THE OCCURRENCE OF FUNGI ON ROOTS AND STEM BASES OF COMMON WHEAT ( Triticum aestivum ssp . vulgare L . ) AND DURUM WHEAT ( Triticum durum Desf . ) GROWN UNDER TWO LEVELS OF CHEMICAL PROTECTION

Investigations were carried out in 2007-2009 on the plots of the Felin Experimental Station belonging to the University of Life Science in Lublin. The studies comprised two cultivation lines of durum wheat (Triticum durum L.): STH 716 and STH 717, as well as the ‘Tonacja’ cultivar of common wheat (T. aestivum ssp. vulgare L.). Two levels of chemical protection were applied in the cultivation: minimal and complex protection. Infection of wheat roots and stem bases was recorded in each growing season at hard dough stage (87 in Tottman’s scale, 1987). After three years of study, the mean disease indexes for the analyzed wheat genotypes in the experimental treatment with minimal protection were 31.13, 30.43 and 38.83 for, respectively, the ‘Tonacja cultivar’ and the cultivation lines of T. durum STH 716 and STH 717. In the experimental combination with complex protection, after three years of study the disease indexes ranged from 25.26 (T. durum STH 716) to 30.83 (T. durum STH 717). The results of mycological analysis of diseased plants showed that Fusarium spp., especially F. culmorum, F. avenaceum as well as Bipolaris sorokiniana and Rhizoctonia solani, caused root rot and necrosis of wheat stem bases. The analyzed chemical protection levels did not significantly influence grain yield of the investigated genotypes of T. aestivum and T. durum.


INTRODUCTION
In recent years, interest in the cultivation of durum wheat (Triticum durum Desf.), which was only of regional character, has been on the increase.Durum wheat is the best raw material to produce pasta (R a c h o ń and S z u m i ł o , 2006).According to the present authors, the quality of the grain of Polish cultivars and lines of T. durum satisfies raw material standards to produce pasta and it does not significantly differ from foreign cultivars (R a c h o ń and S z u m ił o , 2002).Agrotechnical studies by R a c h o ń et al. (2002) on new cultivation lines of durum wheat from the Lublin Centre of the Institute of Genetics, Plant Breeding and Biotechnology point to the possibility of obtaining high and stable yields of this wheat in the climatic conditions of the Lublin region.
The factors decreasing the quality and size of the yield of wheat, including T. durum, are infectious diseases.In the countries where durum wheat is grown in larger areas the crops are infected by the bacterium Clavibacter tritici (C a r l t o n and V i d a v e r ; D av i s et al.; W i e s e , 1998) and by fungi of the genus Fusarium (T a m b u r i n -I l i n i c and G r i f f e y , 2008).Among the pathogens of the assimilation apparatus, a serious role is played by Blumeria graminis DC.Speer f. sp.tritici, Drechslera tritici-repentis (Died.)Shoem.as well as by Puccinia recondita Rob.ex Desm.f. sp.triticina (Eriks.)(W i e s e , 1998: P an a s i e w i c z et al. 2008).
Agrotechnical studies on yields of durum wheat (T.durum) have been conducted at the Department of Plant Cultivation of the University of Life Sciences in Lublin for a few years.Because of considerable importance of common wheat (T.aestivum ssp.vulgare L.) diseases caused by fungi responsible for root and foot rot diseases, research was undertaken on the role of these pathogens in infecting the roots and the stem base of T. aestivum ssp.vulgare L., 'Tonacja' and the cultivation lines of T. durum using two levels of chemical protection.

MATERIALS AND METHODS
The studies were conducted in 2007-2009 in the field of the Felin Experimental Farm belonging to the University of Life Sciences in Lublin (51 o 22' N, 22 o 64'E) on lessive soil made from loess formations, with the granulometric composition of medium loam classified as the good wheat complex, soil quality class II.The content of available forms of P, K and Mg was as follows: P -76.0;K -119.0;Mg -55.5 (mg×kg -1 of soil), and its pH was 6.3 in KCl solution.
The studies comprised two cultivation lines of durum wheat (Triticum durum Desf.):STH 716 and STH 717 as well as the 'Tonacja' cultivar of common wheat (T.aestivum ssp.vulgare L.).Two levels of chemical protection were applied in the cultivation.The first level included minimal protection where the grain was dressed with Oxafun T 75 DS/WS (200 g×100 kg -1 of grain).Chwastox Trio 540 SL in the dose of 2 dm 3 ×ha -1 was used to control weeds.The second level of complex chemical protection included seed dressing with Oxafun T 75 DS/WS at a rate of 200 g×100 kg -1 of grain; weeds were controlled by using two herbicides: Puma Uniwersal 069 EW at a dose of 1.2 dm 3 ×ha -1 and Chwastox Trio 540 SL at 2 dm 3 ×ha -1 .At the tillering stage (20 in Tottman's scale 1987), the fungicide Alert 375 S.C. at a rate of 1.8×ha -1 , the insecticide Decis 2,5 EC at 250 cm 3 ×ha -1 , and Stabilan 750 SL at 1.8 dm 3 ×ha -1 were also applied.In both protection levels, mineral fertilization was applied before sowing at a rate of 26 kg P. ha -1 and 66 kg K×ha -1 .Besides, top dressing was applied twice: using nitrogen at a rate of 70 kg×ha -1 -after the start of growth and 30 kg N×ha -1 at the third node stage.Soil tillage was typical of the plough system.Wheat was sown in the third decade of September in each year.The sowing density was 500 grains per m 2 .
The experiment was carried out in a randomized block design, in four replications in the field after winter rape.The area of the plots was 10 m 2 .
In each growing season, the infection of the wheat roots and stem base was assessed at the hard dough stage of grain (87 in Tottman's scale, 1987).For this propouse, 200 stems (4×50) were taken from each experimental treatment in different places of the plots of the cultivar and cultivation lines.In the laboratory, the proportion of stems with necrotic streaks on the roots and lower internodes was determined and the degree of infection was established according to a 5-grade scale (E n g -C h o n g P u a et al. 1985).Disease indexes were calculated using McKinney's formula (Ł a c i c o w a , 1969).Besides, in each year of the study, after harvest grain yield was determined, on a per hectare basis, from the plots sown with the analyzed wheat genotypes and with two levels of protection.The obtained results were statistically analyzed using Tukey's confidence half-intervals (Ż u k , 1989).
A laboratory mycological analysis was also conducted.The number of plant fragments taken for the analysis and the method of analysis were the same as in earlier studies (K i e c a n a et al., 2003).Fungi of the genus Fusarium were identified according to N e l s o n et al. (1983) as well as L e s l i e and S u m m e r e l l (2006).Other fungi species were determined according to the keys and monographs by E l l i s (1971), D o m s c h et al.(1980), R a m i r e z (1982), and R i f a i (1969).

RESULTS
Plants with the symptoms of root rot and necrotic streaks on the lower internodes occurred in each growing season with minimal and complex protection.In 2007 the disease indexes in the experimental combination with minimal protection ranged from 21.3 (Tonacja) to 27.8 (T. durum STH 716) and they did not differ significantly (Table 1).In 2008 significantly higher values of the disease index were observed in the case of the cultivar Tonacja and the cultivation line T. durum STH 717;they were,respectively,33.7 and 38.7.In 2009 a significantly higher value of the disease index was found for the cultivation line  (Table 1).After three years of study, the disease indexes for the analyzed wheat genotypes in the experimental treatment with minimal protection were 31.13, 30.43 and 38.83 for, respectively, the 'Tonacja cultivar' and the cultivation lines of T. durum STH 716 and STH 717, and they did not differ significantly (Table 1).The disease indexes in the experimental treatment with complex protection ranged in 2007 from 15.6 (T. durum STH 716) to 20 (Tonacja), in 2008 -from 25.2 (T.durum STH 716) to 32.4 (T.durum STH 717), while in 2009 from 31.7 (Tonacja) to 41.5 (T.durum STH 717).In the last two years of the study, the disease indexes for the cultivation line T. durum STH 717 were significantly the highest (Table 1).
The statistical analysis of the disease indexes showed a significant effect of the level of protection on the health of stems in 2007 in relation to the cultivation line T. durum STH 716, while in the years 2007, 2008 and 2009 with respect to the cultivation line STH 717.In the case of the cultivar 'Tonacja' (T.aestivum), no significant effect of the level of chemical protection on the health of plants was observed in any of the years of the study (Table 1).
1280 fungi isolates, including 563 from the roots and 717 from the stem base, were obtained as a result of the mycological analysis of the diseased plants from the experimental treatment with minimal protection (Table 2).In each growing season, colonies of the genus Fusarium spp.were obtained both from the roots and the stem base.In the period 2007-2009, isolates of those fungi accounted for, respectively, 89.2% (489 isolates), 53.7% (187 isolates), and 50.9% (208 isolates) of all isolations (Table 2).In total, after three years of study isolates of fungi of the genus Fusarium constituted 69.1% (884 isolates) of all the obtained fungi isolates (Table 2).Considering the total number of fungi isolates of the genus Fusarium from the diseased plants of durum wheat and common wheat obtained within the three-years study period, the dominating species was F. culmorum whose isolates constituted 51.0% (451 isolates) of all Fusarium spp.(884 isolates).Considerable quantities of F. avenaceum -23.6% (209 isolates) of total Fusarium spp.and F. oxysporum -18.9% (167 isolates) were also isolated (Table 2).
In all the years of the study, the species Alternaria alternata, whose colonies constituted 9.9% (127 isolates) of all isolations, was also isolated from the infected plants (Table 2).
23 isolates of the species Bipolaris sorokiniana were isolated in 2007 and 2008 from the roots and the stem base, which accounts for 1.8% of all isolates during the three years of the study.Besides, Rhizoctonia solani was obtained in 2008 and 2009 from the roots and stem base; isolates of this species constituted, respectively, 5.2% (18 isolates) and 2.7% (11 isolates) of all fungi in those years.In 2007 Aureobasidium pullulans was also obtained; its isolates made up 0.5% (7 isolates) of total isolations (Table 2).
The mycological analysis of common and durum wheat growing on the plots with complex protection provided 911 fungi isolates, including 395 from the roots and 516 from the stem base (Table 2).The colonies isolated during the three years of the study included 83.1% (757 isolates) of fungi of the genus Fusarium.In 2007 88.1% (436 isolates) of the obtained fungi isolates belonged to this species, whereas in the years 2008 and 2009 the colonies of Fusarium spp.constituted, respectively, 79.3% (165 isolates) and 75.0%(156 isolates) of total isolations (Table 2).The dominating species in 2007 was F. culmorum whose isolates accounted for 90.1% (393 isolates) of all isolations of Fusarium spp.(436 isolates) from the roots and the stem base.The other colonies of fungi of the genus Fusarium belonged to the following: F. avenaceum -3.0% (13 isolates), F. graminearum -2.5% (11 isolates), F. sporotrichioides -2.1% (9 isolates), F. crookwellense -1.6 (7 isolates) of all Fusarium spp.isolates (436).Few isolates were obtained from F. poae and F. oxysporum (Table 2).In the growing seasons of 2008 and 2009, considerable numbers of F. avenaceum were obtained both from the roots and the stem base.In 2008 its isolates constituted 41.2% (68 isolates) of all Fusarium spp.isolates (165), whereas in 2009 they made up 46.2% (72 isolates) of all Fusarium spp.isolates (156) isolated from the roots and the stem base (Table 2).Isolates of F. culmorum from the roots and the stem base in the growing seasons of 2008 and 2009 constituted, respectively, 35.2% (58 isolates) and 10.3% (16 isolates) of all Fusarium spp.isolates isolated from the roots and lower internodes.In 2008 the other species from this genus were represented by F. oxysporum -21.2% (35 isolates) and F. sporotrichioides -2.4% (4 isolates), whereas in 2009 by F. oxysporum -42.9% (67 isolates) and F. equiseti -0.6% (1 isolate) of the total number of Fusarium spp.Isolates (Table 2).In 2007 and 2008 the species B. sorokiniana was isolated both from the roots (10 isolates) and the stem base (10 isolates); it accounted for 2.2% of total isolations in the three years of the study.Colonies of A. alternata constituted 3.2% (29 isolates) of all fungi isolates obtained during the three years of the study, A. pullulans 2.0% (18 isolates), while R. solani made up 2.2% (20 isolates) (Table 2).
In the case of both experimental treatments, the significantly highest grain yield, on average during the three-year study period, was observed for cv.'Tonacja'.It was 7.85 t×ha -1 in the treatment with minimum protection and 9.33 t×ha -1 in the one with complex protection (Table 3).On the other hand, the mean grain yield of the cultivation lines STH 716 and STH 717 in the case of minimum protection was, respectively, 4.27 t×ha -1 and 4.65 t×ha -1 , whereas in the case of complex protection it was 5.1 t×ha -1 and 6.10 t×ha -1 .No statistically significant differences were observed in the yield of particular wheat genotypes depending on the level of chemical protection (Table 3).
The temperature in the Lublin region -Felin during the 2006/2007 growing season was higher as compared to the long-term mean in all the months (September 2006-August 2007)

DISCUSSION
The results of the present study, pointing to frequent infection by fungi of the genus Fusarium of the roots and stem base of durum and common wheat cultivated using two levels of chemical protection, confirmed their great importance as pathogens causing root and foot rot of cereals (Ł a c i c o w a et al. 1985; Ł a c i c o w a and P i ę t a , 1998; K i e c a n a and M i e l n i c z u k , 2001; K i e c a n a et al. 2003; 2008; 2009; K u r o w s k i et al. 2007).The species composition and number of Fusarium spp.isolates differed in particular years of the study and it can be assumed that their occurrence was mainly affected by climatic factors, while the level of the applied chemical protection did not have any considerable effect, like in the study by P l ą s k o w s k a and C h r z a n o w s k a --D r o ż d ż (2008).
Special attention should be paid to F. culmorum obtained both from the roots and lower internodes of the stems at both levels of chemical protection, especially in the year 2007 which was characterized by warm humid weather in May, June and July and which proved conducive to the occurrence of this species.According to Ł a c i c o w a and P i ę t a (1998) as well as K i e c a n a et al. (2003), the harmfulness of F. culmorum to cereals is greater in higher temperature.This species plays a big role in injuring wheat, especially its roots, under different conditions of cultivation (Ł a c i c o w a et al. 1985; K u r o w s k i et al. 2007; M a j c h r z a k et al. 2008; F e r n a n d e z et al. 2009).Fusarium culmorum proved to be the most pathogenic to wheat seedlings as compared to F. equiseti, F. reticulatum and F. acuminatum (S t r a u s b a u g h et al. 2004).This fungus colonizes both live and dead tissues in a similar way.This is a so-called indirect way of colonization characterized by fast overgrowth of the tissues, slow utilization of the subsoil and the ability to survive unfavourable periods in the form of chlamydospores (Ł a c i c o w a et al. 1985).Metabolites of F. culmorum contain, among others, inorganic acids, which are highly toxic to cereal seedlings.They cause excessive intake of phosphorus by plants, inhibition of germination and a decrease in root length as well as in the formation of root hairs (K a u t o u l i and M a r c h a n t , 1981).Besides, F. culmorum produces deoxynivalenol, which reduces the content of chlorophyll a and b and caretonoids in leaf tissues (submitted to the effect of this compound at a concentration of 30-90 ppm) (B u s h n e l l et al. 2010).Deoxynivalenol also causes disturbances in the permeability of cell membranes and inhibits the biosynthesis of protein and nucleic acids in plant cells (W o j c i e c h o w s k i et al. 1995); moreover, it increases the level of free fatty acids (U e n o , 1983; K e n d a l and M c K e r s i e , 1989; M i l l e r and E v e n , 1997).Moreover, trichothecenes, including DON, also affect ribosomal L3 (coded by the Rpl 3 gene from rice), which is necessary for the proper functioning of peptide transferase (H a r r i s and G l e d d i e , 2001).
In addition to F. culmorum, the species F. avenaceum, known for its harmfulness to wheat, was obtained from this cereal species under analysis (Ł ac i c o w a et al. 1985; F e r n a n d e z et al. 2009).The presence of F. avenaceum on wheat stems in the growing seasons with different weather conditions is confirmed by the reports about a great tolerance of this fungus to temperature and humidity (K i e c a n a , 1994; M i e l n i c z u k , 2001; K i e c a n a et al. 2003; K i e c a n a and M i e l n i c z u k , 2010).Furthermore, a study on the population of this fungus present in crop residues of wheat grown in Canada (Saskatchewan) pointed to the common occurrence of F. avenaceum in them, since it can constitute a threat to the next crops (F e r n a n d e z et al. 2009).Pathogenicity of F. avenaceum results, for example, from the production of toxic moniliformin (P a c k a , 1997).
The occurrence of the species F. avenaceum and F. culmorum on the roots and the stem base of wheat in both chemical protection treatments and in all the study years confirms the competitive abilities of these fungi, enabling them to live in the soil, and infection of plant roots (Ł a c i c o w a et al. 1985; Ł a c i c o w a and K i e c a n a , 1987).The species F. crookwellense, which is a recognized pathogen of cereals, including wheat, was isolated under different climatic conditions from the infected roots and stem base of wheat in the analyzed cultivation systems (R o s s i et al. 1995; K i e c a n a and M i e l n i c z u k , 2001; K i e c a n a et al. 2003, 2008; S t r a u s b a u g h et al. 2004, 2005).
The applied chemical treatments to control weeds which were hosts to Fusarium spp.decreased the population of species such as F. avenaceum and F. sporotrichioides.Besides, the reduction of the occurrence of weeds by herbicide treatments that decreased moisture in the crop also affected the population of other species colonies of the genus Fusarium (F a r r et al. 1989, according to S h a r , 2003).
It turned out that at both levels of protection, in the conditions when wheat was threatened by Fusarium spp., the cultivation line T. durum STH 717 was most infected by those fungi, which is indicated by the values of the disease indexes.
The fungi accompanying Fusarium spp. in the infection of the roots and stem base of the analysed wheat species were B. sorokiniana and R. solani.Bipolaris sorokiniana constitutes a serious threat to cereals, especially barley and wheat cultivated in various climatic regions of both Americas, the Republic of South Africa and Europe (Ł a c i c o w a et al. 1990; S c o t t , 1995; V a l j a v e c -G r a t i a n and S t ef e n s o n , 1997; A l m g r e n et al. 1999 In the analysed conditions at both chemical protection levels, R. solani should be considered an additional infectious factor causing root and stem base diseases of durum and common wheat, like in the study by Ł a c i c o w a et al . (1990) concerning spring barley.
Similarly to the study by P l ą s k o w s k a and C h r z a n o w s k a -D r o ż d ż (2008), the present research showed that weather conditions had a greater effect on grain yield than the level of chemical protection of plants.

Fungi
isolated from the diseased roots and stem base of T. aestivum and T. durum cultivated under minimal and complex plant protection conditions in 2007chemical protection r -roots, s -stem base ; D u v e i ll e r and G a r c i a A l t a m a r i n o , 2000; F e r n a nd e z et al. 2000).The pathogenicity of B. sorokiniana is related to the production of secondary metabolites, especially prehelminthosporol (P r y c e et al. 1999, according to K u m a r et al. 2001; C a r l s o n et al. 1991, according to A l m g r e n et al. 1999).The latter reduces the efficiency of hydrogen-calcium pumps in a plant cell (O l b e et al. 1995).Besides, common reactions of plants to the effect of toxins of B. sorokiniana include disturbances in the functioning of the cell cytoplasmatic membranes.In the case of barley infection by B. sorokiniana, the effect of increased permeability of cell membranes was found by W i ś n i e w s k a et al. (1998).
from 1.1 o C (August 2006) to 6.2 o C (January 2007).On the other hand, rainfall exceeded the long-term average in November 2006, January, March, May, June and July 2007 by respectively: 5.0; 137.0; 17.0; 39.8; 33.4 and 11.5%.The lowest rainfall was recorded in September 2006 -only 21.1% of the normal level.In 2007/2008 air temperature between September 2007 and August 2008 was lower than the long-term mean in the months of October and November 2007 and May 2008 by respectively 0.3, 1.5 and 0.2 o C, while in the other months it was higher from 0.1 o C (September 2007) to 5.0 o C (February 2008).The rainfall level ranged from 39.4 mm in June 2008 to 251.2 mm in March 2008.In the 2008/2009 growing seasons, air temperature was lower than the long-term mean by 0.3 o C in September 2008 and 0.1 o C in June 2009.On the other hand, rainfall exceeded the long-term mean in the following months: September 2008 by 96.2%, October 2008 by 37.7%, December 2008 by 39.0%, February 2009 by 48.8%, March 2009 by 169.8%,May 2009 by 21.9%, and June 2009 by 90.7%.The lowest amount of rainfall was observed in April of 2009 -7.1% of the normal level (Table 4).

Table 3 .
Grain yield of common wheat and durum wheat in2007-2009 [t×ha -1 ] Means in columns differ significantly (P  0.05) if they are not marked with the same small letter Means in lines differ significantly (P  0.05) if they are not marked with the same capital letter

Table 4 .
Air temperature and rainfall in the 2006Air temperature and rainfall in the  /2007Air temperature and rainfall in the  -2008Air temperature and rainfall in the  /2009growing seasons of winter wheat (T.aestivum,T.durum)
S t r e s z c z e n i e