Plant parasitic nematodes are widespread and cause serious losses to most agricultural crops (Al-Rehiayani and Fouly, 2005). Nematodes are often managed with chemical nematicides which can contaminate agro-eco-systems. Natural antagonistic of nematodes and biocon-trol agents may provide an alternative to the use of pesticides for nematode management. Numerous nematode species are associated with the citrus rhizo-sphere; however few species are known to be of economic importance (El-Banhawy et al., 1997). Many nematode species have been reported to be parasiting the citrus but Tylenchulus semipenetrans (Cobb, 1913) was the most important on worldwide basis (Safdar et al., 2010).Citrus nematode is one of the most important root nematodes of plant trees that have worldwide distribution and cause reduction of crop production and vegetative growth. In addition, this nematode creates slow decline of citrus trees (Ayazpour et al., 2010). Yield reduction by citrus nematode depending of the infection rate, but on average is 10 to 30% (Verdego-Lucas and McKenry, 2004).

Methods commonly employed to control T. semipenetrans depend on local conditions and focus on excluding the pest, minimizing losses through crop management and reducing population of the parasite using nematicides or resistant root stock (El-Banhawy et al., 1997). Considerable information available in the literature has documented the effectiveness of several biological agents to manage plant parasitic nematodes (Al-Rehiayani and Fouly, 2005). The majority of mesos-tigmatid mites in soils are general predators which prey on a range of invertebrates including nematodes (Walter and Lindquist, 1989), although, the minority, are specialized predators feeding on nematodes (Sharma 1971? Habersaat, 1989). Previously, it was found that nymphs and adults of Lasioseiuss scapulatus (Kennet) had the ability to capture, consume and complete its entire life cycle on the root knot nematode Meloidogyne incognita (Kofoid and White Chitwood) (Imbriani and Mankau, 1983).

Also, the suitability of egg masses of Meloidogyne spp. As food source of the ascid mite species Lasioseius dentatus (Fox) was studied where it was found that the predatory mite successfully completed its whole life span on egg masses (Fouly, 1997). Al-Rehiayni and Fouly (2005) was studied effect of adding Cosmolaelaps simplex (Fox) or aldicarb for control of T. semipenetrans on citrus seedlings in greenhouse.

On the other hand, the mesostigmatid mites family Laelapidae Berlese is considered one of the most important groups of soil predators, where it usually feeds on nematodes (Muma, 1975). Predatory laelapids tend to be voracious, polyphagous predators that reproduce quickly and can be reared easily (Beaulieu, 2009). This makes them good candidates for biological control of pests that spend time in the soil or in other plant growing media. The genus Gaeolaelaps Evans and Till is cur-rently one of the largest genera of the family Laelapidae. Some species of this genus, such as Gaeolaelaps aculeifer (Canestrini), G. oreithyiae (Walter and Oliver (1989), and G. gillespiei Beaulieu, are aggressive predators of nematodes and immature arthropods (Kavianpour et al., 2013).

Gaeolaelaps was considered at different taxonomic levels by authors: as a species group (Van Aswegen and Loots, 1970); or as a subgenus of Hypoaspissens. lat. (Karg 1989? Faraji et al., 2008), and as a distinct genus (Lapina, 1976; Hyatt, 1964).

In the present study, we investigated the efficiency of soil-dwelling predatory mite G. aculeifer in biological control of citrus nematode in greenhouse and the effect of adding mite individuals for reduce T. semipenetranson citrus seedlings.

Rearing of predatory mite in laboratory

The predatory laelapid mite G. aculeifer was isolated from soil samples under shrubs of hopbush, Dodonaea viscose (L.) Jgacq. in Fars Science and Research University. After identification, isolated mites were reared in glass jars filled with damp sawdust. Mite samples have been maintained on the acarid mite species Rhizoglyphus robini Claparede, as a food source in rearing units. The acarid mite was maintained in laboratory on onion. All units were kept under normal room temperature at 25 ± 1°C and 65 ± 5% relative humidity.

Extracting nematode T. semipenetrans in laboratory

Nematodes were extracted from soil samples taken with a hand trowel at 15-30 cm beneath the tree canopy in citrus orchard. In this study, Baermann’s technique was used to separating nematode of soil suspention (Goody, 1957).

Greenhouse experiments

A group of 20 plastic pots (25 cm in diameter) containing sand loam sterilized soil and previously transplanted with single 3-month seedlings of key lime (Citrus aurantifolia). The transplanted pots were divided into five groups, four pots each, where the first group was infected with approximately 100 juvenile stages of T. semipenetrans/pot. The second group received the mites at the same time of nematode inoculation, while the third one received 20 individuals of G. aculeifer/pot 15 days after nematode inoculation. The fourth group was infected with nematodes 15 days after adding 20 individuals of G. aculeifer/pot and the fifth one was left without any nematode inoculation and mites. All pots were kept under greenhouse conditions at 27 ± 1°C and 65 ± 5 % relative humidity.

Ninety days after nematode inoculation, citrus seedling that were carefully under consideration where data dealing with shoot length and fresh weight for root system were recorded. Moreover, the second juvenile stage (J2) found in 100 mL of soil samples was estimated for each nematode treatment after extraction by sieving using modified Baermann-funnel method (Goody, 1957). On the other hand, soil samples of about 250 g each were also subjected for mite extraction by the aid of the modified Berlese’s (Tullgern’s) funnels (Berlese, 1905). Where, the average number of mite/250 g of soil was calculated.

In all cases, data were subsequently analyzed by least significance difference (LSD), Duncan’s multiple rang and analysis of variance (ANOVA) testes, where the reproduction index of predatory mite = final mite population (PF)/ initial mite population (PI).

In the study of plant growth, the mite-treated citrus plants showed the better growth as compared to the untreated plants. Significantly enhanced shoot growth was observed in all the mite-treated citrus plants as compared to the T. semipenetrans untreated control (35.70 cm) (Table 1). 

Root weight was slightly enhanced in all mite-treated plants but this response was not significant compared to the plants with nematodes only. The citrus plants without nematodes had the greatest shoot length (58.1 cm). Seedlings with nematode alone as compared to the citrus plants without nematodes showed symptoms such as small and yellowing leaves, low growth, and death of top of branches and defoliate.

Based on the fact that the T. semipenetrans is semi-parasitic nematode; therefore predatory mite feeds on egg masses and juvenile stages of T. semipenetrans. Similarly, it was found before that addition of the predatory mite species L. dentatus either at the same time or 40 days after root-knot nematode inoculation had significant improvement in shoot length, weight and root weight of tomatoes seedlings under greenhouse condition (Mostafa et al., 1997).

The result shows the potential of the predatory mite G. aculeifer in repressing T. semipenetrans reproduction. Compared to the nematode alone, all mite treatments significantly restricted reproduction of T. semipenetrans. Nematode populations ranged from 126 to 161 J2s/100 cm³ for the mite-treated plants compared to 398 - 25 J2s/100 cm³ for the nematode untreated control (Table 2). Nematode population significantly was at lowest level when the predatory mites were added to the treatments at the same time of nematode inoculation.

In spite of a difference means of treatments 2 and 4 in LSD test did not have any significant differences, but tow treatments 1 and 3 showed significant difference (p=1%) according to LSD (Table 3). 

Concerning the reproduction index of G. aculeifer, it was noticed that it was at its highest level when the predatory mites were added to the treatments at the same time of nematode inoculation (PF/PI=42.84) and followed by 31.51 and 24.45 for mites that were added 15 days after and before nematode inoculation, respectively (Table 2).

Also, there were no significant differences between the effect of application time of mites added to citrus seedlings 15 days after nematode inoculation and either mites added at the same time or mites added 15 days before inoculation n the reproduction index of G. aculeifer; whereas, there was significant difference between the effect of adding mites at the same time of inoculation and those that were added before inoculation on the reproduction index of G. aculeifer.

Similarly, it was previously found that the reproduction index of the ascid predatory mite L. dentatus was higher when mite individuals were added to tomato seedlings 40 days after root-knot nematode inoculation (Abou Setta et al., 1986) and similarly, these results significant match with findings (Al-Rehiayani and Fouly, 2005) about reproduction index of the predatory mite species C. simplex on T. semipenetrans under greenhouse condition. That may be due to mite species and its feeding behavior as well as to the biological aspects of nematodes. Therefore, it can be concluded that G. aculeifer had a better response which directly represented by its reproductive potentiality and capability to reducing citrus nematode populations when it was added at the same time of nematode inoculation. In other word, the predatory mite G. aculeifer had the chance to search the developmental individuals of citrus nematodes and feed on them before they can reach the root system and become more difficult for the predator. These results are in harmony with the previous findings where it was found that the developmental stages of root-knot nematodes were eaten by L. scapulatus under laboratory and greenhouse conditions (Imbriani and Mankau, 1983).

Finally, it can be concluded that the predator mite G. aculeifer could be considered as a biological control agent, which may limit populations of citrus nematode. Moreover, mite capability to feed, survive and reproduce on nematodes can be integrated with other control tactics and further field work in this area is highly warranted.

The author(s) have not declared any conflict of interests.