FIRST RECORD OF MYCETOPHAGOUS NEMATODE APHELENCHUS AVENAE IN IRAQ WITH DESCRIPTION AND TESTING THEIR PROPAGATION ON DIFFERENT FUNGUS CULTURE

Aphelenchus avenae was isolated from the wheat crown in Summel distractDuhok, Kurdistan region-Iraq infected by a crown rot disease which is caused by Fusarium spp; wheat's crown culturing on Potato Dextrose Agar (PDA) and incubating at 25°C A. avenae was found associated with fungal culture which meant that fungal nematode was parasitic on crown rot fungi on wheat crown, this species was described for the first time in Iraq. Fungal Nematode incubated with Fusarium graminearum, F. oxysporum and Verticillium dahliae reproduce in both solid and liquid media, best results of nematode reproduction were recorded on F. graminearum followed by F. oxysporum and V. dahlia respectively. The nematode A. avenae did not reproduce on the liquid media of these fungi.


INTRODUCTION
Aphelenchus avenae Bastian, 1865 feeds on a variety range of fungi (Mankau and Mankau, 1963;Giannakis and Sanders, 1989;Okada and Ferris, 2001) and has a wide range of fungal host in both phyto-pathogenic and saprophytic species in different conditions.A. avenae is a mycophagous nematode which can't parasitize on higher plant (Barnes et al., 1981), and there is no record of real parasitism, damage or feeding of this nematode on the plant, thus A. avenae is called fungal nematode.There are many studies on development, growth and feeding of A. avenae on fungi culture media, its growth on Fusarium solani and Rhizoctonia solani has been studied by Barnes et al. (1981).This nematode has been recorded in many countries worldwide in different soil types (Okada and Ferris, 2001).This nematode is capable to the growth and propagation on culture media of fungi grown on PDA in different amount of potato dextrose solution when incubated at 25°C; propagation of A. avenae in Semisolid substrate also studied (Ishibashi et al., 2000).A. avenae was used as a bio-control agent against soil-born fungal pathogen in many studies, using the different population size of A. avenae in infested soil with different pathogenic fungi such as R. solaniAG-4, Fusarium oxysporum f. sp.lagenariae, Pythium sp. and Phytophthora nicotianae var.parasitica showed that this nematode had a noticeable effect on the survival of cucumber plants, thus A. avenae is considered as a good biological control agent for those fungi (Barnes et al., 1981).DOI: http://dx.doi.org/10.26842/binhm.7.2017.14.3.0251The attraction of A. avenae to plant roots also impedes colonization of plant-parasitic nematodes (Matsunaga et al., 1996).The penetration of Pratylenchus coffeae and Meloidogyne incognita of cucumber transformed hairy roots on agar plate decreased the incidence of A. avenae (Ishibashi, 2005).
This study aims to indicate the first record and description of A. avenae in Iraq and to study its growth on different culture media and various soil born-fungi.

MATERIALS AND METHODS
Aphelenchus avenae is collected from wheat crown in Summel distract-Duhok, Kurdistan region-Iraq infected by crown rot disease and is caused by Fusarium spp.after culturing wheat's crown in potato dextrose agar (PDA) and incubated at 25 °C.Nematodes were extracted from culture media and collected according to Grewal et al. (2005) then added again to culture media for reproduction.Then nematode was picked and mounted on slide to study it's for identification.More than 30 slides were made for each of female, male and eggs.Following morph metrical measurements and body, ratios were calculated (Machado et al., 2010): L=total body length.V=distance from vulvae to posterior end.St= total stylet length.F= total esophagus length.v-a= distance between vulvae and tail.T=total tail length.

Study of Aphelenchus avenae reproduction in different fungus culture:
Three pure fungal cultures of F. graminearum, F. oxysporum and V. dahliae were used as host for fungal nematode, two type of medium, used both PDA solid medium and Malt extract liquid media were used with three petri dish for each replication.After calculating number of A. avenae in solution using counting dish, then 50 A. avenae was added by pipette to fully grow fungal culture and was incubated at 25°C for two weeks then nematodes was extracted again from each culture to calculate number of nematode to know the propagation rate of nematode on each fungal culture.Data were analyzed using SAS program and means were compared according to Duncan's Multiple Range test, P=0.05 (SAS, 1999).

RESULTS AND DISCUSSION
Family of Aphelenchidae Fuchs, 1937, Steiner, 1949 Syn.Paraphelenchidae (Goodey, 1960) are characterized by large obvious median bulb (metacarpus) and occupied more than 75% of body width in the located part.Body of A. avenae usually large has plump lips, form flat cap, slightly offset, median bulb round slight elongate nerve ring just behind bulb, stylet long with or without small basal knobs, rectum lumen considerably widened just behind sphincter, anus and rectum can be seen very obviously near to the end of body so A. avenae has short tail.A. avenae fungal feeder nematode can grow and develop on different fungi (Okada and Ferris, 2001).

Aphelenchus avenae Bastian, 1865 description and identification:
Female: (Pl. 1, A, B, E, G) body tapers slightly at head region narrowing abruptly behind vulva, there is no constriction or rarely constriction occurs behind anus.Tail short rounded rarely slightly flattened at terminal, tail is conical.Stylet knob less, junction conical parts is less than 1/2 of its length.Median bulb round to slight elongated, esophageal lumen narrows and widens into intestine, dorsal gland overlapping intestine, usually on left side as can be seen under compound microscope.Anus transverse 1/3 body width, anterior lip slightly protruding.Vulva oval, vagina has thick wall (Bastian, 1865)

Aphelenchus avenae propagation in different fungus culture:
Diagram (1) illustrates that after two weeks of incubation A. avenae propagated and increased in number at the huge level on F. graminearum number of nematode reached to (114233) while F. oxysporum was secondly preferred by A. avenae and number of nematodes were (47013).This means that A. avenae was also propagating well on F. oxysporum and these results agreed with many researchers who stated that A. avenae could feed on different species of Fusrium (Okada, 2006;Karuri et al., 2014).A. avenae incubated in V. dahlia increased in number to 15000, lower nematode reproduction on V. dahlia as compared with two other species of Fusarium is possibly due to the thinner width of V. dahliae hyphae, rather than the length of nematode stylet (Okada, 2006).These results illustrate that A. avenae can propagate very quickly and increase in number to reach a huge number in a short time when incubated on preferable fungi to feed on hyphal tissue (Pl. 3) which make it effective bio-control agents.Number of A. avenae didn't increase and nematodes were dead while incubated on liquid culture of the same fungi after two weeks.This result agreed with the idea that A. avenae could not propagate and living in liquid substrate cause nematode need to perpendicularly attach their stylet to the hyphal tissue to feed (Grewal et al., 2005).In all fungal culture nematode destroyed and hydrolyzed fungal mycelium during their feeding (Pl.4) which means that A. avenae secrete hydrolytic enzymes to dissolve and feed on hyphae tissues (Ishibashi et al., 2005).

Diagram ( 1 ):
Increasing in A. avenae population on Fusarium graminearum, F. oxysporum and Verticillium dahlia cultures grew on PDA after two week of incubation.Plate (1): Aphelenchus avenae: A) Female; B) Anterior portion show esophagus, medium bulb and stylet.C and D) Vulva in ventral and lateral side.E) Tail region show rectum and anal opening.F) Female in egg laying stage, eggs can be seen inside body and vulva opened for egg laying.G) Eggs in different embryogenesis stage.Plate (2): Aphelenchus avenae : A. male; B. C.D. posterior end of male show spicules and tail region.
(Bastian, 1865) is similar to female body shape straight at rest (Pl.2,AB and C).It has two spicules cephalated near to tail.Gubernaculum V shape.Tail is taper; most studies mentioned that male rate is very rare (1:10000) or absent, but in our culture male ratio was approximately 1: 100 females(Bastian, 1865).