The effect of different tillage-fertilization practices on the mycoflora of wheat grains

Wheat (Triticum aestivum L.) is one of the most important cereal crops and a staple food worldwide. In Lithuania, winter wheat prevails and occupies more farmland than any other crop. The production area of spring wheat has increased over the recent years, too. Mouldboard ploughing is a common tillage system in Lithuania; however, direct drilling and other conservation tillage practices are becoming increasingly popular, especially on the more fertile soils of the central part of the country. Reduced tillage has an advantage over the conventional tillage, because of reduced costs (Ribera et al. 2004, Yalcin et al. 2005, Feizienė et al. 2006) and given environmental benefits: reduction in soil erosion, nitrate leaching, fuel use, increasing soil organic matter and activity of soil organisms, improving soil structure and preserving soil moisture (Roldán et al. 2004, Subbulakshmi et al. 2009, Feiza et al. 2010, Bogužas et al. 2010). Nevertheless, no-tillage may result in increased propagule densities of The effect of different tillage-fertilization practices on the mycoflora of wheat grains


Introduction
Wheat (Triticum aestivum L.) is one of the most important cereal crops and a staple food worldwide.In Lithuania, winter wheat prevails and occupies more farmland than any other crop.The production area of spring wheat has increased over the recent years, too.
Mouldboard ploughing is a common tillage system in Lithuania; however, direct drilling and other conservation tillage practices are becoming increasingly popular, especially on the more fertile soils of the central part of the country.Reduced tillage has an advantage over the conventional tillage, because of reduced costs (Ribera et al. 2004, Yalcin et al. 2005, Feizienė et al. 2006) and given environmental benefits: reduction in soil erosion, nitrate leaching, fuel use, increasing soil organic matter and activity of soil organisms, improving soil structure and preserving soil moisture (Roldán et al. 2004, Subbulakshmi et al. 2009, Feiza et al. 2010, Bogužas et al. 2010).Nevertheless, no-tillage may result in increased propagule densities of most of the fungi present in the soil, including plant pathogens such as Rhizoctonia spp.and Pythium spp.(Ploetz et al. 1985).Field experiments verified that non-inversion tillage is a major factor increasing the severity of tan spot (Pyrenophora tritici-repentis) (Jørgensen and Olsen 2007), Septoria leaf blotch (Septoria tritici) (Bailey and Duczek 1995), Stagonospora leaf blotch (Stagonospora avenae) (Elen 2003) compared with conventional ploughing.It may also result in increased Fusarium head blight (FHB) incidence and severity (Dill-Macky and Jones 2000, Fernandez et al. 2005, Lori et al. 2009).
Crop residues left on or near the soil surface in reduced tillage may be a source of inoculum not only for pathogens, but also for a wide range of fungi which may develop on grains and present a potential threat to their quality.Clear and Patrick (1993) indicated at least 59 species representing 35 fungal genera on soft white winter wheat seed from Ontario.Lõiveke et al. (2004) identified 63 fungi species on Estonian spring and winter wheat grains and grain feeds during 1992-1994.Alternaria, Cladosporium, Fusarium, Aspergillus, Penicillium were among the main species identified on wheat grains (Clear and Patrick 1993, Lõiveke et al. 2004, Rajput et al. 2005, Semaškienė et al. 2005, Bakutis et al. 2006, Gohari et al. 2007).Alternaria and Cladosporium are one of the most common species on grains; they are harmless saprophytes of cereals.Some Alternaria species are (opportunistic) plant pathogens that, collectively, cause a range of diseases with economic impact on a large variety of important agronomic host plants including cereals, also well known as post-harvest pathogens (Thomma 2003).Under certain conditions they may produce mycotoxins such us tenuazonic acid, alternariols and others (Scudamore, 2000).Fusarium species are destructive pathogens on cereal crops and produce mycotoxins before, or immediately after, harvest (D 'Mello et al. 1999, Bottalico and Perrone, 2002, Thrane et al. 2004).Certain species of Aspergillus and Penicillium are also plant pathogens, but they are more commonly associated with grain storage (Pitt 2000).Penicillium chrysogenum, Aspergillus flavus and Rhizopus chizopodifarmis may decrease dry matter digestibility, amino acid, vitamin and fat contents in feed during storage (Maciorowski et al. 2007).Aspergillus, Fusarium and Penicillium are the main mycotoxin producers in cereal grains.The most significant mycotoxins in naturally -contaminated foods and feeds are aflatoxins, ochratoxins, zearalenone, T-2 toxin, deoxynivalenol and fumonisins (Surai and Mezes, 2005) produced by the above mentioned fungal species.Mycotoxins, when ingested, may cause a mycotoxicosis which can result in an acute or chronic disease episode (Bryden, 2007).Alternaria, Cladosporium, Aspergillus and Penicillium also are known as air -born allergens causing rhinitis and severe asthma (Breitenbach andSimon-Nobbe 2002, Gomez de Ana et al. 2006).As was shown by Kačergius et al. (2005) the same fungi (Aspergillus, Fusarium, Penicillium, Alternaria and Rhizomucor) which infected grain, vegetable and fruit were also predominant in the air of their storehouses.
Development of fungal infection in cereals has been associated primarily with environmental conditions.Clear and Patrick (1993) recorded yearly differences in the quantity and time of precipitation and the frequency of a number of fungal species (Bipolaris sorokiniana, Pyrenophora triticirepentis, and Septoria nodorum), including a 100 fold increase in the frequency of F. graminearum between 1988 and 1989 in Ontario.In Lithuania, Semaškienė et al. (2005) indicated that host-plant, cultivar and weather conditions influenced organic grain fungal infection level.A survey of the fungal contamination of wheat and barley grains grown in organic and conventional farms was done with a focus on mycotoxin producing fungi (Bakutis et al. 2006).The findings evidenced that fungal contamination on wheat grain from organic farms was by 70.5% (p<0.05)higher than that on conventionally grown grain.
The effect of conventional and reduced tillage systems on FHB and wheat foliar diseases combined with the other agronomic practices is presented by researches; however the information about this effect on the total fungal contamination of cereal grains is rather scanty; the data about fertilization influence are also limited.There is some evidence that FHB can be affected by fertilizer regimes.Teich (1989) and Martin et al. (1991) observed that increasing the amount of nitrogen (N) applied to cereals resulted in increased incidence of FHB or Fusarium-infected grain.However Lori et al. (2009) reported that favourable weather conditions are more important for FHB infection than tillage practice and fertilizer treatments.
This study was designed to explore the influence of different tillage and fertilization practices on winter and spring wheat grain contamination by fungi, potential cereal pathogens, or other contaminants such as mycotoxin producers or allergens for humans and farm livestock.

Materials and methods
The study site is located at the Lithuanian Institute of Agriculture (Central part of Lithuania).Studies were carried out on the basis of two long-term field trials, established in the autumn of 1999 (first trial) and 2000 (second trial) on a loamy Endocalcari -Epihypogleyic Cambisol.The experimental design was a split plot in four blocks (replications), with the tillage treatments: conventional tillage, reduced tillage and no-tillage as the main plots (Table 1).Fertilization rates of mineral NPK (none, moderate and high), designed as subplots, were calculated according to the soil properties and expected crop yield (Švedas and Tarakanovas 2000).Crop rotation was as follows: winter wheat -spring rape -spring wheat -spring barley -pea in both field experiments.
For mycological study we used winter and spring wheat grain samples collected during 2005−2008 (second crop rotation).Because of poor winter wheat over winter survival in 2006, half of the field trial was re-sown with spring wheat (dividing trial plots in two equal parts across all treatments).Therefore in 2006 both winter and spring wheat grain samples were used for assessment.Plant residues of the pre-crops were collected and removed from the experimental field each year after harvest.Using of conventional crop rotation, unfavourable for wheat diseases, we expected to get results influenced only by tillage and fertilization.Each year, 3 weeks after harvesting of previous crop, non-selective herbicide (glyphosate at a dose of 1.44 kg a.i.ha -1 ) was sprayed in no-tillage plots to control weeds and volunteer plants (cereals or oil seed rape).
Grain samples of 1.0 kg for laboratory analyses were taken from each plot at harvesting.Subsamples were stored in plastic jars in a freezer at −20°C to prevent alterations in fungi and mycotoxin contents until the conduct of analyses.Before analyzing, the grain was de-frosted up to room temperature.Grain surface contamination was determined using dilution plating method.Ten grams of each sample was suspended in 100 ml of sterile distilled water.A 1 ml sample of this nutrient suspension was used to prepare a dilution series from 1:1000.Dilution was uniformly dispensed under the surface of acidified malt agar (MA: 300 ml of maltose; 13 g of agar; 1.2 g of citric acid and 700 ml of distilled water) in Petri-dishes and incubated for 3−5 days at 25 o C in the dark.After incubation, the number of fungal colonies was calculated and expressed in colony -forming units per g of grain (cfu g -1 ).
Plating technique was used for internal fungal grain infection estimation.Surface-sterilized (for 3 minutes in 1 % NaOCl solution) grains were plated in Petri-dishes with potato dextrose agar (PDA: 250 g of potato, 10 g of glucose, 14 g of agar and 1 l of distilled water) and incubated for 7−8 days at 26±2 º C (International Rules for Seed Testing 2003).The fungi were identified according to the manuals of Malone and Muskett (1997), Mathur and Kongsdal (2003), Lugauskas et al. (2002), Nelson et al. (1983) and Leslie and Summerell (2006).
The grain fungal infection level per sample was expressed in percent.
Significance of the differences between the means was determined according to the least significant difference (LSD) at 0.05 probability level.The data were processed using the software ANO-VA, SPIL-PLOT from the package SELEKCIJA (Tarakanovas and Raudonius, 2003).Fertilization (factor B) * not fertilized no fertilization moderate rates mineral NPK fertilizers according to soil nutrient status and expected yield (winter wheat 6.5 t ha -1 , spring wheat 4.5 t ha -1 ) high rates mineral NPK fertilizers according to soil nutrient status and expected yield (winter wheat 8.0 t ha -1 , spring wheat 6.0 t ha -1 ) * Fertilization rates were calculated according to the soil properties and expected crop yield, using the Institute of Agriculture -developed computerised program "Tręšimas" (Fertilization) (Švedas and Tarakanovas 2000).

Results
On winter wheat grain surface, we identified 10 fungal genera in 2005 and 6 fungal genera in 2006 (Table 2).Cladosporium and Penicillium were the most frequent species in the winter wheat grain samples.A total of 14 fungal genera were isolated from winter and spring wheat grain surface.Alternaria, Cladosporium, Fusarium and Penicillium species were the most common.Cladosporium spp. was identified in all tested samples.Arthrinium, Botrytis, Pyrenophora, Nigrospora, Trichoderma and Ulocladium were less frequent.Acremonium and Alternaria species were more prevalent on spring wheat grain surface than on winter wheat.Pyrenophora, Nigrospora, Trichoderma and Ulocladium were identified only on spring wheat grains.
The fungal contamination of winter wheat grains ranged from 7.2 × 10 3 to 24.8 × 10 3 cfu g -1 (Table 3).Grains were more contaminated in 2005 than 2006.There were no significant differences between tillage practices.However, high and moderate fertilizer rates in 2005 and high rates in 2006 significantly increased grain surface contamination compared to not fertilized winter wheat.
The fungal contamination of spring wheat grains ranged from 14.8 × 10 3 to 80.3 × 10 3 cfu g -1 (  Cladosporium colony accounted for nearly half of the total fungal colonies formed.As a result, the increase in the total number of colony forming units (cfu g -1 ) in some cases depended on the increase in the count of Cladosporium colonies.A multifactor analysis of variance demonstrated a significant effect of fertilization on the total count of cfu g -1 and on that of Cladosporium spp. in 2005−2006.Without Cladosporium colonies taken into account, fertilization had a significant effect on the fungal count only in spring wheat in 2006 (Table 5).Conversely, a significant tillage effect was established on the amount of the other fungal colonies than Cladosporium on spring wheat grain surface.
A total of 15 fungal genera were detected in surface sterilized wheat grains (Table 6).Internal tissues of wheat kernels were infected mainly by the same fungal species as those found on the grain surface, except for Acremoniella and Gonatobotrys, which occurred in 0.1% − 5.3% and in 1.4% − 14.3% of spring wheat grains, respectively.Alternaria spp.(mostly A. alternata) was the most frequently detected species in the internal tissues of wheat grains, occurring in 46.3% − 96.5% of winter wheat and in nearly 100% of spring wheat grains.Aspergillus spp.occurred in 3.3% (in 2005) and in 2.5− 3.9% (in 2007-2008), Cladosporium spp. in 26.9% − 63.6% and in 35.6% − 77.8% of winter and spring wheat grains, respectively.Pyrenophora spp.infected 1.1% of winter wheat grains in 2005 and 0.3− 4.4% of spring wheat grains in 2007− 2008.Fusarium spp.were detected in 0.9% − 4.7% of winter wheat and in 32.8% − 37.1% of spring wheat grains.Penicillium spp.infected about 2% (1.3− 2.5) of winter wheat and from 1.6% to 11.2% of spring wheat grains.The other fungi (Acremonium, Arthrinium, Botrytis, Mucor, Nigrospora, Trichoderma and Verticillium) occurred only sporadically and at negligible levels (from 0.1% to 5.3%).

Discussion
Fungal species belonging to 16 genera were identified in spring and winter wheat grains during 2005−2008.Very similar fungal complexes devel-oped on the surface and in the internal tissues of grains.Alternaria, Cladosporium, Fusarium and Penicillium were the most frequent in grain samples: Alternaria 46.3% − 99.9%, Cladosporium 26.9% − 77.8%, Fusarium 0.9% − 37.1%, Penicillium 1.3% − 2.5%.Isolation frequency of Aspergillus varied more among years (from 0 to 94.4) compared with that of the above mentioned fungi and, depending on the year, infected grain accounted for from 0 to 3.9% of the grain tested.All these fungi have been previously reported as prevalent in wheat grains (Clear and Patrick 1993, Lõiveke et al. 2004, Rajput et al. 2005, Semaškienė et al. 2005, Bakutis et al. 2006, Gohari et al. 2007).
Most of the identified fungi normally exist as saprophytes or weak plant pathogens.While Fusarium species is mainly associated with FHB, one of most important fungal diseases of wheat in all cropping areas worldwide, which reduce yield, seed quality and causes mycotoxin production in grain (Botalico et al. 1989, Tuite et al. 1990, Argyris et al. 2003).The Alternaria, Fusarium, Peni-  cillium and Aspergillus species may decrease seed germination, induce seed discoloration, chemical and nutritional changes, and form mycotoxins, that constitute a health hazard for humans and animals, under both field and storage conditions (Sauer 1988, Pitt 2000, Bryden 2007, Maciorowski et al. 2007, Malaker et al. 2008).
Our results indicated that spring wheat grain samples had fungal counts higher (14.8 × 10 3 − 80.3 × 10 3 cfu g -1 ) than winter wheat grain samples (7.2 × 10 3 to 24.8 × 10 3 cfu g -1 ) as well as Fusarium infection level: 32.8% − 37.1% and 0.9% − 4.7%, respectively.Similar differences in the Fusarium infection level between winter and spring cereals in Lithuania were reported in previous researches (Semaškienė et al. 2005, Suproniene et al. 2010).The obtained results also agree with those obtained by Kosiak et al. (2007).This might have resulted from some environmental factors: meteorological conditions, time and length of flowering period and source of infection (Suproniene et al. 2010).Yearly differences in fungal counts and infection levels were also indicated during this study, which is in accordance with other investigations (Clear andPatrick 1993, Mankevičienė et al. 2006).
Our investigation showed that the high fertilizer rates may have influence on the increasing of fungal contamination on wheat grain.This agrees with the data reported by Mankevičienė et al. (2006), where the high fertilization level (N 180 P 80 K 140 S 13 ), used for conventionally grown winter wheat 'Ada', increased grain surface contamination with fungi by 75.5% compared with the unfertilized wheat.High fertilizer rates mainly increased the amount of Cladosporium fungi on the wheat grain surface.This can be explained by the investigations done by Last (1955), who has demonstrated a significant increase in spore concentration of Cladosporium spp.and some other fungi in the air in a fertilized (N, P and K) wheat crop.The influence of high fertilizer rates on grain infection with Fusarium and Penicillium fungi was significant only in separate years during our study.Previous investigations also indicate that higher fertilizer rates may increase Fusarium infection in winter wheat (Teich and Nelson 1984, Martin et al. 1991, Lemmens et al. 2004, Krnjaja et al. 2009).
Conservation tillage systems involve leaving all or part of the crop residue on the soil surface after harvest in an effort to reduce soil erosion caused by wind and water runoff (Dill-Macky and Jones 2000).However, a lot of fungal species are capable of surviving saprophytically on plant debris, which might result in an increase in the residueborne diseases of cereals.As was previously shown by Ploetz et al. (1985), in no-tillage plots the mean propagule densities of total fungi in soil were significantly higher than in the plots tilled to 15 cm.Perello (2010) has recently reported that increasing of the foliar diseases caused by necrotrophic pathogens such as Alternaria, Cladosporium, Bipolaris, Pyrenophora and Septoria may also be due to the cultural practices such as conservation tillage, nitrogen fertilization and irrigation as well as conducive weather conditions.It was expected, that removal of the pre-crops' straw from the fields with the purpose of using it for bioenergy could help us to decrease the fungal infection in cereals.However, the obtained results indicated that notillage significantly increased fungal contamination (cfu g -1 ) of spring wheat grains in 2006 and 2007 and winter wheat grain infection with Alternaria (2005Alternaria ( −2006)), Aspergillus (2005) and Cladosporium (2006) species.It is likely that the residues (roots and stalks) left in the field after harvesting are still a relevant source of the fungal infection in cereals in no-tillage treatments.Since wheat precrops were pea and rape, we did not observe any significant increase in wheat pathogenic fungi such as Fusarium and others.However, Alternaria, Aspergillus and Cladosporium species may survive on a wide range of plants including pea and rape (Begum et al. 2004;Brazauskiene and Petraitiene 2006;Brazauskiene et al. 2006).On the other hand, the Verticillium longisporum and V. dahliae are known as Verticillium wilt causal agents of pea (Isaac and Rogers, 1974) and rape (Steventon et al. 2002) grown as pre-crops in our experiment.Small cereals are non-hosts for Verticillium pathogens.However, a greenhouse experiment showed that inoculation of wheat and barley with V. longisporum leads to various-degree stunting at close to the fully ripe stage (Johansson et al. 2006).During our study, Verticillium fungi were detected on the grain surface and in the internal tissues of wheat grains.This confirms Johansson's et al. (2006) suggestions that the plant species outside the Brassicaceae can act as reservoirs of Verticillium inoculum.
Another point to be discussed is use of glyphosate in the fields.Previous glyphosate use was consistently associated with higher FHB levels, while Cochliobolus sativus, the most important common root rot pathogen, was negatively associated, with previous glyphosate use (Fernandez et al. 2009).In our study, glyphosate was used only in no-tillage treatments; however, we did not observe any increase in Fusarium infection in wheat grains.
Our findings suggest that agronomic factors are very important and may increase winter wheat grain fungal contamination.Since the residues of pre-crops were collected and removed from the experimental field and pre-crops were non-hosts for wheat pathogens during the test period, further research is needed to explore the effects of these factors on grain fungal infection level.

Figure 1 .
Figure 1.The influence of no-tillage on winter wheat grain infection with Alternaria, Aspergillus and Cladosporium fungi in 2005-2006.

Figure 2 .
Figure 2. The influence of high fertilizer rates on spring wheat grain infection with Fusarium and Penicillium fungi in 2006-2007.

Table 1 .
Experimental design of different soil tillage-fertilization systems.
Isolation frequency of Aspergillus, Fusarium and Mucor species in winter wheat grain samples was higher in 2005 than that in 2006.Acremonium, Arthrinium, Botrytis and Verticillium were identified only in 2005.Isolation frequency of Penicillium species was similar in both years, while Alternaria was more frequent in 2006.Fungal species of 9−12 genera were identified on spring wheat grain surface.Alternaria, Fusarium and Cladosporium species were detected in all spring wheat grain samples in 2006, Nigrospora and Trichoderma were present only in 2006, however Aspergillus and Pyrenophora only in 2007−2008.Acremonium, Alternaria, Arthrinium, Botrytis and Mucor were less frequent or not detected in 2008.Penicillium and Verticillium were more frequent in 2008 than in 2006−2007.

Table 4 )
. Grains were more contaminated in 2006, than 2007 and 2008.No-tillage significantly increased spring wheat grain surface contamination in 2006 and 2007.High fertilizer rates increased fungal count only in 2006 and moderate rates in 2008.

Table 3 .
The fungal contamination of winter wheat grain as influenced by different tillage-fertilizationtreatments,  2005−2006.

Table 4 .
The fungal contamination of spring wheat grain as influenced by different tillage-fertilizationtreatments,  2006−2008.

Table 5 .
Analysis of variance p values from grain-testing the total count of fungal colony forming units (cfu g -1 ), Cladosporium spp.and other fungal colony forming units (cfu g -1 ) from wheat grain surface,2005−2008.