Fungi colonizing roots of zucchini (Cucurbita pepo L. var. giromontina) plants and pathogenicity of Fusarium spp. to zucchini seedlings

Zucchini is a very valuable vegetable, easy to grow both in the field and under covers but it is often attacked by soil-borne fungi. The investigations were carried out in a farm near Lublin where three zucchini cultivars: Astra, Atena and Soraya, were grown in a tunnel and in the field in 2008-2009. Plants with stem and root rot were collected for laboratory tests. Mycological analysis showed that the predominant fungi were F. culmorum , F. equiseti and F. oxysporum . More Fusarium colonies were isolated from the roots of zucchini grown in the tunnel. In the pathogenicity test, all tested isolates of F. culmorum , F. equiseti and F. oxysporum proved to be pathogenic to zucchini seedlings, causing stunting, stem and root rot. The highest disease index was noticed in the combination with F. culmorum isolate fck61.


INTRODUCTION
Zucchini (Cucurbita pepo L. var.giromontina) is a summer squash cultivated in many countries on all continents.The fruits are low in calories and contain useful amounts of vitamins B, C and PP, provitamin A, carotene, magnesium, potassium as well as flavonoid poly-phenolic antioxidants which help to scavenge harmful oxygen-derived free radicals and reactive oxygen species that play a role in aging and cancer development (M u n t e a n et al. 2006).
Zucchini grown under greenhouse and field conditions is usually infected by pathogens specific for Cucurbitaceae but also by microorganisms pathogenic to other crops (K i m a t i et al. 1997; S u m n e r et al. 1983, 1995).The most important among them are Fusarium spp.surviving in the soil environment as saprotrophic mycelium and chlamydospores (N e l s o n et al. 1983, J a m i o ł k o w s k a , 2009).F. avenaceum and F. culmorum (Smith) Sacc.are known to be pathogens of zucchini and other vegetables causing plant decay due to the colonization of their underground organs (N e l s o n et al. 1983).The most important pathogens seem to be Fusarium oxysporum Schl.responsible for plant wilting and F. solani (Mart.)Sacc.causing stem base and root rot (K w a ś n a et al. 1991; W a g n e r , 2004).
The aim of the study was to investigate the occurrence and diversity of fungi colonizing zucchini plants grown in a plastic tunnel and in the field as well as to evaluate the pathogenicity of some Fusarium spp. to zucchini seedlings.

Mycological analysis.
The experiments were conducted in 2008 and 2009 at a horticultural farm in Łuszczów (15 km north-east from Lublin).The objects of the study were three cultivars of zucchini (Cucurbita pepo L. var.giromontina): Astra, Atena and Soraya.The seedlings were produced at the Felin Experimental Farm in Lublin.They were planted in a plastic tunnel (1st decade of May) and in the field (2nd decade of May) with a spacing of 100 cm in the row and 80 cm between rows.For each cultivar, 40 seedlings in 4 rows were planted.After soil analysis before the experiment, fertilizers were applied according to the recommendation for Cucurbitaceae.Pesticides were not applied and weeds were removed manually.The observations of plant health status were conducted at the beginning of fructification (2nd decade of July) when four plants from each combination were collected for mycological analysis.Roots were precleaned, rinsed with tap water for 20 minutes and then surface disinfected with 50% ethanol and 0.1 % sublimate for 1 minute.The disinfected plant material was rinsed 3 times in sterile distilled water.Next, 3mm fragments were placed on mineral medium in Petri dishes as described by J a m i o ł k o w s k a (2007).For each experimental treatment 10 dishes with plant material, 10 plant fragments per dish, were prepared and incubated at 20-22 o C for 7 days in darkness.The obtained colonies of fungi were transferred to potato-dextrose slants (PDA-Difco) and identified to species with the available monographs.
Pathogenicity test.To estimate the harmfulness of Fusarium spp. to zucchini, the pathogenicity test was conducted using Mańka's method described by W a g n e r (1997).The seeds of cv.Astra (obtained from Krakowska Hodowla i Nasiennictwo Ogrodnicze POLAN sp.z o. o.Kraków) were precleaned, rinsed in tap water for 20 minutes and disinfected with 0.1% sodium hypochlorite for 1 minute.The disinfected seeds were rinsed 3 times in sterilized distilled water, placed into sterilized soil and covered with plastic foil to germinate.The inoculation was carried out in 0.5 l pots filled with sterilized substrate up to 3 cm below the edge.Slices of PDA overgrown with mycelium of single spore cultures of the tested fungi were put on the surface.In the control combination, slices of medium without fungus were used.When cotyledons appeared, healthy looking seeds were placed in pots on medium with or without inoculum and covered by the substrate.Zucchini seedlings were grown in a growth chamber at 24-25 o C and 85% air humidity with 14 hrs photoperiod (from 6 a.m. to 8 p.m.).
In the experiment, the following isolates were tested: -Fusarium culmorum: fck15, fck53, fck61, -Fusarium equiseti: fek16 fek26, fek59, -Fusarium oxysporum: fok1, fok19, fok35.The isolates were selected on the basis of macro-and microscopic properties of fungi isolated in 2009 from the roots of zucchini growing in a tunnel and in the field.For each combination, 15 seedlings were tested in 5 replications.
After four weeks of plant growth in the pots, the seedlings were counted and disease severity was evaluated, using a 4-degree scale: 0 -no symptoms, 1 -small necrotic spots on all lateral roots, no symptoms on leaves or stem, 2 -necrosis on tap-root and stem base, 3 -completely rotten roots and stem base, stunting of aboveground parts of the plant.Then, the number of seedlings in each degree was computed.The disease index was calculated for each replication using Mc Kinney's formula described by W a g n e r (1997).Afterwards, the mean disease index was computed for each combination.To check if the symptoms were caused by the pathogen, the disinfected fragments of the affected stems and roots were placed on PDA.After seven days the growing colonies were identified.
The data were analyzed with Tukey's test at a 5% significance level using the SAS statistical system (SAS Version 9.1, SAS Inst., Cary, N.C., U.S.A.).

RESULTS AND DISCUSSION
Mycological analysis.During field observations, stem base and root rot were noticed (Fig. 1).Mycological analysis resulted in 974 isolates of fungi belonging to 20 species.From the underground parts of zucchini, Fusarium spp.were isolated most frequently.These fungi are important in the pathogenesis of zucchini and other vegetable crops grown under covers and in the field (J a m i o ł k o w s k a , 2009; J a m i o ł k o w s k a and B u c z k o w s k a , 2009).More Fusarium isolates were obtained from the plants grown in the tunnel than from those grown in the field.The conditions in the tunnel are conducive to Fusarium spp.development that depends on such abiotic factors as humidity, temperature and pH (K a c p r z a k and M a ń k a , 2000).The frequency of Fusarium spp. was higher in the roots of cvs.Atena and Astra than in those of cv.Soraya.The roots were colonized mostly by F. culmorum, F. equiseti, and F. oxysporum.The predominant species in the fungal communities from the roots proved to be F. oxysporum (33.2% of total number of colonies) (Table 1) F. equiseti was numerous in the underground parts in 2009, especially in Atena cultivated in the field (Table 1).Several authors (Z a l e w s k a and M a c h o w i c z -S t e f a n i a k , 2004; J a m i o łk o w s k a and W a g n e r , 2005) reported strong pathogenic properties towards some vegetable and spice plants.J a m i o ł k o w s k a (2009) proved the strong pathogenicity of F. equiseti to hot pepper seedlings.The fungus caused root rot and decay of plants.F. equiseti, as other species of the genus, produces toxins and antibiotics, especially trichothecenes and equisetin (W h e e l e r et al. 1999; H e s t b j e r g et al. 2002).F. culmorum, frequently isolated from roots (12% of all colonies) (Table 1), also produces mycotoxins -culmorin and trichothecenes involved in the pathogenesis (L a n g s e t h et al. 2001).Saprotrophic fungi were also isolated: Trichoderma spp., Penicillium spp., Papulaspora irregularis, Mucor hiemalis, Epicoccum purpurascens and Cladosporium fulvum (Table 1).According to some authors (A h m e d et al. 2000; S u a r e z -E s t r e l l a et al. 2007), the presence of these fungi is always a positive phenomenon.
Pathogenicity test.The results confirmed the pathogenic abilities of the tested isolates of F. culmorum, F. equiseti and F. oxysporum (Fig. 2).The affected plants were stunted, with yellow leaves.Necrosis was noticed on the roots and some seedlings were decayed.(Fig. 2).The highest disease index was noticed for F. culmorum isolates fck61 and fck53.In these combinations, all seedlings were affected, showing growth stunting and root rot.Their disease index amounted to 84.44% and 75.55%, respectively, and differed significantly from the control (Table 2).The harmfulness of F. culmorum to many vegetables results very often in damping-off (B u r g i e ł , 2005) and is dangerous due to polyphagous abilities of this fungus (N e l s o n et al. 1983).Root rot was also noticed in the combination with F. oxysporum isolate fok35.The disease index in this combination was 77.77%, differing significantly from the control (Table 2).For other isolates of F. oxysporum, the disease index was lower (51.11% and 55.55%) but significantly different from the control.In the combinations with F. equiseti, necrotic spots were observed on the roots as well as other symptoms.The disease index ranged from 42.22% (fe26) to 66.66% (fek16) and it was significantly higher than in the control (Table 2).F. equiseti is regarded as the cause of damping-off, root and fruit rot not only in Cucurbitaceae (A d a m s et al. 1987; V i g l i o l a , 1994) but also in pepper and other vegetable plants (J a m i o ł k o ws k a and W a g n e r , 2005; J a m i o ł k o w s k a , 2008).The results of the tests depended not only on the species but also on individual isolates.This can be explained by the biodiversity that occurs in every population of organisms (W a g n e r , 1997).
. I t o et al. (2005) report that the infection by F. oxysporum causes wilting and dying of plants due to the colonization of underground organs and xylem.W a g n e r et al. (2001) also proved the harmfulness of F. oxysporum to tomato seedlings indicating the decrease of plant fluorescence resulting from the pathogen presence.The negative impact of F. oxysporum might be the effect of the activity of its toxins.T-2 toxin, HT-2 toxin and diacetoxyscirpenol might have been involved in the pathogenesis (M i r oc h a et al. 1989).

Fig. 1
Fig. 1 Root rot of zucchini stem base (photo by A. Jamiołkowska)

Table 2 .
Disease index for zucchini seedlings inoculated with the investigated isolates of Fusarium spp.
Values followed by the same letters do not significantly differ at 5% error (Tukey's test).