MALTING BARLEY SEED HEALTH DEPENDING ON DIFFERENT FUNGICIDE TREATMENT

Field studies were carried out in the 2004 – 2005 growing seasons. The mycological analysis of malting barley (varieties Prestige and Sezam) grains was performed twice: on seeds stored for 30 days and on seeds stored for fi ve months. The infl uence of fungicide treatment on species diversity and the amount of fungal pathogens on kernels of both varieties of malting barley were determined in the studies. Main fungal pathogens of both varieties of malting barley were fi eld fungal species, such as: Alternaria alternata, Epicocum purpurascens, and fungi of the genus Fusarium. The extension of the grain storage period to fi ve months resulted in an increased share of pathogenic species.


INTRODUCTION
In recent years new technologies have been introduced in cereal growing, designed to create optimal conditions for plant growth and development.Adequate protection against diseases in the growing period enables the production of full-value grain suitable for further technological processing and for animal feed purposes.Cereal grains are colonised by many microorganisms, mainly bacteria and fungi.They are initially infected by the so-called "fi eld fungi" which can be saprotrophs or pathogens.Popular fi eld fungi include the following: Alternaria alternata, Epicoccum purpurascens, Fusarium spp.An important pathogenic species often found on barley grains is the fungus Bipolaris sorokiniana, the culprit of cereal seedling and root canker (K n u d s e n et al. 1995).But on stored seeds, the so-called "storage fungi" develop, belonging to the genus Aspergillus, Penicillium or from the family Mucoraceae (C h r i s t e n s e n , 1972).Grain infection reduces the vigour, restricts germination and is the cause of yield reduction (Š a r i ć et al. 1997).It affects the quality of malt, changes its colour and decreases the taste value.It results from negative changes in the composition of proteins found in the grain.Malting barley grain showing symptoms of strong infection by fungal pathogens cannot be used in the brewing industry; it is only used as animal feed (M a t h r e , 1997).Fungi colonising cereal grains can synthesise numerous secondary metabolites whose presence may be harmful to humans and animals (M u n k v o l d , 2003).Toxic fungal metabolites exhibit cancerogenic, cytogenic, embryogenic, teratogenic and mutagenic effects (P i ę t a , 1997).
The aim of the study was to determine the effect of different intensities of disease protection during the growing period on the health of two varieties of malting barley grain determined after up to 30 days from the harvest and after a 5-month storage period in a fl oor-storage grain store.

MATERIALS AND METHODS
The study material was malting barley (Hordeum vulgare L.) grains, varieties Prestige and Sezam, obtained from a strict fi eld experiment carried out at the Agricultural Experimental Station in Bałcyny near the city of Ostróda.7 methods of barley protection (with different intensity) were used and compared to the untreated control: Doses of preparations were compliant with the recommendations of the Polish Institute of Plant Protection; they were applied at 200 dm -3 ha -1 of water, following good experimental practice.The active substance included: tebuconazol + triozoxid (Dubelt J), fl usilazole and carbendazim (Alert 375 SC), picoxystrobin (Acanto 250 SC), fenpropimorph (Corbel 750 EC), fl usilazole and fenpropimorph (Cerelux Plus), azoxystrobin (Amistar 250 SC), propiconazole and cyproconazole (Artea 330 EC), ethephon (Cerone 480 SL).For weed control, the herbicide Mustang 306 SE was used (s.a.-fl orasulam + 2,4 D).
Malting barley was grown on medium greybrown podzolic soil, belonging to the 3rd agricultural usefulness complex.Sowing was carried out as early as possible, at a spacing of about 370 caryopses per 1 m 2 .45 kg P 2 0 5 , 85 kg K 2 O and 40 kg N were applied before sowing.
Mycological examination was performed on two dates: 1) -up to 30 days after the harvest, 2) -fi ve months after the harvest.The analysis was made using the method designed by N a r k i e w i c z -J o d k o (1991).The grain from the 2004 and 2005 harvest used for analysis was stored in a grain store of the Production and Experimental Facility in Bałcyny, in jute bags.
The pattern of thermal and soil moisture conditions in 2004 was favourable for barley growing.A wet spring guaranteed good water supply for plants at the tillering and shooting stages.But the total rainfall during maturation in July was lower than the long-term mean (Tab.1).The year 2005 was defi nitely too dry, and this was the case throughout the whole critical period.The water shortage was alleviated as late as July.

RESULTS
Following the harvest, 3485 cultures of fungi belonging to 40 species were isolated from the grain of malting barley var.Prestige, in both years of study.The number of isolates in the years of study was different.21.8% more fungal cultures were obtained in 2005 than in 2004.But no signifi cant differences were found in the species composition (29 and 28 species in the respective years).The species Alternaria alternate was isolated in the greatest number, accounting for 45.88% of the total colony (Tab.2).Other fungi colonising the grain in a great number included Epicoccum purpurascens (10.78%),Mucor hiemalis (5.67%) as well as fungi of the genus Fusarium (30.38%), being typical pathogens.Among fungi of the genus Fusarium, F. equiseti (544 col.), F. oxysporum (183 col.) and F. poae (93 col.) were predominant.Among other pathogenic fungi, the species Bipolaris sorokiniana (130 col.-3.73%) accounted for a large share of the total number of isolates.Botrytis cinerea was isolated sporadically.
Barley grains in the combination E, in which two foliar fungicide treatments were applied, were infected most slightly by fungi.Fungi potentially pathogenic to cereals: + Bipolaris sorokiniana, Botrytis cinerea, Cylindrocarpon destructans and representatives of the genus Fusarium, accounted for 28.8% (2004) and 31.3% (2005) in this combination.
After a 5-month storage period, an increase in the number of isolated fungi was observed (Tab.3).3679 colonies were obtained, belonging to 36 species.Among these fungi, the species Alternaria alternata was isolated most frequently, accounting for 43% of the total number of isolates.Cylindrocarpon destructans was also isolated in a great number (9.8%).The ge- nus Fusarium was represented by 13 species, among which F. graminearum, F. equiseti and F. oxysporum dominated.After the 5-month storage period, a slight increase in the number of Epicoccum purpurascens and a marked decline in the number of fungi of the family Mucoraceae, included in typical storage fungi, were observed.Grains from the control combination were most strongly infected by fungi after the two-month storage period, and grains from the combination D and G, in which foliar fungicide treatment was applied, were infected in the slightest degree.However, in both these trial objects pathogenic species: Bipolaris sorokiniana, Botrytis cinerea, Cylindrocarpon destructans, and fungi of the genus Fusarium accounted for over 50% of all isolates.
3260 cultures of fungi belonging to 44 species were isolated from the grain of malting barley var.Sezam (Tab.4) after the harvest.The so-called "fi eld fungi", belonging to the following species: Alternaria alternata (45.0%),Fusarium spp.(29.7%),Epicoccum purpurascens (10.2%) and Bipolaris sorokiniana (6.5%), were isolated in the greatest number.Typical "storage fungi" accounted for 5.0%, and they were represented by the genera Mucor and Rhizopus.Among fungi of the genus Fusarium, the species F. equiseti and F. poae were predominant.
The smallest number of fungi was isolated from barley grains obtained from the control (untreated) and the combination G with full chemical protection in the form of seed dressing and three foliar treatments with the fungicides Corbel, Amistar and Artea.The smallest percentage share of pathogenic fungi was noted on barley grains obtained from the trial object B (only seed dressing) and in the fi rst year of study from grains obtained from the combination G.
Potentially pathogenic fungi were represented most numerously in these combinations by Fusarium equiseti and Bipolaris sorokiniana.The species Alternaria alternata and Epicoccum purpurascens dominated in all the combinations.
Out of the storage fungi, Mucor hiemalis as well as single cultures of Mucor circinelloides and Mucor racemosus were isolated quite frequently.
After the 5-month period of storage of barley grain, the number of fungi colonising it did not change signifi cantly, but the percentage shares of particular species changed.A total of 3232 fungal cultures were isolated (33 species) (Tab.5).A marked increase, by as much as 10%, was found in the number of fungi of the genus Fusarium (from 29.7% up to 39.1%).They were represented by 15 species, among which Fusarium equiseti, F. avenaceum, F. oxysporum were predominant.The number of Epicoccum purpurascens cultures increased.But a signifi cant decline in the number of Alternaria alternata colonies (from 45.0% down to 36.8%) and of fungi of the family Mucoraceae was noted; Bipolaris sorokiniana was also isolated less frequently.
Barley grains from the trial object B, in which only seed dressing was applied, were colonised by fungi in the greatest number.474 fungal cultures from 21 species were isolated.Among them, pathogenic fungi accounted for 42% in 2004 and 20% in 2005, mainly of the genus Fusarium and Bipolaris sorokiniana.The least number of fungal cultures was isolated from grains obtained from the plots on which full threefold protection was applied (trial object G). 344 cultures belonging to 15 species were obtained.Among them, fungi potentially pathogenic to cereals dominated, in particular from the genus Fusarium, and they accounted for 50% in 2004 and up to 85% in 2005.A similar increase in the number of pathogenic fungi was observed in the trial object E where the fungicides Corbel and Amistar were applied.

DISCUSSION
The investigated grain of malting barley, var.Sezam and Prestige, was colonised after the harvest by typical "fi eld" fungi: Alternaria alternata, Epicoccum purpurascens and representatives of the genus Fusarium.In literature, we fi nd reports on the common occurrence of these fungi on cereal grains and seeds of many plants (N a r k i e w i c z -J o d k o , 1986; M a jc h r z a k , 1998; P a r r y et al. 1995).In own study, the species Alternaria alternata was the most frequently isolated fungus, accounting for from 36% up to 45% of total isolates.Alternaria alternata is a facultative parasite which synthesises toxic metabolites: alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT), altertoxin I, II and III (ATX-I, -II, and -III), tenuazonic acid (TeA) (L i et al. 2001 In our study, the greatest number of fungi of the genus Fusarium was found after the application of ½ of a dose of Amistar (azoxystrobin) used at the stage on grains from the plants obtained from the combination B (Dubelt J).
BBCH 42.M a g a n et al. (2002) have found that low doses of azoxystrobin may stimulate the production of mycotoxins both in in vitro conditions and on wheat grains.
Other authors have reported that the application of triadimenol doses sublethal to Fusarium graminearum resulted in increased production of DON and zearalennon (R a m i r e z et al. 2004).In the opinion of R a m i r e z et al. (2004), the application of chemical treatment partially reduces the incidence of typically toxinogenic fungi.
Among potentially pathogenic species, Bipolaris sorokiniana, Botrytis cinerea and Cylindrocarpon destructans were isolated.They are known polyphags affecting many plants.According to Ł a c i c o w a et al. (1990,1992), barley is susceptible to infection by Bipolaris sorokiniana at each growth stage.Grain yield losses as a result of damage caused by this pathogen may reach 30%.The occurrence of Botrytis cinerea, a fungus which is rarely mentioned as a cereal pathogen, on barley is also worth noting.However, in the opinion of the same authors, this species, as an occasional pathogen, is sometimes one of the causes of stem base diseases of barley (Ł a c i c o w a et al. 1990, 1992).According to M i c h a l s k i and H o r o s zk i e w i c z (2003), many pathogens are transferred with plant seeds.

CONCLUSIONS
1. Grains of both studied varieties of malting barley were colonised by typical fi eld fungi, such as: Alternaria alternata, Epicoccum purpurascens, and fungi of the genus Fusarium.2. Among fungi potentially pathogenic to cereals, Fusarium equiseti, F. avenaceum, F. oxysporum and Bipolaris sorokiniana were isolated in the greatest number.3. Grains of malting barley var.Prestige were colonised by fungi more numerously.4.After the 5-month storage period, more pathogenic fungi, mainly of the genus Fusarium, were isolated from barley grains.Among these fungi, the species F. equiseti accounted for the largest percentage share on stored grains.5.The largest number of fungi of the genus Fusarium was isolated from both malting barley varieties from the trial objects on which ½ of azoxystrobin dose was applied (trial object F). 6. Fungi of the genus Fusarium occurred least frequently on malting barley grains in the combination G (Dubelt + Corbel + Amistar + Artea).7. The application of the fungicide Amistar (azoxystrobin) reduced most strongly the occurrence of the species Alternaria alternate; this species occurred
; S c o t t 2001).Toxic metabolites produced by this species may inhibit the growth of cereal shoots, as well as they exhibit a toxic effect on cells of mammals (N a r k i e w i c z -J o dk o , 1986; S i l v a n a d a M o t t i a and S o a r e s , 2000; W a t a n a b e et al. 2007).The harmfulness of fungal toxins from the genus Alternaria is related to their very common occurrence in the environment, primarily on affected grains and fruit (W a t a n a b e et al. 2007).As reported by B r a z a u s k i e n ė , storage of grain at a temperature of 25°C for a period of 6-8 months reduces seed infection by Alternaria spp.by 50% (B r a z a u s k i e n ė et al. 2006).But the lowering of storage temperature does not reduce the level of infection by fungi belonging to this genus, since they are tolerant to low temperatures (M a j c h r z a k , 1998).Therefore, seed infection by fungi of the genus Alternaria can be of great signifi cance in regions characterised by a cool climate (S m i t h et al. 1988).In our study, fungi of the genus Fusarium were also isolated in a great number.They belong to widely distributed plant pathogens inducing many plant diseases and producing a number of phytotoxic compounds (B r o w n et al. 2006; N i e s s e n , 2007).Fusarium equiseti was the species which occurred most numerously on malting barley grains.As reported by A i g b e and F a w o l e (1999), this fungus occurs most frequently in regions characterised by a warm climate, though its presence has been observed on grains of cereals grown in the former USSR countries as well as in Norway and France (M a r a s a s et al. 1987, M o r r i s o n et al. 2002, I o o s et al. 2004).In Poland the sporadic occurrence of this species on barley grains has been described by P e r k o w s k i et al. (1997).According to many researchers, this fungus is not included in the Fusarium complex inducing ear fusariosis (L o g r i e c o et al. 2003), but it may cause root diseases, decay of tubers, fruit and seeds of different plants, including cereals (A i g b e and F a w o l e 1999; D e m i r c i and D a n e 2003; F e d e l -M o e n and H a r r i s , 1987).F. equiseti synthesises numerous secondary metabolites of different toxicity, among others, trichotecens A and B, butenolide, equisetin, zearalenon and fusarochromanone (M o r r i s o n et al. 2002).

Table 2
Fungi isolated from grains of spring barley, variety Prestige, 30 days after harvest.

Table 3
Fungi isolated from grains of winter barley, variety Prestige, 5 months after harvest.

Table 4
Fungi isolated from grains of winter barley, variety Sezam, 30 days after harvest.

Table 5
Fungi isolated from grains of winter barley, variety Sezam, 5 months after harvest.