Effects of maize residues on the Fusarium spp. infection and deoxynivalenol (DON) contamination of wheat grain

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Crop Protection 27 (2008) 182–188 www.elsevier.com/locate/cropro

Effects of maize residues on the Fusarium spp. infection and deoxynivalenol (DON) contamination of wheat grain Andrea Maiorano, Massimo Blandino, Amedeo Reyneri, Francesca Vanara Department of Agronomy, Forest and Land Management, University of Turin, via Leonardo da Vinci 44, 10095 Grugliasco (TO), Italy Received 8 May 2007; accepted 9 May 2007

Abstract Fusarium head blight (FHB) of small grains is a worldwide spread disease that reduces yield, causes mycotoxin production in grain and reduces seed quality. Previous crop residues such as maize stalks and grain, and straw of barley, wheat, and other cereals are considered the principal inoculum sources for Fusarium graminearum and Fusarium culmorum, the most important Fusarium spp. causing FHB in Europe. The residues present on the soil surface and in the first 10 cm of soil in tilled and not tilled fields were quantified and their relative influence on Fusaria infection and deoxynivalenol contamination was evaluated. The total amount of residues in the first layer of the soil (10 cm) and on its surface was found to be correlated with DON contamination (R2 ¼ 0.848), but ANOVA showed that tillage was not significant (P40.05) and that the major role in Fusarium spp. infection and DON contamination was played mainly by the residues lying on the surface of the soil (Po0.05). These results were used to evaluate management strategies of four different previous crop residues by comparing their effectiveness in reducing crop residues from the surface of the soil and the consequent contamination and their costs. r 2007 Elsevier Ltd. All rights reserved. Keywords: Deoxynivalenol; Previous crop residues; Fusarium head blight; Wheat; Tillage

1. Introduction Fusarium head blight (FHB) of small grains is a fungal disease caused by Fusarium spp., which reduces yield, causes mycotoxin production in grain, and reduces seed quality in several cereal areas worldwide. In Europe, Fusarium graminearum and Fusarium culmorum are the most important and spread agents of FHB of wheat (Teich, 1989; Birzele et al., 2002; Gilchrist and Dubin, 2002). These Fusaria produce deoxynivalenol (DON), a mycotoxin of the trichotecenes group, one of the most spread mycotoxin in cereal, which can be synthesized in the field in winter cereal and in maize grain (Chelkowski, 1989; Schaafsma et al., 1993). Corresponding author. Tel.: +39 011 670 8928; fax: +31 011 670 8798. E-mail address: [email protected] (A. Maiorano).

0261-2194/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2007.05.004

Trichotecenes are associated with serious mycotoxicosis in humans and animals. They have cytotoxic activity (protein synthesis inhibition, effects on DNA and RNA synthesis, mitochondrial function inhibition, effects on cellular membranes and on cellular correct division, and apoptosis) and immunosuppressor effect, which reduce the resistance to microbial infections (Rotter et al, 1996; Shifrin and Anderson, 1999; Minervini et al., 2004). The EC Reg. 1881/2006 has fixed the Fusarium toxin limits in winter cereals and has consequently increased the need for an agricultural technique modernization aimed at obtaining higher hygienic and sanitary standards for grain lots, above all for those destined for human consumption. It has already been stressed (Cleveland et al., 2003; Edwards, 2004; Ramirez et al., 2004; Koch et al., 2006) that the hygienic and sanitary qualities of cereal are

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determined starting from the field. In fact, in all the following post-harvest stages, fungi development and mycotoxin synthesis can be well controlled, respecting the maximum limits of relative humidity (Miller, 1995; Bottalico, 1998). Moreover, while the environmental conditions during the storage can all be potentially controlled, the field stage interactions between plants, pedoclimatic conditions, pests, and agronomic techniques led to this complex system being carefully managed with the choice of the best cultural practices (Bilgrami and Choudhary, 1998; Schaafsma et al., 2001; Rossi et al., 2002; Doohan et al., 2003; Aldred and Magan, 2004). Soil and seeds can be sources of inoculum, but previous crop residues like maize stalks and grain, and straw of barley, wheat, and other cereals are considered the main inoculum sources for F. graminearum and F. culmorum (Atanasoff, 1920; Khonga and Sutton, 1988; Dilantha, 1999; Xu, 2003). Consequently, management of previous crop residues is one of the most important cultural practices which can contribute to the reduction of FHB (Pirgozliev et al., 2003). However, in Italy and in all of Europe in the previous years, a trend to reducing tillage was observed for environmental (avoiding erosion and increasing organic matter in the soil) and economical (costs of tillage) reasons; consequently, minimum tillage and no-tillage (direct drilling) systems increased, without taking into account the consequences on the sanity of the products. The aims of the present work were (i) to assess the amount of maize residues on the surface and in the first layer (10 cm) of the soil in tilled and not tilled fields, (ii) to quantify the relative amount of crop residues moved by ploughing from the more superficial to the deeper layers of the soil, (iii) to compare the relative importance of the residues on the surface and in the first layer (10 cm) of the soil in Fusarium spp. infection and DON contamination, (iv) to evaluate different management strategies of previous crop residues, and (v) to stress on the general importance of the management of previous crop residues as one of the tool to increase quality of yields at a time in which minimum tillage and no-tillage strategies are preferred.

183

2. Materials and methods 2.1. Experimental fields and trials The experiments were carried out in three different locations during 2004–2006. The agronomical and geographical information about the experimental fields are given in Table 1. Planting was held in 12 cm wide rows in October at a seeding rate of 450 seeds m2. Nitrogen fertilization (150 kg ha1) was applied in the form of ammonium nitrate. In all sites, 50% of nitrogen was applied at wheat tillering (GS 31: Zadocks growth stage; Zadocks et al., 1974) and the remaining 50% at the end of wheat stem elongation (GS 39). Each year, two adjacent experimental fields were prepared:

 

Not tilled field: planting the seed directly into the soil after maize, with the residue remaining on the soil surface Tilled field: planting after an autumn ploughing (30 cm) and disc harrowing to prepare a proper seedbed

The fields’ dimensions were between 8000 and 9000 m2. The following treatments were applied, in not inoculated condition: Not tilled field:

 

No removing of previous crop residues. Normal not tilled field (NTR+) Manual removing of previous crop residues from the soil surface (NTR) Tilled field:

 

Replacing of the residues removed from NTR (TR+) Normal tilled field, without any replacing of residues. (TR) Here NT ¼ not tilled; T ¼ tilled; R+ ¼ presence of previous crop residues in the soil surface;

Table 1 Geographical and agronomic information about the experimental fields Year

Location

Geographic coordinate

Soil

Altitude (m)

Wheat variety

Previous crop

2004

Ternavasso Poirino

Bolero

Maize

Valle San Bartolomeo

Deep and acid loamy soil Aquic frugiudalf Deep loamy soil Typic Dystrochrepts

282

2005

Lat. 441 510 0000 , Long. 071 500 5800 Lat. 441 570 1900 , Long. 081 380 3300

138

Isengrain

Maize

2006

Riva presso Chieri

Lat. 441 590 900 , Long 071 520 2400

Deep and acid loamy soil Typic Dystrochrepts

262

Serio

Maize

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R ¼ absence of previous crop residues on the soil surface. Residues from NTR to TR+ were moved at the beginning of stem elongation (GS 30). The treatments were assigned to experimental units using a randomized complete block split-plot design with three replications for each sub-plot, in which the experimental block factor was the effect year/variety/ location, the main plot factor was the effect of tillage (NT vs. T), and the sub-plot factor was the effect of the crop residues on the soil surface (R vs. R+). Each sub-plot was 16 m2 and they were harvested in early–mid July with a Walter Winterstaiger cereal plot combine harvester. The harvested grains were accurately mixed and milled, and samples were taken to perform mycological and toxicological analysis. The samples for mycological analyses were refrigerated at 4 1C, while those for mycotoxin content analyses were frozen at a temperature of 8 1C.

into eight classes, classified according to codes 1–7, which correspond to a percentage of surface showing symptoms (1 ¼ 0–5%, 2 ¼ 5–15%, 3 ¼ 15–30%, 4 ¼ 30–50%, 5 ¼ 50–75%, 6 ¼ 75–90%, 7 ¼ 90– 100%). Then, the percentage of ear infection was calculated. 2.4. Mycological analysis Evaluation of the fungal infections was carried out using 10 ears per sub-plot randomly collected at harvesting. The wheat kernel was surface disinfested for 3 min in a 0.5% solution of sodium hypochlorite, then rinsed three times with sterile water. The kernels were placed in Petri dishes containing dicloran chloramfenicol peptone (DCPA) and incubated at 20 1C. The Fusarium colonies were identified after 7–10 days by colony and conidial morphology, as reported by Nelson et al. (1983). 2.5. Mycotoxin analysis

2.2. Determination of residues’ weight in the tilled and not tilled fields Each year, the previous crop residues in the first 10 cm of the soil in the tilled field (corresponding to the TR treatment) and not tilled field (corresponding to the NTR+ treatment) were weighed after manual harvesting from five randomized 4 m2 plots from each field. The residues were harvested and weighed after dividing them into two groups: (a) superficial residues: from the soil surface, easily manually harvestable; (b) deep residues: partially incorporated in the soil structure, down the soil surface, in the first 10 cm. Then, the residues in NTR and TR+ were easily calculated as follows:

A 5 kg representative sample of grain from each subplot was freeze-dried and milled. Samples of 25 g were extracted by shaking for at least 30 min with 100 ml of MilliQ water containing 5 g of polyethylene glycol 8000. The supernatant was filtered through filter paper (0.45 mm) and was cleaned using a Donprep column (R-Biopharms Rhone Ltd.) by rinsing with 5 ml of MilliQ water. The DON was then eluted using 1.5 ml of methanol and 250 ml of water/acetonitrile 90/10. A 25 ml solution was injected into an HPLC column (Synergi 4m Fusion-RP80A Phenomenex column, 1 ml min1; detector DAD-UV, 220–225 nm). Toxin quantification was performed using external standards and peak height measurements. The detection limit was 10 mg kg1.



2.6. Statistical analysis

  

deep residues present in NTR ¼ deep residues present in NTR+; superficial residues in NTR ¼ absent for manual harvesting; superficial residues in TR+ ¼ superficial residues in TR7superficial residues in NTR+; deep residues in TR+ ¼ deep residues in TR.

2.3. Determination of the percentage of Fusariuminfected heads The FHB incidence and severity were recorded for each plot, carrying out visual evaluations of the disease at soft dough (GS 85). The FHB incidence was calculated as the percentage of plants with symptoms that were noted on analyzing 200 ears per plot. The FHB severity was computed as the percentage of kernels per ear with symptoms. The affected areas were grouped

Normal distribution and homogeneity of variances were verified by performing, respectively, the Kolmogorov–Smirnov normality test and the Bartlett– Box test. Difference between the weights of the superficial and deep residues was tested for the tilled andnot tilled fields by analysis of variance (ANOVA) (SPSS Inc., Chicago, IL, Version 13.0) using a completely randomized block design in which the weight of the residues was the independent variableand the factor year/variety/location was the random factor. The effect of treatments on FHB incidence and severity, fungal infection, and DON content was tested by ANOVA (SPSS Inc., Chicago, IL, Version 13.0) using a randomized complete block split-plot design in which the experimental block factor was the effect year/ variety/location, the main plot factor was the effect of

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tillage (NT vs. T), and the sub-plot factor was the effect of the crop residues on the soil surface (R vs. R+). When significant, the block means and the means of the interaction between mean-plot and sub-plot factors were compared using Bonferroni’s test at P(F)p0.05. The incidence and severity values of FHB were previously transformed using y0 ¼ arcsenOx, as percentage data derived from counting.

Table 3 Weights of the superficial and deep residues for each treatment Dataa

Field

Treatmentb Residues

Measured Not tilled NTR+

Tilled

TR

3. Results The 3 years showed different meteorological trends from the shooting stage to harvesting (Table 2). Meteorological conditions in 2004 and 2005 were very similar for both rainfall and mean temperature, while in 2006 there was a limited rainfall with higher temperatures above all during the final ripening period (June and July). The highest rainfall was registered in 2003– 2004. 3.1. Crop residues Both in the not tilled and tilled fields the amount of deep residues was significantly higher (Po0.01) than the amount of superficial residues, representing in both fields 61% of the total amount of crop residues (Table 3). No significant effect of the factor year/variety/ location on the weight of previous crop residues was observed for the tilled or not tilled field (P40.05). Ploughing reduced the amount of previous crop residues by 94% both on the surface and in the first 10 cm of soil. Hence, the amount of total residues was on average 17 times higher for NTR+ compared with TR; for NTR and TR+ residues were similar but mainly located in the first 10 cm of the soil for the first treatment and on soil the surface for the second one (Table 3).

Table 2 Rainfall and mean temperatures from October to July (2003–2006) at the experimental field

Total rainfall (mm) October–February March–July Mean temperature (1C) October–February March–July

2003–2004

2004–2005

2005–2006

Ternavasso Poirino

Valle san Bartolomeo

Riva presso Chieri

417.8 237.4

239.2 225.6

236.4 140.2

5 15.1

5.1 14.8

3.7 15.5

(z) data recorded until 15 July of each year.

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Calculated Not tilled NTR

Tilled

TR+

Residues weight (g m2)c Mean

s.m.e.d

Superficial Deep Total Superficial Deep Total

602a 939b 1541 35a 55b 90

95 92

Superficial Deep Total Superficial Deep Total

0 939 939 637 55 692

2 5

Means followed by different letters are significantly different (Po0.01). The analysis of variance was performed only for the measured weights. a Measured ¼ determination after manual harvesting. Calculated: deep residues present in NTR ¼ deep residues present in NTR+; superficial residues in NTR ¼ absent for manual harvesting; superficial residues in TR+ ¼ superficial residues in TR7superficial residues in NTR+; deep residues in TR+ ¼ deep residues in TR. b NTR+ ¼ not tilled, no removal of previous crop residues; TR ¼ tilled, without any replacing of residues; NTR ¼ not tilled, manual removing of previous crop residues from the soil surface; TR+ ¼ tilled, replacing of the residues removed from NTR. c Dry matter. d s.m.e. ¼ standard mean error.

3.2. Mycological analysis The most frequently isolated fungal species in the samples from the field were F. graminearum and F. culmorum at harvesting in all the years. In 2006, the mean contamination was significantly higher than in 2004 and 2005, when similar low percentages of kernels infected by DON-producing Fusarium spp. were found. No effect of tillage was observed; on the contrary, the presence of the residues on the surface of the soil (R+) determined a significant (Po0,05) higher infection (Table 4). 3.3. FHB incidence and severity Incidence and severity of FHB symptoms registered during the visual evaluations were very similar in 2004 and 2005 and significantly higher in 2006 (Po0.001). Neither the tillage (main factor) nor the residues on the soil surface (sub-plot factor) showed significant effects (P40,05), but their interaction was significant (Po0,05). However, a significant higher incidence for the treatment NTR+ was evidenced; moreover, there was a higher severity for NTR+ and also for

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Table 4 Effects of previous crop residue treatments on FHB symptoms (incidence and severity) and on Fusarium spp. infection and DON production Factor

Source of variationa

Incidenceb (%)

Severityc (%)

Fusarium spp.d (%)

DON (mg kg1)

Sub-plot

R R+ P(F)

44 43 0.907

7 6 0.721

7a 10b 0.047*

837a 1343b 0.032*

Block

2004 2005 2006 P(F)

32a 31a 68b 0.000***

2a 4a 12b 0.000***

5a 6a 16b 0.000***

1381a 54b 1834a 0.000***

Main  sub-plot

NTR NTR+ TR TR+ P(F)

46a 53b 42a 34a 0.041*

4a 7b 9b 4a 0.031*

8 12 6 9 0.917

976 1809 698 876 0.155

Main

T NT P(F)

38 50 0.154

7 6 0.766

8 10 0.433

787 1393 0.245

Means followed by different letters are significantly different (the level of significance is shown in the table). a NT ¼ not tilled; T ¼ tilled; R+ ¼ presence of previous crop residues on the soil surface; R ¼ absence of previous crop residues on the soil surface. b FHB incidence was calculated as the percentage per plot of the ears with symptoms of the disease at the stage of soft dough (Zadocks GS 83). Values are based on four replications. c FHB severity was calculated as the percentage of kernels per ear showing symptoms of the disease at the stage of soft dough (Zadocks GS 83). Values are based on four replications. d Fusarium spp. ¼ percentage of kernels infected by F. graminearum (F. gram)+F. culmorum (F. culm).

TR, which unexpectedly showed the highest mean severity value (Table 4).

3.4. DON contamination All samples were positive to DON contamination over the 3 years. In 2004, DON concentration varied from 125 to 4750 mg kg1. In 2005, the lowest contaminations were registered from 25 to 130 mg kg1. The year 2006 had the highest mean concentration, with DON contamination varying from 1192 to 2660 mg kg1. Over the 3 years, the minimum contaminations were always found in TR samples and the maximum ones in NTR+ samples. The mean of the total amount of previous crop residues in each sub-plot showed a positive correlation with the DON contamination (R2 ¼ 0.848) (Fig. 1). ANOVA showed a significant effect of the block factor (Po0.001) due to the great variability over the 3 years (Table 4). As for fungi infection, any effect of tillage was observed. However, a significant effect of the presence of residues on the soil surface was clearly evidenced; their presence determined a significant (Po0.05) higher DON contamination compared with the mean of the plots without residues on the soil surface, whose contamination was 38% lower.

4. Discussion and conclusions The results showed a high variation of Fusarium infection and DON contamination over the 3 years. Despite this high variation, experimentation confirmed the important role of the agronomic technique in FHB of wheat, in particular tillage and the management of previous crop residues. Visual evaluation of the symptoms did not give significant and clear results as was pointed out by Blandino et al. (2006). As expected, incidence was found higher for the treatment NTR+, and in general for the not tilled treatments (NT). On the contrary, severity gave an unexpected result, showing higher value for the treatment TR. The hypothesis is that the elements included in the block factor (year, wheat variety and location) could have more influence on the symptoms than the effect of the treatments. Tillage did not show any significant effect on Fusarium spp. contamination and DON concentration, but the differences between tilled and not tilled fields were remarkable and stressed on the importance of ploughing as an instrument to minimizing the source of inoculum; in fact, the total amount of previous crop residues showed a positive correlation with the DON contamination. However, the results of ANOVA showed the major role played by the residues lying on the surface of the soil both on Fusarium spp. infection

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Fig. 1. Relation between the amount of previous crop residues on the surface of the soil and in the first 10 cm layer and DON concentration. NT ¼ not tilled; T ¼ tilled; R+ ¼ presence of previous crop residues on the soil surface; R ¼ absence of previous crop residues on the soil surface. The amount of residues is expressed as g m2 of dry matter.

and on the subsequent DON contamination. These results have a triple explanation:







The litter layer formed by previous crop residues protects the soil from rain and consequently from erosion but breaks down into organic matter, enriching the surface of the soil in carbon and organic forms of nitrogen, and modifying its chemical and biological characteristics. Stores of inorganic carbon increase, the supply of soil mineral nitrogen decreases very slowly and the mineralization process is delayed. The microbial biomass (bacteria and fungi) increases at the surface and its turnover rate increases in the first 10 cm of soil. Humus is thus less well degraded in this zone, which rapidly becomes more acidic, favoring the development of fungi over that of bacteria. Thus, about 90% of the Fusarium spp. population is located in the first 10 cm of soil (Champeil et al., 2004). The residues keep the water on the surface of the soil favouring the release of the spores of Fusarium spp., which are contained in mucilage that may require some wetting to necessitate their release (Ho¨rberg, 2002). The mechanic of the splash dispersal of Fusarium spp. conidia and spores is enhanced by the presence of straw on the surface of the soil (Walklate et al., 1989).

Thus, the decrease in the density of residues on the surface of the soil in the sub-plots TR and NTR helped to decrease (i) the production of inoculum, (ii) the quantity of spores available for dispersal, and (iii) the dispersal itself. Each sub-plot in this trial can easily find its correspondence with four different management strategies of previous crop residues. Direct drilling, corresponding to the NTR+ sub-plot, has no costs of residues management but, determining a high density of crop residues on the surface of the soil, provokes a

critical Fusarium spp. infection and DON contamination, and consequently the expected sanitary quality level of grain is very low. The TR+ sub-plot can be related to a minimum tillage with only a partial and very superficial burying of the residues, with a consequent high Fusarium spp. infection and DON contamination and low sanitary quality of grain. Stalk bailing, simulated by the NTR sub-plot, removing the residues from the surface of the soil can be an effective agronomic management strategy to assure a medium– high-quality level of grain and it can represent a good compromise between grain safety and management costs. The sanitary quality level of grain is assessed as ‘‘medium–high’’ because the effectiveness of this strategy cannot be completely compared with that of a manual, more effective, harvesting of the residues from the surface of the soil. Thus, more residues are expected to lie on the surface of the soil than those on the subplot NTR. Ploughing, represented by the TR subplot, is the more effective and safe previous crop residues management strategy because it remove the residues from the first layers of the soil to a depth of almost 15–30 cm, and thus it highly influences the amount of inoculum source available for the development of the disease (Miller et al., 1998; Xu, 2003). The negative aspect of ploughing is represented by the higher costs of this agronomic technique. In conclusion, the trial clearly underlined the importance of previous crop residues management for winter cereals on determining the sanity of the grain, above all if they are in rotation after maize, whose debris are the principal substrate where the process of infection starts its cycle: wheat cultivated after maize is in average more contaminated than wheat in succession with all other crops due to the larger amount of residues that maize leaves on the soil (Sutton, 1982; Dill-Macky and Jones, 2000; Barrier-Guillot, 2006), which can represent an ideal source of inoculum on the soil up to the second year (Khonga and Sutton, 1988).

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