THE USE OF BIOTECHNOLOGICAL METHODS FOR SELECTION OF SUGAR BEET BIOTYPES RESISTANT TO FUSARIUM SP.

Využití biotechnologických metod pro selekci biotypů cukrovky, odolných proti Fusarium sp.

V. Redko, Ŕ. Nurmukhammedov, Ň. Nedyak, Ŕ. Syumka

The Institute of Sugar Beet Research UAAS, Ukraine

Summary: The article suggests methods of in vitro evaluation and selection of sugar beet breeding materials resistant to the fungi of Fusarium Lk:Fr. genus (F. culmorum, F. oxysporum, F. solani, F. gibbosum) with the help of double cultures and selective media. A significant correlative dependence (r = 0,76-0,91) between resistance of the materials in vitro and in vivo was established. The resistance reaction of sugar beet was shown to depend on both pathogen species and genotype of the host plant which is a donor of explants.

Key words: sugar beet, Fusarium Lk:Fr., double culture, selective medium

Souhrn: Článek uvádí metody “in vitro” hodnocení a selekce šlechtitelských materiálů cukrovky resistentních k houbám Fusarium Lk:Fr. rod (F. culmorum, F. oxysporum, F. solani, F. gibbosum) pomocí zdvojených kultur a selektivního media. Významná korelační závislost (r = 0,76 - 0,91) byla zjištěna u rezistence mezi materiály “in vitro” a “in vivo”. Ukázalo se, že reakce v odolnosti cukrovky závisí jak na druhu patogenu, tak na genotypu hostitelské rostliny, z níž byly odebrány explantáty.

Klíčová slova: cukrovka, , Fusarium Lk:Fr., zdvojené kultury, selektivní medium

Introduction

Fungi of the Fusarium genus belong to the most wide-spread causative agents of black led and root rots - they amount to 50 % among black led causative agents and the number of plants affected by Fusarium root rots caused by the above mentioned fungi amounts sometimes to 15-20 % [6, 9].

The use of resistant varieties and hybrids is the most perspective and reliable method of control of Fusarium sp. In this connection, the use of biotechnological methods can considerably increase the efficiency of selection owing to uniformity and specificity of infectious load and can shorten the time needed for obtaining a resistant initial breeding material [1, 4, 5].

The article gives the result of development of express-methods of evaluation and selection of sugar beet breeding materials resistant to fungi of Fusarium genus.

Material and methods

Ten genotypes of beet of different origins were introduced in a sterile culture. The genotypes were propagated through in vitro cloning in a modified nutrient medium of Gamborg and Eveleigh B5 (C200) [2, 3, 7]. Explants of petioles and leaf blades of these plants were used for obtaining calluses. For induction of callus-genesis, 4 variants of nutrient media were used on the MS basis with different concentrations of 2,4-D acid and BAP. Culturing was carried out under conditions of: temperature 24± 2oC, lightning 2 klux, 16 hour photoperiod and 70% relative air humidity. Then, 5-7 mm size portions of the young callus with active growth were transferred to Petri dishes and cultured there for 3-4 weeks [8].

Isolated buds of sterile plants were transferred to selective media prepared on the C200 basis.

Pure cultures of the fungi of Fusarium Lk:Fr. genus (Fusarium culmorum (Sm.) Sacc., Fusarium oxysporum Schlecht. emend. Snyd. et Hans., Fusarium solani (Mart.) App. et Wr., Fusarium gibbosum App. et Wr. emend. Bilai) were kindly given to us from the collection of the Institute of Microbiology and Virology of NAS of Ukraine.

The evaluation of resistance of the breeding materials was carried out with the two following methods:

1. According to the linear growth of a fungus hypha on callus surfaces (double culture). For this purpose, calluses, when they reached the diameter of 2 cm, were inoculated with pure cultures of the fungi (F. culmorum, F. oxysporum, F. solani). A small portion of the inoculum of 1 mm2 in size was placed on the callus surface and the infected calluses were incubated in the dark with 22oC. Calculations of infection were carried out on the 3rd day of co-incubation [8].

2. According to the bud reaction to a selective factor being in this case the extract from the pure cultures of Fusarium genus fungi. For this purpose, the species of the fungi studied cultivated in stationary mattrasses for 15 days at 26oC on Chapek medium with the addition of beet broth (50 ml/l). Chloroform extraction from the fungi was carried out twice for 15 minutes each time in the proportion 1:5. The extract was evaporated and dried at 60oC. Calculations of beet bud growth on selective media with different concentrations of the extract were carried out on the 15th and 25th days. The survived buds were transplanted to a standard medium C200 and then, after 25 days, their ability to restore their growth processes and bud formation was evaluated.

Rooting of the materials selected for their resistance was carried out on C203 - a modified Gamborg and Eveleigh B5 medium. The plants obtained were transplanted to perlit and later on to soil, where they grew for 2 months. After this, the material was evaluated on an artificial infectious background.

Results

By using a double culture method, we investigated resistance of 10 callus lines of beet of different origins. According to the data obtained, the differentiation of the material shown by the index of resistance to the pathogen appeared to be insignificant (Fig. 1). Some resistance to all the Fusarium species studied was shown by the K 1/4 and K 1/10 lines. Besides this, it was found that the reaction of one and the same callus material to different species of Fusarium was not identical. Thus, in K 1/10 number, the greatest growth on the callus surface was found with F. culmorum (11.8 mm) and the poorest growth - with F. solani (5.4 mm) while the growth of F. oxysporum was 9.8 mm.

Investigations on selective media have shown that a F. culmorum extract has a relatively high phytotoxic action. When applying this species for studying beet resistance, we failed to obtain any living plants after their transplanting to C200 (Table 1). The most suitable for development of selective media to obtain beet cultures resistant to fungi extracts appeared to be F. oxysporum. An extract, obtained from this species has a relatively high toxicity for beet meristems and makes it possible to clearly differentiate any breeding material according to its resistance. It should be noted that callus lines and buds of sterile plants of the Ivanivskiy MS 33 hybrid and of the selection numbers K XXIII/1 and K 1/6 have displayed higher resistance to Fusarium sp. being evaluated by both double culture method and selective medium method.

The rooted plants of the six lines which displayed higher resistance when being evaluated by biotechnological methods were transplanted on the infectious background of F. oxysporum in the glasshouse. The selection numbers K1/4 and K1/10 appeared to be the best ones which corresponded to the results of in vitro evaluation.

It has been established that the correlation coefficient between the evaluation of sugar beet resistance by the double culture method and selective medium method amounts to +0.91+++ and the correspondence of these two methods with the results of evaluating on the infectious background was +0.76++ and 0.82++, respectively.

Conclusions

The investigations carried out have made it possible for us to develop and to propose two schemes of evaluation and selection of sugar beet breeding materials, resistant to Fusarium sp. genus fungi by using biotechnological methods: a double culture of sugar beet calluses and fungi; and a selective medium containing extracts of Fusarium sp. genus fungi. The use of these schemes allows to increase the efficiency of selection of resistant biotypes and to considerably accelerate the breeding process.

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Adresa autora

Table 1. Reaction of beet buds to a selective medium with a certain addition of Fusarium sp. extracts and the restoration of growth processes on the control medium (Ń200)

Designations used:

I - selective medium - 15 days of growth;

II - selective medium - 25 days of growth;

III - Ń 200 medium - 25 days of growth;

+++ - growth and development correspond to the control;

++ - growth and development have become slower;

- - the bud parished