Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2013
Volume 16
Issue 4
Topic:
Agronomy
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Packa D. , Graban Ł. , Lajszner W. , Załuski D. , Ho¶cik M. 2013. ALPHA-AMYLASE ACTIVITY IN THE KERNELS OF HULLED SPRING WHEAT AFTER HEAD INOCULATION WITH FUSARIUM CULMORUM (W.G.SMITH) SACC., EJPAU 16(4), #06.
Available Online: http://www.ejpau.media.pl/volume16/issue4/art-06.html

ALPHA-AMYLASE ACTIVITY IN THE KERNELS OF HULLED SPRING WHEAT AFTER HEAD INOCULATION WITH FUSARIUM CULMORUM (W.G.SMITH) SACC.

Danuta Packa, Łukasz Graban, Waldemar Lajszner, Dariusz Załuski, Michał Ho¶cik
Department of Plant Breeding and Seed Production, University of Warmia and Mazury, Olsztyn, Poland

 

ABSTRACT

Alpha-amylase activity was studied in the kernels of four wheat species, including 11 accessions of T. monococcum L., 11 accessions of T. dicoccum (Schrank)Schuebl., 4 accessions of T. spelta L. and 4 cultivars of T. aestivum L., after head inoculation with a spore suspension of Fusarium culmorum. Alpha-amylase activity was determined using the Ceralpha Method. This method is recommended for measuring the activity of alpha-amylase of both plant and microbial origin. Kernels from inoculated treatments showed increased levels of alpha-amylase activity in all tested species except for T. spelta in 2008. T. aestivum was characterized by the lowest alpha-amylase activity, both in non-inoculated (control) and inoculated treatments.

Key words: Ceralpha Method, hulled wheats, Triticum spp.

INTRODUCTION

Necrotrophic pathogens of the genus Fusarium infect and colonize host plants by synthesizing extracellular hydrolytic enzymes and phytotoxic compounds [9]. Fusarium culmorum, one of the causal agents of Fusarium head blight (FHB) in north-eastern Poland, produces cellulases, xylanases and pectinases which degrade cell wall components [7, 8, 30] as well as proteases which decompose endosperm reserves [14, 18, 28]. Structural damage to the endosperm of common wheat [6], triticale [15], hulled wheat [16] and barley [20] observed under a scanning electron microscope provides further evidence of the activity of hydrolytic enzymes in cereal grain infected by Fusarium spp. The results of biochemical analyses of wheat and barley grain infected by Fusarium pathogens also suggest the activity of hydrolytic enzymes of fungal origin [1, 22]. The objective of this study was to determine alpha-amylase activity levels in mature kernels of four wheat species after head inoculation with Fusarium culmorum.

MATERIALS AND METHODS

The aim of this study was to determine the response of three hulled wheat species of the genus Triticum, T. monococcum 2n=2x=14, T. dicoccum 2n=4x=28 and T. spelta 2n=6x=42, to Fusarium culmorum (W.G. Smith) Sacc. infection, as compared with the common wheat Triticum aestivum 2n=6x=42 represented by four spring cultivars (Table 1). Two of the studied spring wheat cultivars Parabola (quality wheat, FHB-7.5° in 9° scale) and Torka (elite wheat, FHB-8° in 9° scale) are admitted to the Polish National List, while Frontana and Sumai-3 are standard cultivars used for resistance tests to FHB. The field experiment was conducted in 2007 and 2008 at the Experimental Station of the University of Warmia and Mazury in Olsztyn, located in Bałcyny near Ostróda (53°36'N, 19°51'E). Plot area was 6.0 m2, NPK fertilizers were applied at the rates of 40/25/80 kg∙ha-1, and chemical control was not applied except for herbicide treatment. In the field experiment, wheat stands at the full flowering stage (65 BBCH) were inoculated with an aqueous spore suspension of Fusarium culmorum – DON chemotype, with the use of a manual sprayer, 600 mL-1 of the suspension with a concentration of 500 000 spores per mL-1 was applied per plot each time. Inoculation was performed twice, on two consecutive days, in the evening, towards the end of June and in the first week of July. Non-inoculated plants served as control. The Fusarium culmorum isolate used for inoculation was obtained from naturally infected grain of common spring wheat grown in north-eastern Poland.

Table 1. Wheat species and accessions
Accession
Species
Accession No.
Origin*
K-1
T. monococcum L.
PL 020790
NCPGR
K-4
T. monococcum L.
PL 024068
NCPGR
K-5
T. monococcum L.
PL 020751
NCPGR
K-6
T. monococcum L.
PI 290511
NGRL
K-8
T. monococcum L.
PI 326317
NGRL
K-9
T. monococcum L.
PI 330551
NGRL
K-12
T. monococcum L.
PI 352479
NGRL
K-14
T. monococcum L.
PI 418587
NGRL
K-15
T. monococcum L.
PI 584654
NGRL
K-16
T. monococcum L.
PI 428171
NGRL
Terzino
T. monococcum L.
cultivar
Germany
K-17
Triticum spelta L.
PL 021981
NCPGR
K-18
Triticum spelta L.
TRI 17506
IPK
K-20
Triticum spelta L.
TRI 3419
IPK
K-21
Triticum spelta L.
TRI 982
IPK
K-25
Triticum dicoccum (Schrank)Schuebl.
PI 191390
NGRL
K-30
Triticum dicoccum (Schrank)Schuebl.
PI 94621
NGRL
K-33
Triticum dicoccum (Schrank)Schuebl.
PL 020761
NCPGR
K-36
Triticum dicoccum (Schrank)Schuebl.
PL 021799
NCPGR
K-38
Triticum dicoccum (Schrank)Schuebl.
PL 021984
NCPGR
K-39
Triticum dicoccum (Schrank)Schuebl.
PL 022482
NCPGR
K-40
Triticum dicoccum (Schrank)Schuebl.
PL 022863
NCPGR
K-46
Triticum dicoccum (Schrank)Schuebl.
TRI 18117
IPK
K-48
Triticum dicoccum (Schrank)Schuebl.
TRI 18219
IPK
K-49
Triticum dicoccum (Schrank)Schuebl.
TRI 2020
IPK
K-50
Triticum dicoccum (Schrank)Schuebl.
TRI 2246
IPK
Parabola
Triticum aestivum L.
cultivar
PBC
Torka
Triticum aestivum L.
cultivar
PBC
Sumai-3
Triticum aestivum L.
cultivar
PBAI
Frontana
Triticum aestivum L.
cultivar
PBAI
*NCPGR – National Centre for Plant Genetic Resources (Poland, Radzików)
*NGRL – National Germplasm Resources Laboratory (USA)
*IPK – Institut für Pflanzengenetik und Kulturpflanzenforschung (Germany)
*PBC – Plant Breeding Company (Poland)
*PBAI – Plant Breeding and Acclimatization Institute (Poland, Radzików)

Alpha-amylase activity was investigated in two growing seasons. In both years 2007 and 2008, spring wheat was sown in the second half of April, and grain was harvested in the second half of August. After harvest, control and inoculated spikes were threshed in the Wintersteiger LD 180 laboratory thresher. Grain was ground in the Cyclotec 1093 mill prior to analysis. The activity of alpha-amylase in wheat kernels was determined after harvest based on the Ceralpha Method [13] which is used to measure the activity of alpha-amylase of plant and microbial origin. Alpha-amylase activity levels were determined with the use of Megazyme reagents (Ceralpha Method, Amylase HR Assay Reagent). Alpha-amylase activity is expressed in Ceralpha Units (CU) per g of flour – one unit of activity (CU) is defined as the amount of enzyme required to release 1 mmol of p-nitrophenol (in the presence of excess a-glucosidase) per minute at 40°C. Flour with alpha-amylase activity of 0.08 CU g-1 was used as a reference standard. Alpha-amylase activity was determined in two replications. The results were processed statistically using the Statistica v. 10.0 application. Two-way ANOVA was performed for each year of the study, and the significance of differences between control and inoculated kernels of each accession line was evaluated with Student's t-test at p < 0.01 (**) and p < 0.001 (***).

RESULTS

Meteorological conditions in the studied years
Weather conditions varied between the analyzed years of 2007 and 2008. The wheat flowering and ripening season in 2007 was characterized by warm and wet weather, whereas higher temperatures and lower levels of precipitation were noted in 2008, in particular in June and July. Total precipitation during the flowering and ripening season was higher in 2007, at 318.3 mm than in 2008 when it reached 177.9 mm. In both years of the study, the greatest differences in precipitation levels were reported in July at 176.5 mm and 47.0 mm in 2007 and 2008, respectively (Fig. 1). During wheat inoculation (the end of June and the first week of July) total precipitation reached 128.3 mm in 2007 and only 27.4 mm in 2008. August precipitation levels were higher in 2008 at 103.1 mm, compared with 81.0 mm in 2007 (Fig. 1).

Fig. 1. Weather conditions during wheat flowering, ripening and harvest over two years of the study, in comparison with the long-term average of 1961–2000 (Experimental Station of UWM, in Bałcyny).

Alpha-amylase activity in wheat kernels
In both years of the study, higher levels of alpha-amylase activity were observed in kernels sampled from heads inoculated with Fusarium culmorum, except for Triticum spelta where both higher and lower activity levels of the analyzed enzyme were reported relative to control (Figs 2 and 3, Tab. 2). The analysis of variance revealed that the interaction between wheat accessions and their response to inoculation was significant at p < 0.001.

Fig. 2. Alpha-amylase activity in kernels of 30 wheat accessions in 2007 (CU g-1).

** – p < 0.01
*** – p < 0.001
ns – not significant

Fig. 3. Alpha-amylase activity in kernels of 26 wheat accessions in 2008 (CU g-1).
** – p < 0.01
*** – p < 0.001
ns – not significant

Table 2. Average alpha-amylase activity in kernels of four wheat species (CU g-1)
Wheat species
Year
Control ± SD
Inoculation ± SD
T. monococcum (2n=2x=14)
2007
0.237 ± 0.055
0.379 ± 0.160
2008
0.218 ± 0.112
0.271 ± 0.089
T. dicoccum (2n=4x=28)
2007
0.241 ± 0.190
0.321 ± 0.195
2008
0.308 ± 0.160
0.501 ± 0.198
T. spelta (2n=6x=42)
2007
0.255 ± 0.011
0.291 ± 0.048
2008
0.376 ± 0.154
0.335 ± 0.142
T. aestivum (2n=6x=42)
2007
0.189 ± 0.057
0.252 ± 0.067
2008
0.164 ± 0.058
0.219 ± 0.145

The average activity of alpha-amylase in control samples of Triticum monococcum kernels was similar in both years of the study at 0.237 CU g-1 (0.174–0.331) in 2007 and 0.218 CU g-1 (0.117–0.412) in 2008. After inoculation, alpha-amylase activity levels increased to 0.379 CU g-1 (0.225–0.702) in 2007 and 0.271 CU g-1 (0.132–0.417) in 2008 (Tab. 2). In comparison with control, the average increase in alpha-amylase activity in inoculated grain of Triticum monococcum reached 60.0% in 2007 and 24.3% in 2008. The response of the studied accessions varied between years (Figs 2 and 3).

The average activity of alpha-amylase in control samples of Triticum dicoccum kernels varied between the two years of the study, and it reached 0.241 CU g-1 (0.118–0.776) in 2007 and 0.308 CU g-1 (0.161–0.730) in 2008. After inoculation, average alpha-amylase activity levels increased to 0.321 CU g-1 (0.146–0.837) in 2007 and to 0.501 CU g-1 (0.246–0.876) in 2008 (Tab. 2). In comparison with control, alpha-amylase activity in inoculated grain of Triticum dicoccum increased by 33.2% in 2007 and by 62.7% in 2008 on average. The response of the studied accessions varied between years (Figs 2 and 3).

The average activity of alpha-amylase in control samples of Triticum spelta kernels varied between the analyzed years, and it was determined at 0.255 CU g-1 (0.243–0.267) in 2007 and 0.376 CU g-1 (0.147–0.565) in 2008. After inoculation, alpha-amylase activity levels both increased and decreased in comparison with control, reaching 0.291 CU g-1 (0.222–0.332) in 2007 and 0.335 CU g-1 (0.147–0.485) in 2008 (Tab. 2). In comparison with control, alpha-amylase activity in inoculated grain of Triticum spelta increased by 14.1% in 2007 and it decreased by 10.9% in 2008 on average. The response of the studied accessions varied between years (Figs 2 and 3).

The average activity of alpha-amylase in control samples of Triticum aestivum kernels was similar in both years of the study at 0.189 CU g-1 in 2007 and 0.164 CU g-1 in 2008. Alpha-amylase activity levels increased after inoculation to reach 0.252 CU g-1 in 2007 and 0.219 CU g-1 in 2008 on average (Tab. 2). A similar increase in alpha-amylase activity in inoculated grain of Triticum aestivum was observed in both years of the study, reaching 33.3% in 2007 and 33.5% in 2008 on average in comparison with control.

In both years of the study, the lowest levels of alpha-amylase activity were noted in both control and inoculated samples of T. aestivum (Tab. 2). In the group of control samples, the highest alpha-amylase activity was noted in T. spelta kernels in 2008, and in the group of inoculated samples – in T. dicoccum (2008) and T. monococcum (2007) kernels. The response of the analyzed accessions and cultivars (within wheat species) to inoculation differed subject to the prevalent weather conditions in each year of the study.

DISCUSSION

Starch is the main reserve material accumulated in the endosperm of grain kernels, and it accounts for nearly 70% (w/w) of kernel weight in rice, wheat, triticale, corn, barley, sorghum, oat and rye [25]. Starch granules deposited in endosperm cells are composed of two glucose polymers: amylose with a linear arrangement of glucose molecules and amylopectin with branched glucose chains. The amylose to amylopectin ratio in cereal grain ranges from 22–35% to 68–75% [3, 19]. Analyses of amylose and starch content of hulled wheat kernels point to both interspecies and intraspecies variations which could affect the rate at which starch is decomposed by endogenous and exogenous amylolytic enzymes.

Wheat grain infections caused by Fusarium culmorum reduce starch levels, but the observed decrease was insignificant in comparison with the general drop in yield. In a study by Matthäus et al. [12], the starch content of inoculated grain decreased by 1% DM (71.3% DM /70.3% DM), and the yield from experimental plots accounted for only 54% of the yield noted in control treatments (45.1 dt∙ha-1 / 83.4 dt∙ha-1). The authors attributed the minor decrease in the starch content of inoculated grain to differences in the starch content of individual kernels, which varied subject to the severity of infection. Wang et al. [27] did not observe any correlations between starch content and infection severity expressed in terms of the Fusarium protein equivalent (FPE) value. In a study investigating the starch content of fractioned batches of common wheat kernels subjected to light, moderate and severe infection, Siuda et al. [23] observed an 8% drop in the starch content of the most severely infected kernels. In our previous microscopic analyses of common wheat and triticale kernels infected by Fusarium culmorum, we observed severe damage of starch granules leading to their complete disappearance. In strongly infected kernels, the endosperm was replaced by extensive mycelium [6, 15]. Damaged starch granules were also found in hulled wheat kernels infected by Fusarium culmorum [16].

In cereal kernels, alpha-amylase is an enzyme which decomposes starch and makes sugar available for germ development during sprouting. The activity of alpha-amylase is low in mature wheat grain, but it can be intensified by way of selected mechanisms, including pre-harvest sprouting (PHS) and PHS-independent synthesis of alpha-amylase in a maturing kernel, referred to as late maturity alpha-amylase (LMA) [10, 11]. Both mechanisms (PHS and LMA) are complex quantitative traits which are strongly modified by environmental factors before and during harvest. The variations in alpha-amylase activity in the control grain of the four analyzed wheat species are a product of their genetic traits and modifying environmental impacts. The average levels of alpha-amylase activity in T. monococcum kernels were similar to those noted in wheat cultivated in Italy [2], whereas the results reported for spring spelt correspond to the values noted in winter spelt [31]. The activity of alpha-amylase in five winter spelt cultivars was significantly lower than in common winter wheat [4]. Due to a general scarcity of data regarding hulled wheat, our results cannot be fully compared with the findings of other authors.

Infections of common wheat kernels caused by fungi of the genus Fusarium are generally accompanied by enhanced activity of proteolytic and amylolytic enzymes [12, 17, 26, 27, 28, 29], but the presented results concern individual cultivars of common wheat. A similar phenomenon was observed by Schwarz et al. [21] in barley grain infected by F. graminearum and F. poae. Necrotrophic pathogens of the genus Fusarium use hydrolytic enzymes to effectively colonize kernels and acquire nitrogen and carbon from endosperm reserves. According to Wang et al. [27], alpha-amylase in Fusarium culmorum remains active in a wide range of temperatures, and the resulting damage to starch granules may adversely influence flour performance and bread quality.

Head inoculation stimulated alpha-amylase activity in kernels of the analyzed wheat species. In 2007, the highest increase in alpha-amylase activity was noted in T. monococcum (60.0%), followed by T. aestivum and T. dicoccum (33.3 and 33.2%), whereas the lowest increase was reported in T. spelta (14.1%). In 2008, alpha-amylase activity increased most significantly in T. dicoccum (62.7%), followed by T. aestivum and T. monococcum (33.5 and 24.3%), whereas in T. spelta, the activity of the studied enzyme decreased by 10.9% in comparison with control. The research of Hidalgo et al. [5] confirming the view that alpha-amylase activity is strongly influenced by the environmental conditions.

The response of T. spelta and T. dicoccum accessions, manifested by a minor increase or decrease in alpha-amylase activity in inoculated grain, could be attributed to the activity of exogenous/endogenous amylase inhibitors in the endosperm which control alpha-amylase activity and participate in defence against pathogens and pests [24]. Alpha-amylase inhibitors may be activated in response to infections caused by pathogens which synthesize extracellular hydrolytic enzymes. The above mechanism could be part of the active resistance of plants to necrotrophic pathogens, but further interdisciplinary studies are needed to validate this hypothesis.

CONCLUSIONS

  1. The inoculation of wheat spikes with an aqueous suspension of F. culmorum spores under field conditions increased alpha-amylase activity in Triticum monococcum, T. dicoccum and T. aestivum kernels in both years of the experiment. The only exception was T. spelta which was characterized by both an increase and a decrease in alpha-amylase activity relative to the control treatment (non-inoculated spikes).
  2. Environmental factors modified alpha-amylase activity in the kernels of the analyzed wheat species.
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Accepted for print: 13.12.2013
Danuta Packa
Department of Plant Breeding and Seed Production,
University of Warmia and Mazury, Olsztyn, Poland
tel. 89 524 53 29
pl. Łodzki 3, 10-724 Olsztyn, Poland
email: packa@uwm.edu.pl

Łukasz Graban
Department of Plant Breeding and Seed Production,
University of Warmia and Mazury, Olsztyn, Poland
pl. Łodzki 3, 10-724 Olsztyn, Poland

Waldemar Lajszner
Department of Plant Breeding and Seed Production,
University of Warmia and Mazury, Olsztyn, Poland
pl. Łodzki 3, 10-724 Olsztyn, Poland

Dariusz Załuski
Department of Plant Breeding and Seed Production,
University of Warmia and Mazury, Olsztyn, Poland
pl. Łodzki 3, 10-724 Olsztyn, Poland
email: dariusz.zaluski@uwm.edu.pl

Michał Ho¶cik
Department of Plant Breeding and Seed Production,
University of Warmia and Mazury, Olsztyn, Poland
pl. Łodzki 3, 10-724 Olsztyn, Poland

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