Maydica 54 (2009): 263-267
RESISTANCE OF FLINT AND DENT MAIZE FORMS FOR COLONIZATION BY FUSARIUM SPP. AND MYCOTOXINS CONTAMINATION E. Czembor1,*, P. Ochodzki2 1
Grasses and Legumes Department, Plant Breeding and Acclimatization Institute, Radzikow, 05-870 Blonie, Poland 2 Plant Pathology Department, Plant Breeding and Acclimatization Institute, Radzikow, 05-870 Blonie, Poland
Received June 15, 2009
ABSTRACT - This paper considers the aspect of ear rot caused by Fusarium graminearuum and/or by F. verticillioides in relation to flint and dent forms of maize. Flint and dent inbred lines were inoculated with F. graminearum and F. verticillioides. Significant differences in resistance level and toxin content were determined in all treatments: after silk-channel inoculation with F. graminearum and after tooth-pick inoculation with F. graminearum and F. verticillioides. The flint forms were more susceptible to F. verticillioides after tooth-pick inoculation than dent forms. The relationship between phenotype score of disease severity and deoxynivalenol (DON) and fumonisins - FUM (FB1, FB2 and FB3) was determined. The flint kernels have been more resistant than the dent ones to pre or- postharvest attack by Fusarium spp. KEY WORDS: Maize; Flint; Dent; Fusarium; Resistance.
INTRODUCTION In maize, one of the most important diseases of maize is produced by Fusarium spp. including Giberella ear rot (“red ear rot”) caused by F. graminearum and Fusarium ear rot (“pink ear rot”) caused by Gibberella fujikuroi complex such as F. verticillioides, F. proliferatum and F. subglutinans. Infection by Fusarium spp. results not only in yield reduction but also in contamination of mycotoxins such as deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEA) which are produced by F. graminearum and fumonisins (FUM), moniliformin (MON) and beauvericin produced by F. verticillioides (MOLTO et al., 1997; MIEDANER, 2004; SUTTON, 1982; MAGG et al., 2002, 2003). Because mycotoxins are resistant to high temperatures and chemicals, they can be accumulated in grains and heavily contaminate grain-based food and feed that may result * For correspondence (e.mail:
[email protected]).
in various diseases after ingestion in animals and humans (CHISTENSEN et al., 1986). In recent years the EU set maximum levels for DON, FUM and ZEA (1.750, 4.000 and 350 μg kg-1, respectively) in unprocessed maize for human consumption [Commission regulation (EC) No 1126/2007]. For animal feeding the limits were set between 2.000 and 8.000 μg kg-1 for DON and FUM and 250-500 μg kg-1 for ZEA depending on the feedingstuff and an animal. Two types of resistance to ear rot were identified in maize (REID et al., 1992; CHUNGU et al., 1996; REID et al., 1996a). Silk channel resistance prevents the fungus invading through silk channel down to the kernels, while kernel resistance blocks the spread of the fungus from kernel to kernel. The most effective method of control is to use resistant hybrids. The resistance to Fusarium spp. is quantitatively inherited, but the fully resistant maize genotype is not still known. The relationships between resistance and mycotoxin contamination were reported by a number of reports (GENDLOFF et al., 1986; REID et al., 1996a; CLEMENTS et al., 2004; ROBERTSON-HOYT et al., 2006). Additionally, the incidence and severity of the diseases is strongly influenced by environmental factors, such as temperature and humidity (REF). The aim of this paper was to find differences between flint and dent maize forms in resistance to F. graminearum and F. verticillioides and for tolerance to DON and FUM accumulation after silk-channel and toothpick inoculation. Additionally the correlation between ear rot rating and mycotoxin concentration was established. MATERIALS AND METHODS Field experiment For the field experiment the RCBD (randomized complete block design) model was used. About 25 plants of flint and dent form of maize inbred lines (Tab. 1) were grown in one row in two replications under field conditions.
264
E. CZEMBOR, P. OCHODZKI
TABLE 1 - Description of tested inbreed lines. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Kernel type FAO Origin Ears, kernel and cob description ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– flint 210 - 220 developed from the crosses long cylindrical ears with 14 kernels FF7*GK72-54 (F2, EP1 related) rows, yellow kernels and white cob ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– dent 230 developed from CGP (Canadian Gene conical ears with 14-16 kernel rows, Pool) with Iowa Dent background yellow kernels and red cob ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– dent 290 developed from an experimental hybrid P3306 yellow kernels and red cob of Iowa Dent/Stiff Stalk Synthetic background long, cylindrical ears with 16 kernels rows ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– dent 240 developed from the cross VID*S41791 long cylindrical ears with 14 kernel of Iowa Dent/Stiff Stalk Synthetic background rows; yellow kernels and white cob –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Primary ears of 84 plants were silk-channel or tooth-pick inoculated following the method of REID et al. (1996b) with F. graminearum and F. verticillioides. With F. graminearum were inoculated 28 plants using silk-channel method and 28 plants using tooth-pick method. With F. verticillioides 28 plants were inoculated using tooth-pick method. As a control, 28 plants of each inbreed line which grown in 2 locations with natural infection were used. For silk-channel inoculation 1 ml inoculum per ear in 1 x 105 spore concentration was used. For tooth-pick inoculation with 1 ml inoculum per ear in 1 x 106 spore concentration was used. Inoculation was done 5-6 or 8-9 days after each plant female flowering (end of R1 stage and beginning of R2 stage) using silkchannel or tooth-pick inoculation, respectively. The ears were
dehusked at normal grain harvest and immediately afterwards the surface of ear covered by mycelium was rated in according to 1 - 7 scale. Ears were hand-picked, and air dried to approximately 15% grain moisture. Mycotoxin analysis Deoxynivalenol (DON) analysis - 5 g of ground maize kernels was extracted with 25 ml of aqueous acetonitrile (84:16 v/v) solution overnight on orbital shaker and centrifuged 3500 rpm, 5 min. 6 ml of supernatant was purified on Multisep 227 Trich + multifunctional column (Romer Labs) acc. to producer’s manual. 4 ml of purified extrat was evaporated to dryness, and residues were redissolved in 500 µl of mobile phase. 20 µl of the sample was injected onto Pinnacle II, C18, 5um HPLC column, 150 x 4,6
FB1
FB2
FIGURE 1 - Chromatograms of HPLC analysis of fumonisins (for details see text above).
MYCOTOXINS CONTAMINATION
265
FIGURE 2 - The weather conditions in experimental field at Radzikow.
TABLE 2 - Mean squares from analysis of variance of ear rot disease scores and mycotoxin content in corn samples. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Fusarium verticillioides Fusarium graminearum Source –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– ear rot FB1 FB2 FB3 FUM ear rot DON ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– treatment 7.70*** 1033.56*** 164.37*** 15.08*** 2386.63*** 39.43*** 5271.87*** kernel type 2.77* 512.86** 76.48** 11.92*** 1214.16** 1.53 982.26 treatment x kernel type 0.36 333.47** 45.39** 6.86*** 762.78** 1.37 1581.53* error 0.28 43.07 8.30 0.48 101.82 0.85 531.49 ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– *, **: Significant at 0.05, 0.01 and 0.001 probability level, respectively.
mm with apropriate guard column. The used mobile phase consisted of acetonitrile:water 8:92 v/v, at flow rate 1 ml/min. DON was detectd by UV detector at wavelength 236 nm. Fumonisins analysis - Ground maize kernels (5 g) were extracted overnight with methanol:water (3:1) solution (25 ml) on orbital shaker. Subsequently pH of an aliquot was adjusted to 6.2, applied to conditioned SAX column, washed with 5 ml of methanol:water (3:1) and 3 ml of methanol and finally eluted with 12 ml of 1% acetic acid in methanol and evaporated to dryness. Prior separation on HPLC column, sample were redissolved in 0.5 ml of MeOH and derivatized with OPA solution. FB1 was eluted with mobile phase methanol: 0,1 M phosphate buffer (77:23), pH 3.35 at a flow rate 1 ml*min-1 and detected with fluorimetric detector. Fluorescence of the OPAderivatives was recorded at excitation and emission wavelengths 335 nm and 440 nm, respectively. FB1 was identified and quantified by comparison of retention time and peak area with reference standard solution (Fig.1). Blotter test Under laboratory conditions two hundred seeds per replication were evaluated by the blotter test, according ISTA rules. One hundred seed were tested without inocaltion, the another one hundred seeds were inoculated with F. graminearum and F. verticilioides. The percent of infected seeds by Fusarium spp. was
determined in the treatment with non inoculated samples and aboundance of mycelium which was growing on seeds was scored in the treatment with inoculation. Weather conditions were obtained from meteo station at Radzikow (Fig. 2).
RESULTS AND DISCUSSION All inoculations resulted in visible disease symptoms. After inoculation with F. graminearum the differences in the disease severity and DON contamination between treatments were found. However, differences between dent and flint forms were statistically not significant (Tab. 2). After silk-channel inoculation in the end of R1 silking stage the disease severity was lower than after tooth-pick method inoculation in the early plants R2 stage. The visual symptoms of the disease and DON content were significantly higher in dent forms than in the flint ones (Tab. 3).
266
E. CZEMBOR, P. OCHODZKI
TABLE 3 - Ear rot resistance of tested inbreed lines and mycotoxin contamination after silk-channel and tooth-pick inoculation with Fusarium spp. and after natural infection. –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Infection type Trait Dent forms Flint forms –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Natural infection ear rot 1.6 2.0 DON 0.34 0.99 FB1 0.27 1.93 FB2 0.11 0.87 FB3 0.03 0.35 FUM 0.41 3.14 –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Silk-channel inoculation ear rot 3.3 4.5 with F. graminearum DON 17.5 24.1 –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Tooth-pick inoculation ear rot 6,0 5.6 with F. graminearum DON 68.5 20.1 –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Tooth-pick inoculation ear rot 2.8 3.8 with F. verticillioides FB1 8.06 30.1 FB2 3.57 11.8 FB3 0.74 3.97 FUM 12.3 45.8 ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Another relationship between dent and flint forms was demonstrated after inoculation with F. verticillioides. Visual symptoms of the disease were significantly lower on dent forms than on flint forms, and it was confirmed by FUM contamination.
FIGURE 3 - Colonization of dent form after inoculation with Fusarium spp. under laboratory conditions.
Similar results were obtained in another studies (REID et al., 1999; MIEDANER et. al., 2008) where F. verticillioides caused a lower level of disease and toxins content than F. graminearum. High correlation coefficient between visual symptoms of disease and DON and FUM content was found (0.76 and 0.78, respectively) what was confirmed in another study (ROBERTSON et al., 2006). The natural colonization of kernels by Fusarium spp. was evaluated using Blotter test. In the flint group 24 % of kernels were infected by Fusarium spp. compared to10% in the dent group. After kernels inoculation it was possible to observe that the flint kernels have been more resistant than the dent ones to pre or - postharvest attack by Fusarium spp. After inoculation by F. graminearum and F.
FIGURE 4a, b - Colonization of flint form after inoculation with Fusarium spp. under laboratory conditions.
MYCOTOXINS CONTAMINATION
verticillioides on the dent kernels the mycelium was aboundant and grown quickly. The differences between genotypes were not observed (Fig. 3). On the flint forms the mycelium not grown or grown much lower, and the differences between genotypes were observed (Fig. 4a, b).
CONCLUSIONS Visual screening is cheaper than mycotoxin analyses and the correlation between ear rot rating and the respective mycotoxin exists. However mycotoxin analysis in the preliminary breeding program is also necessary. The different response to Fusarium spp. of flint and dent genotypes under field and laboratory conditions could indicate some kind of coadaptation between host and pathogen related to the origin site.
REFERENCES COMISSION REGULATION (EC), 2007 of 28 September 2007 Amending regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards Fusarium toxins in maize and maize products. Official J. European Union 1126: 14-17. CLEMENTS M.J., C.A. MARAGOS, J.K. PATAKY, D.G. WHITE, 2004 Sources of resistance to fumonisim accumulation in grain and fusarium ear and kernel rot of corn. Phytopatol. 94: 251-260.
267
genic Bt maize hybrids, their isogenic counterparts, and commercial varieties. Plant Breed. 121: 146-154. MAGG T., M. BOHN, D. KLEIN, V. MERITAJ, A.E. MELCHINGER, 2003 Concentratrion of moniliformin produce by Fuasarium species in grains of transgenic Bt maize hybrids compared to their isogenic counterparts and commercial varieties under European corn borer pressure. Plant Breed. 122: 322-327. MIEDANER T., 2004 Plant breeding as a tool for reducing mycotoxins in cereals. pp. 89-111. In: D. Barug, H. van Egmond, R. Lopez-Garcia, R. van Osenbruggen, A. Visconti (Eds.), Meeting the Mycotoxin Menance. Wagengingen Acad. Publ., The Netherlands. MIEDANER T., M. LOFFLER, CH. BOLDUAN, B. KESSEL, M. OUZUNOVA, V. MIRDITA, A.E. MELCHINGER, 2008 Genetic variaton for resistance and mycotoxin content of European maize inoculated with fusarium graminearum and F. verticilliodies. Cereal Res. Comm. 36: 45-48. REID L.M., D.W. STEWART, R.I. HAMILTON, 1996a A 4-year study of the association between Gibberella ear rot severity and deoxynivaleol concentration. J. Phytopathol. 144: 431-436. REID L.M., R.I. HAMILTON, D.E. MATHER, 1996b Screening maize for resistance to Gibberella ear rot. Technical Bulletin 19965E. Eastern Careal and Oilseed Research Centre. Research Branch, Agriculture and Agri-Food Canada, Ottawa, Canada, 40 pp. REID L.M., R.W. NICOL, T. OUELLET, M. SAVARD, J.D. MILLER, J.C. YOUNG, D.W. STEWART, A.W. SCHAAFSMA, 1999 Interaction of Fusarium graminearum and F. moniliforme in maize ears: disease progress, fungal biomass, and mycotoxin accumulation. Phytopathol. 89: 1028-1037. ROBERTSON L.A., C.E. KLEISCHMIDT, D.G. WHITE, G.A. PAYNE, C.M. MARAGOS, J.B. HOLLAND, 2006 Heritabilities and correlations of ear rot resistance and fumonisin contamination resistance in two maize populations. Crop Sci. 46: 195-2009.
GENDLOFF E.H., E.C. ROSSMAN, W.L. CASALE, T.G. ISLEIB, L.P. HART, 1986 Components of resistance to Fusarium ear rot in field corn. Phytopathol. 76: 684-688.
ROBERTSON-HOYT L.A., M.P. JIINES, P.J. BALINT-KURTI, C.E. KLEINSCHMIT, D.G. WHITE, G.A. PAYNE, C.M. MARAGOS, T.L. MOLNAR, J.B. HOLLAND, 2006 QTL mapping for fusarium ear rot and fumonisim contamination resistance in two maize populations. Crop Sci. 46: 1734-1743.
MAGG T., A.E. MELCHINGER, D. KLEIN, M. BOHN, 2002 Relationship between European corn borer resistance and concentration of mycotoxins produced by Fusarium spp. in grains of trans-
SUTTON J.C., 1982 Epidemiology of wheat head blight and maize ear rot caused by Fusarium graminearum. Can. J. Plant Pathol. 4: 195-209.
