Taxonomic diversity of Fungi Associated with Some PCN Populations from Serbia

Violeta Oro1, Jelena Bošković2, Slobodan Milenković2 and Solveig Tosi3 1Institute for Plant Protection and Environment, Department of Plant Pathology, Nematology Laboratory, Teodora Drajzera 9, 11000 Belgrade, Serbia (viooro@yahoo.com) 2Megatrend University, Faculty of Biofarming, Maršala Tita 39, 24300 Bačka Topola, Serbia 3University of Pavia, Department of Earth Science and Environment, Mycology Laboratory, Pavia, Italy Received: October 10, 2011 Accepted: February 24, 2012


INTRodUCTIoN
Increased public concern over the significant presence of pesticides in food chain and its consequences resulted in the development of biological agents as alternative methods of control invasive organisms.
The potato cyst nematodes (PCN) -Globodera pallida and G. rostochiensis belong to the group of the most important parasites-the quarantine organisms and their control is complex due to their long persistance in soil.
The fungal antagonists of nematodes consist of a great variety of organisms which include the nematode-trapping or predacious fungi, endoparasitic fungi, parasites of nematode eggs and cysts and fungi which produce metabolites toxic to nematodes.The fungal spore has undergone some remarkable adaptation and specialization to capture or penetrate nematodes.Among the lower fungi, motile zoospores appear to have positive tropism toward nematodes.Catenaria anguillulae spores most often accumulate around the natural body orifices of the nematode cuticle (Mankau, 1980).Nematophthora gynophila causes total destruction of Heterodera avenae cysts in less than seven days (Kerry, 1980).Verticillium chlamydosporium, Acremonium strictum and Fusarium oxysporum are the main parasites in eggs of H. schachtii (Dackman and Nordbring-Hertz, 1985).
Flavipin, a low molecular weight compound, a metabolite of the fungus Chaetomim globosum is responsible for most of the nematode-antagonistic activity (Nitao et al., 2002).Combined application of Paecilomyces lilacinus and Monacrosporium lysipagum reduced 65% of Heterodera avenae cysts (Khan et al., 2006).There is a commercialised biopesticide based on Paecilomyces lilacinus (Kerry, 2000).In our country, fungal antagonists of potato cyst nematodes have not been investigated so far.The aim of the study was to identify taxonomic diversity of fungi associated with some PCN populations from Serbia.

MATERIAL ANd METHodS
Mycobiota of Globodera cysts from Kladnica, Šanac, Gojna Gora and Milatovići populations which are morphologically and molecularly identified (Oro et al., 2010) was investigated.The cysts (3) and eggs of a single cyst were placed on potato-dextrose agar (PDA), containing antibiotics (bensylpenicillin K + bensylpenicillin-procaine: 200.000 i.u.+600.000i.u., 200 mg/l), and incubated for two weeks at 25 a o C. The cysts and eggs were previously disinfected according to Heungens et al., 1996.For identification purpose fungi were maintained on MEA (Malt Extract Agar) at room temperature.The isolates were identified by morpho-dimensional analysis using slide-cultures of the fungi.Only Mycelia Sterilia was identified by biomolecular analysis.

RESULTS ANd dISCUSSIoN
The external and internal area of a cyst harbor numerous fungal and bacterial species competing for substrate.In the process of identification, the following fungal species were found: Fusarium oxysporum on cysts of the Kladnica population, Oxyporus latemarginatus on eggs of the same population, Botrytis cinerea and Aspergillus fumigatus, on eggs of the Šanac population, Geotrichum candidum on cysts of the Gojna Gora population, Oxyporus latemarginatus on eggs of the same population, Geotrichum candidum on cysts of the Milatovići population and Aspergillus fumigatus was isolated on eggs of the Milatovići population.The found species are harmless saprobes and they are relatively abundant members of the soil microbial community.Nearly all of them performed antagonistic activity toward potato cyst nematodes and have the potential to be used as biocontrol agents (results are not shown).The morphological view of isolated fungi is presented in photos.

Fungi
Dikarya Ascomycota Pezizomycotina Sordariomycetes Hypocreales Nectriaceae Fusarium Teleomorph: Gibberella Fusarium is a large genus of filamentous fungi widely distributed in soil and in association with plants (Ohara et al., 2004).Many F. oxysporum isolates appear to be host specific, which has resulted in the subdivision of the species into formae speciales and races that reflect the apparent plant pathogenic specialization.Fusarium oxysporum and its various formae speciales have been characterized as causing the following symptoms: vascular wilt, yellows, corm rot, root rot, damping-off etc. (Kim et al., 2002).In solid media culture, such as potato dextrose agar (PDA), different special forms of F. oxysporum can have varying appearances.On PDA, growth is rapid and the white aerial mycelium may become tinged with purple or be submerged by the blue color of the sclerotia or by the cream to tan to orange sporodochia when they are abundant (Nelson et al., 1983;Leslie and Summerell, 2006).Fusarium oxysporum produces three types of asexual spores: microconidia, macroconidia, and chlamydospores (Ruiz-Roldan et al., 2010).Mycelium of Fusarium oxysporum on PDA and under a light microscope (LM) are shown in figures 1 and 2 respectively.

Fungi
Dikarya Basidiomycota Agaricomycotina Agaricomycetes Polyporales Polyporaceae Oxyporus Oxyporus latemarginatus is widely distributed in Europe; it induces stem and root rot of Fagus and Quercus species.The fungus colonizes and survives on dead wood or roots in the soil, forming white mycelial mats and dirty white to pale yellow fruiting bodies, annual, resupinate, rather soft when fresh, 2-5 (10) mm thick (Ryvard and Gilbertson, 1994).Mycelium of Oxyporus latemarginatus on PDA and under a dissecting microscope (Di) are shown in figures 3 and 4 respectively.

Botrytis cinerea (de Bary) Whetzel
Fungi Dikarya Ascomycota Pezizomycotina Leotiomycetes Helotiales Sclerotiniaceae Botrytis Teleomorph: Botryotinia Botrytis cinerea has a worldwide distribution but occurs mainly in humid temperate and subtropical regions; it is very common in the phylloplane of plants but does not attack healthy leaves; it can be facultative parasite on a wide range of plants causing blight or rot of leaves, flowers and fruits, the so-called gray mold (Domsch et al., 1980).Botrytis cinerea causes infections characterized by rapid destruction (maceration, necrosis) of the tissues of its plant host as it colonies it (necrotrophy).
The fungus overwinter as mycelia or black resting structures called sclerotia that may be evident on dead plant tissue in late summer.Sclerotia germinate in the spring, or grows out of infected debris and conidia (infectious spores) develop (Williamson et al., 2007).
Mycelium of Botrytis cinerea on PDA and under a light microscope (LM) are shown in figures 5 and 6 respectively.

Fungi
Dikarya Ascomycota Saccharomycotina Saccharomycetes Saccharomycetales Endomycetaceae Geotrichum Teleomorph: Dipodascus Geotrichum candidum is an extremely common fungus with a worldwide distribution, occurring mainly in soil, air, water, sewage, various plant substrates and very frequently on milk and milk products (Domsch et al., 1980).It can be plant pathogen that causes sour rot on peach, nectarine, tomato and carrot.Species of the genus Geotrichum produce chains of hyaline, smooth, onecelled, subglobose to cylindrical, slimy arthroconidia (Gente et al., 2006).Mycelium of Geotrichum candidum on PDA and under a light microscope (LM) are shown in figures 9 and 10 respectively.The mode of antagonistic action of fungi on potato cyst nematodes is very complex and involves numerous toxins, metabolites, lytic enzimes and other antihelmintic compounds.
Botrytis cinerea utilizes a large range of pathogenic factors (lytic enzymes, activated oxygen forms, toxins, plant hormones) to attack its host plants but it was not reported as nematode antagonist.The fungus is a member of Sclerotiniaceae family of Ascomycota.
When the fermentation broth of Aspergillus fumigatus was screened, a number of indolic alkaloids with antimitotic properties were discovered such as tryprostatins, with spirotryprostatin B being of special interest as an anticancer drug (Cui et al., 1996).Aspergillus fumigatus grown on certain building materials can produce genotoxic and cytotoxic mycotoxins, such as gliotoxin (Nieminen et al., 2002), it can also produce antibiotics such as sideramines fusigen, ferricrocin and fumigacin (Domsch et al., 1980).Aspergillus fumigatus had immobilized 51-75% juvenils of Meloidogyne spp. in three days (Mubyana-John and Wright, 2011).Aspergillus belongs to Trichocomaceae family of Ascomycota.
The process of cyst colonisation by fungi is enhanced by cystogenesis.
In shape, cysts are spherical sacs in which 500-600 eggs can be laid.There are specific ecological conditions within the cyst (temperature, relative humidity, pH, presence of different chemical compounds) which make it an ideal medium for microbial growth.Generally, eggs and larvae are not infected while cysts are intact.In process of hatching, the first larva appears leaving the vulva open and allowing fungal colonization.When detached from root tissue, mature cysts often lack a head leaving the cyst open on the other side, so that in this way antagonistic microorganisms can invade from both directions.In this study, different fungal species colonize external and internal side of a cyst.Although the majority of mentioned fungal species are occasionally found in association with other nematode species, Oxyporus latemarginatus is for the first time found to be associated with PCNs.The diversity of PCN fungal antagonists is not reflected only at the species level but also at the level of higher taxonomic categories i.e. in existence of different phyla such as Ascomycota and Basidiomycota.Thus, the cyst can be considered as specific "microcosm" containing a spectrum of different fungi inside and outside of the cyst.

Figure 5 .
Figure 5. Mycelium of Botrytis cinerea (PDA) EmericellaAspergillus fumigatus, a saprotroph widespread in nature, is typically found in soil and decaying organic matter, where it plays an essential role in carbon and nitrogen cycling.Colonies of the fungus produce from conidiophores thousands of minute grey-green conidia (2-3 μm) that readily become airborne.For many years, A. fumigatus was thought to reproduce only asexually, as neither mating nor meiosis had ever been observed.O´Gorman et al., 2009 proved A. fumigatus possess a fully functional sexual reproductive cycle.The fungus is capable of growth at 37 o C up to 50 o C, with conidia surviving at 70 o C. Mycelium of Aspergillus fumigatus on PDA and under a light microscope (LM) are shown in figures 7 and 8 respectively.