FUNGI COLONIZING THE SOIL AND ROOTS OF TOMATO (Lycopersicum esculentum Mill.) PLANTS TREATED WITH BIOLOGICAL CONTROL AGENTS

Tomato plants, cv. Rumba Ożarowska, grown in the greenhouse of the University of Warmia and Mazury, were protected in the form of alternate spraying (twice) and watering (twice) with 5% aqueous extracts of the following plant species: Aloe vulgaris Lam., Achillea millefolium L., Mentha piperita L., Polygonum aviculare L., Equisetum arvense L., Juglans regia L. and Urtica dioica L. Plants not treated with the extracts served as control. After fruit harvest, samples of roots and soil were collected. The roots were disinfected and next placed on PDA medium. Soil-colonizing fungi were cultured on Martin medium. Fungi were identified microscopically after incubation. Pathogenic fungal species, Colletotrichum coccodes, Fusarium equiseti, F. oxysporum and F. poae, accounted for over 60% of all isolates obtained from the roots of tomato plants. The soil fungal community was dominated by yeast-like fungi (75.4%), whereas pathogenic fungi were present in low numbers. The applied 5% aqueous plant extracts effectively reduced the abundance of fungi, including pathogenic species, colonizing tomato plants and soil. The extract from P. aviculare showed the highest efficacy, while the extract from J. regia was least effective. Fungi showing antagonistic activity against pathogens (Paecilomyces roseum and species of the genus Trichoderma) were isolated in greatest abundance from the soil and the roots of tomato plants treated with A. millefolium, M. piperita and U. dioica extracts.


INTRODUCTION
Plants extracts show potential for biocontrol of the following pathogenic species: Colletotrichum capsici (O b a g w u et al. 1997), Rhizoctonia solani (S r i v a s t o v a and L a l , 1997; S e h a j p a l et al. 2009), Phytophthora infestans (S c h m i t t et.al. 2005), Erwinia carotovora ssp.carotovora (B d l i y a and D a h i r u , 2006), Fusarium oxysporum f. sp.ciceris (S a h a y a r a j et al. 2006) and Verticillium dahliae (S h o h o u g et al. 2009).In a pot experiment carried out by B u r g i e ł et al . (2008), extracts from plants of the family Apiaceae provided effective control of powdery mildew Erysiphe spp. on marigolds Calendula officinalis.S l u s a r e n k o et al. (2008) and P o r t z et al. (2008) demonstrated that greenhouse vegetables may be protected against fungus-like organisms (e.g.Phytophthora and Pseudoperonospora) with allicin from Allium sativum.Extracts from Paeonia suffruticosa, Hedera helix (R o h n e r et al. 2004), plants of the genus Cameroon (G o u f o et al. 2008) and A. sativum (P o r t z et al. 2008) showed high efficacy in late blight control in tomatoes.According to K o n s t a n t i n i d o u -D o l t s i n i s et al. (2006), Reynoutria sachalinensis extract has a fungistatic effect on Leveillula taurica, the causal agent of powdery mildew of tomatoes.
The aim of this study was to determine the quantitative and qualitative composition of fungal communities colonizing the soil and roots of tomato plants treated with aqueous plant extracts.

MATERIALS AND METHODS
Tomato plants Lycopersicum esculentum Mill., cv.Rumba Ożarowska, were grown in the greenhouse of the University of Warmia and Mazury in Olsztyn in 2006-2007.Tomato seedlings were planted in pots (one seedling per pot) filled with peat substrate and garden soil at a ratio of 1:3 (total 9 dm 3 of substrate, without inoculum).The experiment involved biological control (three weeks after seedling planting) in the form of alternate spraying (twice) and watering (twice) at 10-day intervals with aqueous extracts of the following plant species: Aloe vulgaris Lam., Achillea millefolium L., Mentha piperita L., Polygonum aviculare L., Equisetum arvense L., Juglans regia L. and Urtica dioica L. (45 ml/plant).Aqueous extracts were prepared from dry plant material (50 g) mixed with water (1 l), except for aloe whose fresh leaves were crushed in a mortar to obtain 5% extract.The experiment was performed in six replications (six pots).Plants not treated with the extracts served as control.After fruit harvest, samples of roots and soil were collected from each pot.Following disinfection (with 50% ethanol and 1% sodium hypochlorite), pieces (0.5 cm) were cut from the roots and placed on PDA medium (in 5 replications per treatment -5 Petri dishes x 6 pieces of roots).Soil samples collected from under each plant (at a depth of up to 5 cm) were placed in dishes and mixed with a rotary motion.149 g fine sand was mixed thoroughly with 1 g of the resultant fraction in a flask with a rotary motion (for 10 minutes).300 mm 3 of the mixture was poured with Martin medium (50 o C) (M a ń k a , 1974).Colonies of yeast-like fungi were counted.After 5 days of incubation at 22 o C, fungal colonies were transferred to PDA slants for species identification (B o o t h , 1971; E l l i s , 1971; S k i r g i e ł ł o et al. 1979).

RESULTS AND DISCUSSION
The fungal soil community was more abundant and diverse than the community colonizing the roots of tomato plants.The applied biological control, which involved alternate spraying and watering with aqueous extracts of seven plant species, effectively reduced the abundance of fungi, including pathogenic species, colonizing tomato plants and soil.Fifteen species of filamentous fungi were isolated from the tomato roots (Table 1).Yeast-like fungi accounted for 6.4% of all isolates (Fig. 1a).Among saprotrophic fungi, species of the order Mucorales were isolated in relatively high numbers (Mortierella alpina, M. isabelina, M. zonata and Rhizopus nigricans -over 14% of all isolates).Fungi showing antagonistic activity against pathogens were represented by P. roseum, Trichoderma hamatum and T. harzianum.They were isolated in greatest abundance from the roots of tomato plants treated with A. millefolium, M. piperita, E. arvense and U. dioica extracts (Fig. 1b).
In a study by J a m i o ł k o w s k a and W ag n e r (2007), the essential oil from Thymus vulgaris, containing thymol and carvacrol, increased the abundance of fungi of the genera Trichoderma and Mucor on the roots of pepper plants, but it also inhibited the growth of Alternaria alternata and F. oxysporum on the aboveground parts of plants.According to O s o r i o et al. (2009), the development of pathogens such as A. alternata, C. coccodes, F. oxysporum, F. sambucinum, F. solani and R. solani may be inhibited by phenolic extracts from Carya illineonsis, Punica granatum and Larrea tridentate.
The abundance of pathogenic fungi isolated from the roots of tomato plants ranged from 32% in the treatment with P. aviculare extract (a significantly lower number of pathogens was isolated from the tomato roots in this treatment, compared with other treatments -Table 3) to 72% in the control treatment and in the treatment with J. regia extract.In all treatments, the predominant species was C. coccodes -the only pathogen that colonized the roots of tomato plants treated with A. vulgaris 2004), extract from the seeds of Heracleum sosnowskyi was characterized by the highest activity against F. culmorum, due to high concentrations of esters found also in other species of this genus.In an earlier study, W o l s k i et al. (1996) attributed the fungistatic effects of H. sosnowskyi extracts to the production of secondary metabolites such as coumarins and furanocoumarins.
The soil fungal community was dominated by yeast-like fungi, which accounted for 75.4% of all isolates from all treatments (Fig. 2a).Among 27 species of filamentous fungi (Table 2), members of the genus Penicillium were found in relatively large numbers, in particular in treatments with J. regia and U. dioica extracts (approximately 18% of all isolates in each treatment).Fungi of the order Mucorales (M.alpina, M. isabelina, M. vinacea, Mucor hiemalis and R. nigricans) and antagonists of plant pathogens (P.roseum, T. hamatum, T. harzianum, T. koningii, T. polysporum and T. viride) had a 3.9% and 4.5% share of all isolates, respectively.The latter were most frequently isolated from soil samples collected from under tomato plants sprayed with A. millefolium, M. piperita and U. dioica extracts (Fig. 2b).
Pathogenic fungal species, including B. cinerea, C. coccodes, F. oxysporum and F. solani, were seldom isolated from the soil.They were not found in treatments with P. aviculare and E. arvense extracts.According to S a s -P i o t r o w s k a and P i o t r o ws k i (1995,2003), the fungicidal properties of extracts from plants of the family Polygonaceae against Fusarium species result from high concentrations of phenolic acids.

CONCLUSIONS
The applied biological control, which involved alternate spraying and watering with aqueous extracts of seven plant species, effectively reduced the abundance of fungi, including pathogenic species, colonizing tomato plants and soil.
The extract from Polygonum aviculare showed the highest efficacy.
C. coccodes and Fusarium spp.were isolated in great abundance (over 70%) from the roots of control tomato plants and tomato plants treated with an aqueous extract from Juglans regia.
The soil fungal community was dominated by yeast-like fungi, whereas pathogenic fungi were present in low numbers.

Fig. 1 .
Fig. 1.Fungi isolated from the roots of tomato plants during the investigation period (%) B d l i y a B .S ., D a h i r u B ., 2006.Efficacy of some plant extracts on the control of potato tubers soft rot caused by Erwinia carotovora ssp.carotovora.J. Plant Prot.Res.46 (3): 285-294.B o o t h T .C ., 1971.The genus Fusarium.Commonwealth Mycological Institute Kew Surrey, England.B u r g i e ł Z .J ., To m a s z k i e w i c z -P o t ę p a A . , Vo g t O ., B u r g i e ł M .M ., 2008.Fungistatyczne własności ekstraktów z nasion wybranych roślin należących do rodziny Apiaceae./Fungistatic properties of extracts from seeds of selected species of apiaceaeaus plants.Progr.Plant Protection/ Post.Ochr.Roślin, 48 (2): 701-705.(in Polish).By r n e J. M ., H a u s b e c k M .K ., H a m m e r s c h m i d t R ., 1997.Conidial germination and appressorium formation of Colletotrichum coccodes on tomato foliage.Pl.Dis.81: 715-718.E l l i s M .B ., 1971.Dematiaceus hyphomycetes.Commonwealth Mycological Institute Kew Surrey, England.J a m i o ł k o w s k a A . , W a g n e r A . , 2007.Próby zastosowania olejku tymiankowego do ochrony papryki uprawianej w polu przed grzybami chorobotwórczymi./Effect of thyme oil in protection against fungal diseases on Grzyby kolonizujące korzenie oraz glebę spod uprawy pomidora (Lycopersicum esculentum Mill.) chronionego biologicznie , M. piperita, P. aviculare and J. regia extracts.Fungi of the genus Fusarium were isolated in highest numbers from the roots of control tomato plants (34.5%).Those pathogens are the causal agents of tomato root rot (B y r n e et al. 1997; V a tc h e v and H a d j i d i m i t r o v , 2006).In an in vitro experiment, B u r g i e ł et al. (2008) noted an inhibitory effect of extracts from plants of the family Apiaceae on the growth of A. alternata, Botrytis cinerea and Fusarium culmorum.As demonstrated by S e f i d k o n et al. (

Table 1
Fungi isolated from the roots of tomato plants during the investigation period (% of all isolates) Aloe vulgaris Lam., Am -Achillea millefolium L., Mp -Mentha piperita L., Pa -Polygonum aviculare L., Ea -Equisetum arvense L., Jr -Juglans regia L. i Ud -Urtica dioica L. * -pathogenic fungi, ** -antagonistic fungi Table2Fungi isolated from soil samples collected from under tomato plants during the investigation period (% of all isolates)

Table 3
Fungi isolated most frequently from tomato organs and soil (mean number of isolates in years) Explanations as inTable 1,Path -pathogens, Antag -antagonists, Muc -Mucorales, Spor -Sporotrichum olivaceum, Yeastyeast-like, Pen -Penicillium spp.* means with the same letter do not differ significantly (Duncan's test, p=0.01) a. fungi isolated most frequently b. pathogenic and antagonistic fungi isolated in individual treatments