crops is observed in different countries. For example, in the USA the yield shortage caused by this disease resulted in an economic detriment accounting 1 billion $ in 1993 and 500 million $ in 1994. Not less annual economic losses are observed in China, Canada, countries of South America, and also in Ukraine, where damage of agricultural crops by this pathogens amounts up 70-80 % [1,2, 3, 4].

Harmfulness of cereals affection by Fusarium graminearum L. consists in considerable yield loss and decline of sowing properties and nourishing grain quality. The displays of sick plant pathogenesis are physiological signs that show up in thinning and in browning plant stems (defeat at early growth stages) or in plant withering or fading (the day before or in the period of flowering). As a result of plant vessels corking by fungal mycelium and excretions by its toxic substances and ethylene the vascular system (fusarial fading) and plant tissues (rot of roots, fruit and seed) are struck. Except it the plant pathologies are dangerous for the health of man and agricultural animals due to mycotoxin accumulation in cereals and plant stems, which can not be used on a feed to the cattle for the receiving of high-quality meat, milk and products after its processing [5]. Diseases worsen the bread baker's properties of flour, and the bread baked from fusarial grain has subzero quality, toxic and dopey properties.

F. graminearum L. produces several dangerous mycotoxins, including deoxynivalenol (DON) and derivatives, zearalenone, fusarin C, and aurofusarin  [3, 5-10]. DON is a potent protein biosynthesis inhibitor affecting the digestive system and major organ function in humans and animals when ingested in sufficient quantities [6]. DON levels are regulated in food supplies of many countries; for example, the European Community limits DON levels to 0.5 µg/g  for cereals and the United States limits DON levels to 1 µg/g  for finished products for human consumption [3, 6]. The presence of F. graminearum in maize has resulted in high levels of zearalenone and other mycotoxins in the "distillers' dried grains with solids," remnants from maize - based ethanol production fed to cattle and pigs [8]. Zearalenone  increses the estrogen level in animals, including humans, and levels of this mycotoxin in food products are regulated in some countries of European Community [3, 10]. Although zearalenone is of concern to the U.S. Food and Drug Administration, there are currently no regulatory standards limiting its levels in grain [3, 6].

Another widespread pathogenic micromycete of Fusarium L. genusis Fusarium oxysporum f. ciceris that causes fusariosis of leguminous culture chickpea (Cicer arietinum L.), which is important for agriculture of Western and Middle Asia, North Africa, and North America countries [1, 11, 12].

In the Ukraine chickpea is widely cultivated in south areas: in Kherson, Odessa, Mykolaiv and on the south coast of Crimea. There are different pathological signs of disease, which causes this pathogen at plants such as: turning yellow of leaves (chlorosis), drying out and necrosis of leaves due to a defeat by fungal mycelium of vessel system and enzyme cleavage of cellular wall, that results in corking and blockade of plant transport system [11, 12]. According to FAO data, the damage of agricultural sowing of chickpea in different countries arrives at 50 %.

Large labiality of micromycetes of Fusarium L. genus to the environmental factors and out-of-control application of highly toxic fungicides of chemical origin have resulted in the increase of different varieties of Fusarium due to deterioration of the competitive activity between the different varieties of pathogens.

A problem of agricultural plant protection against fusariosis is very actual for Ukraine. At the Institute of Biorganic Chemistry and Petrochemistry NAS of Ukraine in cooperation with the National Enterprise Interdepartmental Science & Technology Center "Agrobiotech" NAS and MES of Ukraine the new ecologically safe composition polycomponent preparations with a bioprotective effect Biolan, Regoplant and Stimpo were created. They contain metabolism products in culture in vitro ofroot fungus-endophyte from ginseng Panax Ginsed M roots as well as of soil streptomycete Streptomyces avermitilis metabolites with phytostimulating, antiparasitic and antipathogenic action[1].  

Our molecular-genetic experiments [13, 14] have showed that these preparations considerably promoted plant resistance to the different pathogens due to stimulation in the plant cells synthesis of small regulatory si/miRNA, which in the process of posttranscriptional gene silencing (PTGS) either block the translation of imperfect on the structure own mRNA and complementary mRNA of pathogens or destroy them jointly with the enzymes of RISC complex (RNA-induced silencing complex) by the way of enzymatic degradation [15, 16].

The purpose of this work was the investigation of molecular-genetic mechanisms of increasing of immune-protective properties and resistance of wheat and chickpea to pathogenic micromycetes of Fusarium L. genus by means of plant growth regulators Biolan, Regoplant and Stimpo.

Materials and methods. We determined the positive or negative consequences of treatment with growth regulators Biolan, Regoplant and Stimpo of first generation wheat and chickpea plants grown on an infection background (at presence of pathogenic micromycetes Fusarium graminearum and Fusarium oxysporum Ciceriae) on growth and molecular-genetic indexes of next generation plants, which were grown without treatment with growth regulators on infection background (thus the consequences of post-action of growth regulators as immune-modulators were investigated). We used in experiments 2 different varieties of chickpea and wheat seeds that were kindly provided by O. V. Babayants, Doctor of Biological Sciences, Selection & Genetic Institute – the National Centre of Seed-growing and Variety Study of NAAS of Ukraine, Odessa.

Plant seeds were sprouted in Petri-dishes on a filter paper moistened by the distilled water on infection background. We determined:

1) the integral indexes of growth and development of seedlings;2) the molecular-genetic indexes – percent of homology of basic constituents of plant immune system - si/miRNA (isolated

from seedlings of the 2nd generation of wheat and chickpea plants grown on infection background obtained from seeds of the 1st

generation of plants treated with growth regulators and grown on an infection background, i.e. in the presence of Fusarium graminearum and Fusarium oxysporum f. ciceris) to mRNA of control seedlings of the 2nd generation of plants that were grown on infection background obtained from seeds of the 1st generation of plants grown on infection background without treatment by growth regulators. Small regulatory si/miRNAs were isolated from plants by our earlier elaborated and published method [14]. Specificity of growth regulators’ post-action was determined according to difference in the degree of homology between si/miRNA and mRNA of the control and experimental wheat and chickpea plants of different varieties.

Results and discussion. As one can see from Fig. 1 and Fig. 2  the seedlings obtained from seeds of both varieties of chickpea plants, infected by pathogenic micromycete F. oxysporum f. ciceris, and seedlings obtained from seeds of both varieties of wheat plants, infected by pathogenic micromycete F. graminearum, which were treated with growth regulators Biolan,

Regoplant and Stimpo, according to integral indexes of seed germination speed and growth, passed ahead sharply the growth and development of the control seedlings, obtained from the seeds of plants that were grown without regulators on an infectious background.   Already, starting from the 5th day to 7th day from the beginning of germination the growth indexes of the experimental seedlings exceeded almost three times indexes of control seedlings. It testifies about increasing viability of new generation of plants due to their treatment with growth regulators and about high biological potential of seeds obtained from chickpea and wheat plants, which were grown on an infectious background and treated with growth regulators, as compared to plants of first generation grown on an infectious background without treatment with growth regulators.

For greater demonstrativeness the typical sizes of control and experimental chickpea seedlings taken from Petri-dishes A and C (Fig. 2) are showed on Fig. 3.

The explanation of this difference on growth indexes between control and experimental plants can be results of molecular-biological experiments that are presented in a Table. 

The differences of morphological indexes showed in a Table testify in favor of the circumstance that seedlings obtained from the seed of first generation plants which during the vegetation stage were treated with the growth regulators Biolan, Regoplant and Stimpo on infection background (in the presence of pathogenic micromycetes F. graminearum and F. oxysporum f. ciceris) according to integral indexes of germination, growth and development have higher viability in comparison with control seedlings (obtained from seeds of infected plants of first generation which were not treated with growth regulators).

A considerable difference was found also on molecular-genetic indexes, namely in all the experimental variants the percent of homology between immune-protective si/miRNA (that was isolated from seedlings obtained from seeds of infected and treated with growth regulators plants) and mRNA (isolated from control seedlings) differs considerably. It is possible to see also varietals differences in the effects of post-action of different growth regulators (on differences in the homology level between si/miRNA and mRNA of the control and experimental wheat and chickpea plants of different varieties). Obtained difference on the above-mentioned indexes confirms circumstance that in the embryogenesis during the period of forming of plant embryos the genome reprogramming occurs by a way of switching of one adaptation genes on others in gene families of the same functional purpose,

but specific to concrete physiology conditions which are formed under action of growth stimulants. Results that were obtained in our earlier experiments [17, 18] as well as data which suggests an existence of the flexible system of plant cell genome reprogramming under action of growth regulators both on the different stages of ontogenesis [19] and during the adaptation mechanisms’ formation of plant resistance to the different pathogenic organisms [20] confirm this supposition.

Conclusions. The results of conducted experiments testify in favor of the mechanism of epigenetic inheritance of resistance to pathogenic micromycetes of Fusarium L. genus by the investigated wheat and chickpea plants of next generation obtained from seeds of first generation plants which were treated on infection background by growth regulators with bioprotective properties Biolan, Regoplant and Stimpo. We concluded that at the embryogenesis during forming of plant seeds there is observed the process of reprogramming genome of plant seed embryos through the way of “switching on” genes of the antipathogenic si/miRNA which synthesis is induced under PGRs’ action.

 

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5. Geraldo M.R., Tessmann D.J., Kemmelmerier C. Production of mycotoxins by Fusarium graminearum isolated from small cereals (wheat, triticale and barley) affected with scab disease in Southern Brazil // Brazilian J. Microbiol. – 2006. – V. 37. – P. 58 – 63.6. Mycotoxins: Risks in Plant, Animal and Human Systems, Task Force Report / Council for Agricultural Science and Technology, Ames, IA – 2003.7. Starkey D.E., Ward T.J., Aoki T., Gale L.R. et al. Global molecular surveillance reveals novel Fusarium head blight species and trichothecene toxin diversity // Fungal Genet. Biol.– 2007.– V. 44. – P. 1191–1204.8. Wu F., Munkvold G.P. Mycotoxins in ethanol co-products: modeling economic impacts on the livestock industry and management strategies // J. Agr. Food Chem. – 2008. – V. 56. – P. 3900 – 3911.9. Voigt C.A., Schafer W., Salomon S. A secreted lipase of Fusarium graminearum is a virulence factor required for infection of cereals // Plant J. – 2005. – V. 42. – P. 364 – 375.10. Utermark J., Karlovsky P.  Role of zearalenone lactonase in protection of Gliocladium roseum from fungitoxic effects of the mycotoxin zearalenone // Appl. Environ. Microbiol. – 2007. – V. 73. – P. 637 – 642.11. Couteaudier Y., Alabouvette C. Survival and inoculums’ potential of conidia and chlamydospores of Fusarium oxysporum f. sp. lini in soil// Can. J. Microbiol. – 1990. – V. 36. – P. 551 – 556.12. Leslie J.F., Summerell B.A. The Fusarium Laboratory manual / Blackwell Publishing: Iowa, USA. – 2006. – 372 p.13. Tsygankova V. A., Galkin A. P., Galkina L. O. et al. Gene expression under regulators’ stimulation of plant growth and development.  In Chapter 3 of the Monograph «New plant growth regulators: basic research and technologies of application» / Ed. Ponomarenko S. P., Iutynska H. O. – Kyiv: Nichlava, 2011. – P. 94 – 152.14. Tsygankova V.A., Andrusevich Ya.V., Blume Ya.B. Isolation from plant cells the small regulatory si/miRNA with antinematode activity // Dopovidi Academii Nauk Ukrainy. -2011. – № 9. – P. 159 - 164.15. Filipowicz W., Jaskiewicz. L., Kolb F.A. and Pillai R.S. Post-transcriptional gene silencing by siRNAs and miRNAs // Curr. Opin. Struct. Biol. – 2005. – V. 15. – P. 331–341.16. Voinnet O. Post-transcriptional RNA silencing in plant-microbe interactions: a touch of robustness and versatility // Curr. Opin. PlantBiol. - 2008. – V. 11, № 4. – P. 464 - 470.

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Fig. 1. The 7-day old seedlings of wheat plants (variety “Lastivka”):A, B – seedlings, obtained from seeds of plants, untreated growth regulators (control);C, D -   seedlings, obtained from seeds of plants, treated with Stimpo;E, F - seedlings, obtained from seeds of plants, treated with Regoplant 

 

 Fig. 2. The 7-day old seedlings of Cicer arietinum L. (variety “Triumph”):

A – seedlings, obtained from seeds of plants, untreated growth regulators (control);B - seedlings, obtained from seeds of plants, treated with Stimpo;C - seedlings, obtained from seeds of plants, treated with Regoplant;D - seedlings, obtained from seeds of plants, treated with Biolan

  

                                                                                   A                          B

 Fig. 3. Comparative sizes of 7 day old Cicer arietinum L. seedlings, obtained from seeds of plants that during the vegetation stage were treated (A) and were not treated (B) by growth regulator Regoplant on infectious background (Fusarium oxysporum f. ciceris)

Table№ Plant  varieties and growth regulators

 

 

1

Wheat plant varieties 

«Lastivka» variety (variants of experiments) «Princess Olga» variety (variants of experiments)

Control (si/mi

RNA and mRNA

from control

plants)

Treatment  with

Regoplant

Treatment 

with

Stimpo

Control (si/mi RNA

and mRNA from

control plants)

Treatment  with

Regoplant

Treatment  with

Stimpo

98 ± 1.4 82 ± 1.6* (~16%) 86 ± 1.2* (~12%) 98 ± 1.6 84 ± 1.4* (~14%) 88 ± 0.96* (~10%)

 

 

2

Chickpea plant varieties 

«Rosanna» variety (variants of experiments) «Triumph» variety (variants of experiments)

Control

(si/mi RNA and

mRNA from control

plants)

Treat-ment 

with Biolan

Treat-ment wi

th Regoplant

Treatment with

Stimpo

Control

(si/miRNA and

mRNA from

control plants)

Treat-ment w

ith Biolan

Treat-ment wit

h Rego-plant

Treatment with

Stimpo

98 ± 1.2 86 ± 1.6*

(~12%)

78 ± 1.4*

(~20%)

88 ± 0.98*

(~10%)

98 ± 1.4 82 ± 1.8*

(~16%)

80 ± 1.6*

(~18%)

76 ± 2.42* (~22%)