The activity of nitrate reductase and nitrite reductase in winter triticale cultivars of different susceptibility to cereal aphids

47The activity of nitrate reductase and nitrite reductase in winter triticale cultivars...

Pestycydy, 2007, (1-2), 47-54.ISSN 0208-8703

The activity of nitrate reductase and nitrite reductase in winter triticale cultivars of different susceptibility

to cereal aphids

Cezary SEMPRUCH, Antoni P. CIEPIELA. , Wiesława ZAWADZKA, Agnieszka FRAŃCZUK

Department of Molecular Biology and Biophysics, University of Podlasie,12 B. Prusa St., 08-110 Siedlce, Poland,

e-mail: [email protected]

Abstract: Nitrate reductase (NR) and nitrite reductase (NiR) considerably affected the rate of the absorption of soil forms of nitrogen by plants. Nitrogen, besides its great importance for plant biology, also determines their nutritive value for aphids. The NR and NiR activity was determined in fresh ears, shoots and roots of three winter triticale cultivars (x Triticosecale Wittm.) of different susceptibility to cereal aphids Sitobion avenae F. and Rhopalosiphum padi L. Statistical analyses showed significant positive correlations between the density of cereal aphid populations and NR activity in shoots and NiR activity in shoots and ears of the analyzed plants.

Keywords: nitrate reductase, nitrite reductase, winter triticale, Sitobion avenae, Rhopalosiphum padi

INTRODUCTION

Nitrate reductase (E.C. 1.6.6.1-3; NR) and nitrite reductase (E.C. 1.7.7.1; NiR) are enzymatic components of the system of the assimilative reduction of nitrates, responsible for the assimilation of inorganic forms of nitrogen by plants. This two-step process of great biological importance includes 2-electronic reduction of NO3

- to NO2- catalyzed by NR, and 6-electronic reduction of NO2

- to NH4+ by

NiR [1]. Nitrogen plays an important role in the interaction between plants and

48 C. Sempruch, A. P. Ciepiela., W. Zawadzka, A. Frańczuk

herbivores. It determines the nutritive value of host plants which increases with the increase in the quantity of nitrogenous compounds, and with the decrease in the level of defensive substances the structure of which includes carbonic chains and rings [2]. The increase in the content of nitrogenous compounds, especially low-molecular ones, accelerates the development and increases the fecundity of various aphid species [3-5]. Therefore, the aim of the present study was the qualification of the NR and NiR activities in ears, shoots with leaves, and roots of winter triticale cultivars (x Triticosecale Wittm.) of different susceptibility to grain aphid (Sitobion avenae F.), and bird cherry-oat aphid (Rhopalosiphum padi L.).

MATERIALS AND METHODS

The culture of plants, and entomological observations were carried out under natural field conditions in Agricultural Experimental Station in Zawady near Siedlce. Three winter triticale cultivars: Janko, Tornado and Witon were used in the study. Experiments were conducted in three replicates for each cultivar.

The densities of cereal aphid populations were estimated in random block arrangements, according to the method by Wratten et al. [6] and Lykouressis [7]. The observations were carried out from the moment of the aphid raid, until the population disappearance (G.S.52 – 88 in scale of Tottman and Broad [8]), in one-week intervals, applying the technique of counting the aphids on 50 randomly selected plants, diagonally across the field. The obtained results were used to calculate the dynamics and the density of the population S. avenae and R. padi.

NR and NiR were isolated from fresh ears, shoots and roots of plants void of aphids, collected at the early milk (G.S.73), and homogenized with 0.1 M buffer Tris-HCl pH 7.5, with addition of l mM EDTA, 5 mM of the cysteine, and 2.5% of casein. The obtained homogenates were filtered through two layers of blotting-cloth, and then centrifuged at 20000 x g for 15 minutes at 4 °C. Supernatants were collected as the extract to enzymatic analyses. The determination of the activity of NR and NiR was performed according to Sherrard and Dalling [9] method, and Vega et al. [10] method, respectively. The activities of enzymes were expressed in µM of nitrites produced or converted by 1 mg of enzymatic protein per 1 hour (the specific activity). The nitrite concentration was determined with the spectrophotometer UV-Vis, HP 8453 type (made by Hewlett-Packard). The content of protein in enzymatic extracts was analyzed according to Lowry et al. [11].

Differences in aphid numbers and enzyme activity in the triticale cultivars were analyzed using ANOVA followed by Duncan’s test. In addition, Person’s


correlation coefficients between density of aphid population, and the enzyme activity were also calculated.

RESULTS AND DISCUSSION

The obtained results revealed that both aphid species on triticale cultivars under study showed similar population dynamics (Figures 1 and 2). Both, S. avenae and R. padi were observed for the first time during fluorescence emergence (G.S.52). In the next developmental phases the number of aphids decreased, until the moment of maximum abundance at early milk (G.S.73). At the phase of the late milk (G.S.78), the density of cereal aphid populations decreased, until their complete disappearance at hard dough (G.S.88). The only exception was the cultivar Witon, on which R. padi population reached the maximum density at half-way anthesis (G.S.65) (Figure 2).

0

1

2

3

4

5

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7

8

G.S.52 G.S.65 G.S.71 G.S.73 G.S.78 G.S.82 G.S.88

Growth stage*

Num

ber o

f ind

ivid

uals

per

bla

de

Janko

Tornado

Witon

Figure 1. Dynamics of grain aphid population on the studied winter triticale cultivars.

*the growth stage was showed in Tottman and Brad [13] scale.


0

1

2

3

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5

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7

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G.S.52 G.S.65 G.S.71 G.S.73 G.S.78 G.S.82 G.S.88

Growth stage

Num

ber o

f ind

ivid

uals

per

bla

de

Janko

Tornado

Witon

Figure 2. Dynamics of bird cherry-oat aphid population on the studied winter triticale cultivars.

The dynamics of the S. avenae population was similar to that observed in the previous study on the winter wheat with the maximum density at the phase of the milk maturity [12]. However, the bird cherry-oat aphid reached the peak density on the winter at complete anthesis (G.S.69) [13]. According to Ruszkowska [14] over the last years, significant changes took place in the biology of R. padi resulting in different time of mass abundance, different way of the colonization of plants, different share of sexual morphs in the population, and colonization of winter crops during autumn. Therefore, it is possible that differences in the dynamics of the population of this species comparing to the earlier observations, are related to the changes in its biology.

The statistical analysis showed significant differences in the number of S. avenae and R. padi individuals and in the percentage of infested plants among the cultivars (Tables 1 and 2). Populations of both cereal aphid species attained the greatest density on Tornado cultivar, and the lowest number on Witon cultivar. These differences concern both: results calculated as average for entire vegetative season, as well as results obtained during maximum abundance of cereal aphids at early milk stage.


Table 1. The density of grain aphid population on studied winter triticale cultivars

Cultivar

Number of individuals per blade Percent of infested plants

Early milk(G.S.73)

Average invegetation

seasonEarly milk(G.S.73)

An average invegetation

seasonJanko 3.60b 0.80b 58.00b 14.86bTornado 7.06a 1.65a 72.00a 22.29aWiton 1.88c 0.54c 48.00c 15.14b

Values in columns followed by different letters are significantly different at P<0.01 (Duncan’s test).

Table 2. The density of bird cherry-oat aphid population on studied winter triticale cultivars

Cultivar

Number of individuals per blade Percent of infested plants

Early milk(G.S.73)


seasonEarly milk(G.S.73)


seasonJanko 1.62b 0.28b 16.00b 4.57bTornado 6.92a 1.98a 72.00a 24.00aWiton 0.00c 0.09c 0.00c 4.86b

The comparison of the number of individuals of studied species of cereal aphids on the selected organs of winter triticale plants proved that S. avenae preferred ears as the place of feeding, however R. padi occurred abundantly on shoots (Figure 3). The obtained data fully confirm the results of the previous study [13] which revealed that lower parts of leaves, and the stem are the main place of feeding of bird cherry-oat aphid. Only after drying out of the bottom parts of plants some individuals of this species moved on the ear. The grain aphid abundantly settled flag leaves in the first stage of the migration. After emergence, 90% populations of this species move on generative organ.

Statistically significant differences in the NR activity appeared only in vegetative parts of studied cultivars (Table 3). In the case of shoots, this enzyme was the most active in Tornado cultivar, and least active in Janko cultivar. In roots, the highest activity of NR was observed in Witon cultivar.


0

0,2

0,4

0,6

0,8

1

Num

ber o

f ind

ivid

uals

per

bla

de

Sitobion avenae Rhopalosiphum padi

Ears

Shoots

Figure 3. Density of both species of cereal aphids on selected organs of winter triticale plants.

Table 3. Activity of nitrate reductase and nitrite reductase (in µg of nitrite/mg of protein/ hour) in analyzed organs of studied winter triticale cultivars

CultivarActivity of enzymes

Nitrate reductase Nitrite reductaseEars Shoots Roots Ears Shoots Roots

Janko 0.078a 0.039c 0.071b 0.750b 1.340c 1.550bcTornado 0.061ab 0.148a 0.050b 1.570a 5.300a 2.940aWiton 0.067ab 0.062b 0.115a 0.480b 2.070b 2.280ab

The ears of Tornado showed higher NiR activity in comparison with the same organs of remaining cultivars (Table 3). Similar results were obtained for the shoots in which the enzyme showed the highest activity in Tornado cultivar and lowest in Janko triticale. No statistically significant differences in NiR activity in the roots occurred among the triticale cultivars under study.

The analysis of the relationships between the population densities of cereal aphids, and the activity of enzymes revealed positive correlations between the NR activity in shoots of all winter triticale cultivars, and the number of individuals


of S. avenae (r = 0.821, P≤0.05) and R. padi (r = 0.910, P≤0.05). Negative correlations were found between the number of grain aphid (r = -0.924, P≤0.01) and bird cherry-oat aphid (r = -0.878, P≤0.05), and NR activity in the roots. The activity of NiR in ears and shoots positively correlated with the S. avenae number (r = 0.996, P≤0.01 for both organs) and R. padi number (r = 0.999, P≤0.01 for both organs).

The analysis of the relations between the population density of cereal aphids, and the activity of analyzed enzymes showed positive correlations between the NR activity in shoots, and the number of S. avenae (r = 0.821, P≤0.05) and R. padi individuals (r = 0.910, P≤0.05). Negative correlations were found between the number of grain aphid (r = -0.924, P≤0.01) and bird cherry-oat aphid (r = -0.878, P≤0.05), and NR activity in the roots. The activity of NiR in ears and shoots positively correlated with the S. avenae number (r = 0.996, P≤0.01 for both organs) and R. padi number (r = 0.999, P≤0.01 for both organs).

Differences in the activity of both enzymes in studied cultivars show that both biocatalyzers play an important role in the interactions between winter triticale and cereal aphids species. Positive values of correlation coefficients prove that especially the rate of NR function in shoots and NiR in shoots and ears of plants is essential for these interactions. High activity of both enzymes in overground organs of plants numerously settled by aphids, undoubtedly contributes to the increase of nitrate assimilation rate, and in consequence - an increase of the level of nitrogenous compounds in plant tissues which results in an increase in nutritive value of host plants for herbivores. Similar conclusion was formulated by Throop and Lerdau [2] who stated that a decrease in nitrogen level results in a reduction of concentration of nitrogenous compound, and on the other hand, an increase in the level of defensive substances containing carbon chains and rings.

Presented results are consistent with findings of other authors. According to Strong et al. [15], the positive correlation exists between the growth and the development of cereal aphids, and the concentration of nitrogen in leaves of cereals. Soluble nitrogen plays the key role in this relationship, concentration of which depends on the content of such important nutrients as free amino acids [4].

Moreover, the results of present study showed a significant correlation between NR activity in roots of analyzed plants, and the number of cereal aphids on its overground parts. However, negative values of these coefficients indicate more complex nature of these relationships the mechanisms of which are difficult to explain, especially that literature data concerning this issue are lacking.


REFERENCES

[1] Hoff T., Stumian B.M., Hennigsen K.W., Physiol. Plant., 1992, 84, 616-624. [2] Throop H., Lerdau M., Ecosystem, 2004, 7, 109-133. [3] Brodbeck B.V., Stavisky J., Funderburg J.E., Anderson P.C., Olson S.M., Entomol.

Exp. Appl., 2001, 99, 165-172. [4] Sempruch C., Ciepiela A.P., Ann. Agric. Sci, 1999, 28, 29-35. [5] Morales H., Perfect I., Ferguson B., Agric. Ecos. Envir., 2001, 84, 145-155. [6] Wratten S.D., Lee G., Stevens D.J., Proc. Brit. Crop. Protec. Conf. - Pests and

Diseases, 1979, 1-8. [7] Lykouressis D., Z. Ang. Ent., 1984, 97, 77-84. [8] Tottman D.R., Broad H., Ann. Appl. Biol., 1987, 110, 441-454. [9] Sherrard J.H., Dalling M.J., Plant Physiol., 1979, 63, 346-353. [10] Vega I.M., Carderas I., Losada M.T., Meth. Enzymol., 1980, 69, 255-270. [11] Lowry I.O.H., Rossenbrough W.J., Farr A.L., Randal R.J., J. Biol. Chem., 1951,

57, 355-364. [12] Kąkol E., Miętkiewski R., Aphids and Other Homopterous Insects, 2001, 8,

169-173. [13] Leszczyński B., Bąkowski T., Marciniuk M., Niraz S., Zesz. Probl. Post. Nauk Rol.,

1990, 392, 21-33. [14] Ruszkowska M., Przekształcenia cyklicznej partenogenezy mszycy Rhopalosiphum

padi L. (Homoptera; Aphidoidea) - znaczenie zjawiska w adaptacji środowiskowej, Wydawnictwo Instytutu Ochrony Roślin, Poznań 2002, pp. 1-63.

[15] Strong D.R., Lawton H.J., Southwood R., Insect of plant-community pattern and mechanisms, Blackwell Sci. Publ., Oxford 1984, pp. 1-313.

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The activity of nitrate reductase and nitrite reductase in winter triticale cultivars of different susceptibility to cereal aphids