Heterodera Avenae

Study of natural antagonists of cereals cyst nematode "Heterodera avenae ." in Algeria

Righi Kada* 1, Assia Righi Fatiha 1, Mokabli Aissa 2, Madani F. Zohra 1

1Laboratory of research on biologic systems and Geomantic. Department of Agronomy Sciences. Faculty of Science .University of Mascara, Algeria 2- Department of Agronomy Sciences Faculty of Science .University of Khemis Meliana, Algeria.

Received: February 1, 2016 Accepted: April 12, 2016 ABSTRACT

This study aims to highlight the fungi associated with cysts of Heterodera taken in cereal plots in the Mascara region of western "Algeria. Macroscopic and microscopic examination revealed the presence of a fungal flora associated with cysts of Heterodera obtained from different regions. 10 orders have been isolated from the cysts which 70% belongs to Pleosporaceae, Mucoraceae and Trichocomaceae with respectively 30, 20 and 20%. These fungi are ( Pythium sp ), Zygomycetes ( Rhizopus sp ) and several Deuteromycetes such as Alternaria sp , Aspergillus sp , Diplodia sp , Drechslera sp , Fusarium sp , Penicillium sp , Periconia sp , Pithomyces sp and Trichothecium sp . The substances that make up the two metabolites of fungi ( F. solani and Penicillium sp ) selected in our study are volatile fatty acids (monocarboxyliques) identified by chromatography (CPG): formic acid (45.10 -2mol / L and 51 10 -2mole / L), acetic acid (5.20 mol / L and 3.25 mol / L) and butyric acid (85. 10 - 2mol / L and 55 10 -2mole / L) which are respectively produced by F. solani and Penicillium sp. KEY WORDS : Fungi , Heterodera avenae, inhibitory effect, cereals, chromatography.

1. INTRODUCTION

Cereals are considered the most cultivated cultures by man and have long been prominent in human nutrition. By the importance of occupied areas and its role in the food security of the country, cereal crops occupies a prominent place in both agricultural production and agri-food Algeria. In Algeria cereal consumption represents 25% of food expenditures and 230 kg / year [1]. Although these cultures have benefited from some development programs but national production, did not know the expected improvement. The causes of this are mainly of technical orders, and phytosanitary of these, nematodes induce 10% loss of global production, [2]. For cereals, the complex Heterodera avenae : cereal cyst nematode is by far the most to fear. It is present in temperate cereal regions, [3; 4; 5]. This wide distribution follows the expansion of cereal surfaces. The Heterodera complex is represented by a twelve species in the world. However, the specific composition varies from one region to another. In Algeria, Heterodera group was the subject of several investigations, the results of which revealed the presence of several species in cereal plots, [6; 7 ]. There the use of synthetic nematicidal was unsatisfactory in addition to their adverse effects on the flora and fauna that have led to their reduction. Thus, new solutions must be found quickly to solve the nematodes problems. Among these means, the use of natural antagonists including fungal metabolites are an interesting way forward, easy to use and environmentally friendly. In this concordance many fungi are known to produce nematicidal compounds or nematostatic. Ankeet al.1995 [8 ; 9 ; 10 ; 11 ; 12 ; 13 ]. Thus secondary metabolites of Fusarium oxysporum are toxic to Meloidogyne incognita . So many toxins of Fusarium sp , reduce the viability of nematodes, [14; 11; 15 ]. Other studies have shown the inhibitory action of hatching eggs or nematicidal from some fungi isolated from soil nematodes and plants [16; 17; 18; 19; 20; 21; 22; 9; 23 ]. Our contribution aims to reveal the identity of fungi that are responsible for the low viability of cysts, eggs and larvae of Heterodera during our earlier work that mean the preliminary research on natural antagonists cyst nematodes cereals " Heterodera avenae ” in Algeria and the research of the active principles of the toxic metabolites’ by chromatography (CPG) of the two selected nematicidal fungi in our study (Fusarium solani and Penicillium sp ).

2. MATERIALS AND METHODS

Cysts involved in this study were obtained from samples of soil communities in the area of Mascara Western “Algeria”. They are collected during the period from February to April 2014on cereal crops attacked by H. avenae .

*Corresponding Author: Righi Kada, 1Laboratory of research on biologic systems and Geomantic. Department of Agronomy Sciences. Faculty of Science .University of Mascara, Algeria. [email protected]

28 Righi et al., 2016

2.1 Isolation and identification of fungi . Isolation of fungi was performed on cysts disinfected in a solution of NaOCl in 1% for one minute and rinsed five times with sterile distilled water. These are then placed in Petri dishes of 90 mm diameter on WA agar medium (20 g of agar in one liter of distilled water) at a rate of 5 to 10 cysts per box in availability cysts. Four repetitions are performed for each plot. All operations are performed under laminar flow hood to avoid external contamination. After 72 hours of incubation at 22 ° C, the dishes are examined every 48 h for the first ten days and once a week later. After a month, the percentage of infection of the cysts by fungi was calculated. Fungi, which developed from the cysts were isolated and purified on agar medium. From these isolates monosporales cultures are carried out on PDA medium and incubated at 22 ° C for identification. Microscopic observations are made on agar containing fruiting fragments forms stained with blue cotton, placed between slides and cover slide and fixed with a quick pass on the flame of a spirit lamp. The different orders of fungi isolated are identified according to the key of [24 ].

2.2 Methods of filtrate cultures: A cutting taken from the mycelia growth area of a culture of each antagonist fungi ( Fusarium solani and Penicillium sp ) is placed in flasks of 500 ml Erlen Meyer containing 300 ml GYP liquid nutrient medium. These flasks were incubated for 3 weeks in the dark at 26° C. After this period, the mycelium is separated from the culture medium by filtration on different diameters [11; 12; 5; 13 ]. This avoids the passage of all contaminating spores.

2.3 Analysis of filtrates Culture: The identification of toxic metabolites in the filtrate culture of F. solani and Penicillium sp is realized by chromatography (CPG). (Vector gas Hydrogen (H2), flow rate 0.2-0.3ml / min; H2 (leak) = 18ml / min; H2 flame = 30ml / min; column length (25m): DT.0.3mn, DE0.35mn; capillary column FFAP (liquid phase); integrator speed 0.4cm / min; injector temperature 250 ° C; column temperature (programming) 60 ° C (2min) 120 ° C 4 °C/ min. The chromatography is performed at the laboratory of the national company of maritime transport of oil and chemicals (Arzew), Algeria. Thus a quantity of samples 1(µl) of the filtrate is introduced at the column top, it is driven by an inert hydrogen gas H2 (mobile phase). At the output of the column a detector receives the components driven at variable speeds and separated. The components are thus identified by an integrator in the form of peaks.

3. RESULTS AND DISCUSSION

3.1 Distribution of fungi isolated : Macroscopic and microscopic examination revealed the presence of a fungal flora associated with cysts of Heterodera obtained from different regions. In addition, 10 orders have been isolated from the cysts which 70% belongs to Pleosporaceae, Mucoraceae and Trichocomaceae with respectively 30, 20 and 20%. (Tab.1, Fig .1).

Table1: Fungi isolated from the cysts of different populations of Heterodera sp . Genus Family Order Aspergillus Trichocomaceae Eurotiales Alternaria Pleosporaceae Pleosporales Stemphylium Pleosporaceae Pleosporales Epicoccum Pleosporaceae Pleosporales Cladosporium Davidiellaceae Capnodiales Chaetomium Chaetomiaceae Sordariales Mucor Mucoraceae Mucorales Rhizoctonia Ceratobasidiaceae Cantharellales Rhizopus Mucoraceae Mucorales Penicillium Trichocomaceae Eurotiales

29 J. Appl. Environ. Biol. Sci. , 6(6)28-35, 2016

Ceratobasidiaceae 10%

Trichocomaceae 20%

Mucoraceae 20%

Pleosporaceae Chaetomiaceae 30% 10%

Davidiellaceae 10% . Figure1: Rate distribution of isolated fungi from cysts of Heterodera sp .

The macro and microscopic appearance of some fungi isolated: The Aspergillus sp conidiophore ends with a bulge provided with sterigmata with unicellular and globoid chains of conidia. Rhizopus sp is characterized by non-septate hyphae, conidiophores continued growth in that give rise to sporangia. The identification of Alternaria sp was made on the morphology of multi-cellular conidia elongated dark generally wider on one side than the other and closed at the base by a pedicel. The partitions are formed in both the longitudinal and transverse direction. Penicillium sp is characterized by prolonged phialides conidiophores which carry at their apex, long chains of conidia unicellular globoid to ovoid form. The genus Mucor is very close to Rhizopus genus but unlike Mucor is more upright; the spices do not produce solons and rhizoids (Fig.2).

a b c

d e f

30 Righi et al., 2016

g h i

j k

Figure2 a :Alternaria sp, b: Aspergillus sp , c :Rhizopus sp , d : Aspergillus niger , e: Chaetomium sp f: Stemphylium sp , g: Penicillium sp., h: d’ Epicoccum sp , i: Rhizoctonia sp, j: Cladosporium sp , k: Mucor sp.

3.2 Frequencies of occurrence of fungi isolated. Fungal strains of different species have been identified within 150 cysts of Heterodera different populations with varying frequency of occurrence in different populations. (Fig.3). The genus Aspergillu s which is represented by two species in the occurrence Aspergillus sp and A. Niger , records a frequency of 48.71%. This genus has been detected on several phytophagous nematodes. [25 ] who isolated it from females and cysts of Heterodera avenae . [26 ] have observed it on Meloidogyne in India. The second kind is Rhizopus which is common in 35.89% of the populations of Heterodera . Similarly Penicillium sp , we have isolated from several populations of Heterodera , its frequency is 17.94%. Some metabolites of this fungus have an inhibitory effect on egg hatch of Heterodera avenae [27]. Many studies have demonstrated the presence of this fungus in the cysts of Heterodera and other phytophagous nematodes. [25; 26 ], confirmed that an isolate of Penicillium shows promising results as inhibitor eggs of Meloidogyne .

48.71 50 45 40 35.89 35 30 25 17.94 20 Frequncy% 15 7.69 10 5.12 5.12 2.56 2.56 2.56 2.56 5 0

Genus

Figure3: Occurrence frequency of fungi associated with cysts of Heterodera sp

31 J. Appl. Environ. Biol. Sci. , 6(6)28-35, 2016

Rhizoctonia sp was detected in 7.69% of the populations studied cysts, achieving fourth place. [28] recommended that the isolation of Rhizoctonia sp is best directly from the ground. The same authors showed that this fungus colonizes only females and is known as mycorrhizal endotrophic fungus, suggesting that colonizes females of Meloidogyne from infected plant cells rather than egg masses. The species Mucor sp and Stemphylium sp have also shown colonizers agents of Heterodera avenae cysts. The frequency values are 5.12% for each of these two species. The frequency of 2.56% was recorded for each of the following genus: Epicoccum, Alternaria, Cladosporium , and Chaetomium . The species Chaetomium sp , is known by its inhibitory hatching action of nematode eggs. According to [29 ], the culture filtrate of Chaetomium globosum has an inhibitory action against hatching of both M. incognita and H. glycines . [33], observed reduced mobility of larvae (L2) under the action of the activity of the culture filtrate of Chaetomium murorum after 24 and 48 hours. This fungus produces a variety of biologically active compounds as chaetoglobosin, chaetomugilin, and chaetoviridin [30; 31]. Among the substances tested antagonists in vitro by [29 ] on the eggs of Meloidogyne is flavipine. So its presence in the populations cysts study in our region can justified eggs mortality rates registered. The Flavipine is produced by several species of Aspergillus , Epicoccum and Chaetomium globosum . [32 ]. Also [34] isolated Flavipine from Epicoccum nigrum witch has the power to inhibit hatching eggs of Meloidogyne incognita and Heterodera glycine

3.3 Characteristic of the active principal The chromatograms obtained by the technique of chromatography (CPG), allow to high light the majority peaks. The peaks observed at different retention times are characteristic of certain volatile fatty acids of low molecular weight, this is confirmed by comparison with standard chromatograms of acetic acid, formic acid and butyric acid. The acid concentration in the two filtrates of Fusarium solani (Fig. 4) and Penicillium sp . (Fig. 5) is determined by measuring the peak areas compared with the reference acids. The concentrations of formic acid, acetic acid and butyric acid are 45.10 -2mol / L; 5.20 mol / L 85. 10 - 2mol / L; and 51.10 -2mole / l; 55.10 -2mole / L respectively for F. solani and Penicillium sp . According [35 ], analyzes nematicidal toxins produced by fungi, showed the presence of fatty acid monocarboxylic which is acetic acid.

Figure 4 : Chromatogram of Fusarium solani filtrate

32 Righi et al., 2016

Figure5: Chromatogram of Penicillium sp filtrate

Our results are consistent with those obtained by [36 ]. These authors showed that the purified extract of Penicillium sp ; Penicillium oxalicum and Penicillium anatolicum Stolk has a nematicidal activity at 100 and 200ppm.

4 Conclusion: In conclusion, 11 fungi belonging to different genus were identified from the cysts of H. avenae populations from several cereal producing regions. These fungi belong to six family and six orders with different frequency. These are opportunistic [ 37; 38 ]. However some of them were the subject of many studies and showed inhibitory action on the mobility of infective larvae (L2) of roots nods or cysts nematodes. The study of toxic substances in the filtrate of Penicillium sp and Fusarium solani ; confirmed the significant efficacy of the two filtrates on the mortality of eggs of Heterodera avenae demonstrated in the anterior work of [27]. [39; 40] estimate that the rate of cysts, eggs, and female cyst nematodes colonized by fungi in agricultural soils ranged of 10 - 90% with about 50% being the most common. At the level of soil, populations of opportunistic fungi associated with nematodes can be significantly higher than the populations of obligatory parasitic fungi [41]. The importance of the regulation of the populations of plant parasitic nematodes in the soil is positively related to the population of all fungal parasites, including opportunistic [42]. All the results obtained shows that it is now possible to envisage a biological control against cyst nematodes " Heterodera " by fungus F. solani and Penicillium sp and while respecting certain precautions. First assessment of fungal growth so that they have time to secrete substances metabolites and thus act effectively against the eggs of phytophagous nematodes. Regarding soil protection, we are moving towards the use of saprophytes fungi.

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