Abstract: A total of 28 species belonging to 16 genera of fungi parasitic on root-knot nematodes were isolated and identified. Aspergillus terreus, A. nidulans, A. tamarri and Fusarium anthophellum are appeared to be new report on root-knot nematode and not reported hitherto. Of the fungi isolated, Aspergillus flavus, A. fumigatus, A. niger, Aspergillus sp., Acremonium butyri, Alternaria alternata, Catenaria sp. Cephalosporium sp., Cladosporium cladosporoides, Cladosporium sp., Cunninghamella elegance, Curvularia lunata, Fusarium exosporium, Fusarium sp., Ulocladium atrum and a sterile fungus are new records on root-knot nematodes in Pakistan. Natural infestation of Paecilomyces lilacinus and Arthrobotrys sp. is also recorded first time from Pakistan.
Introduction
The control of plant parasitic nematodes using trapping or parasitic fungi is of great interest because the use of nematicides or soil sterilent brings about various ecological and toxicological dangers (Jatala, 1986). At least four products have been developed commercially for the control of plant parasitic nematodes but non has used widely because control has tend to be erratic and practical application rate (Kerry, 1992). Despite such difficulties positive results obtained with biocontrol fungi of nematode in a number of other studies indicates that continued isolation and testing of fungi should lead to fungal agent that will add in nematode control (Morgan-Jones et al., 1981; Susan et al., 1990). Although a wide literature concerning fungi parasitic on root-knot nematodes is available worldwide (Kim et al., 1992; Ma et al., 1991; Jatala et al., 1979; Morgan-Jones et al., 1984; Viaene and Abawi, 1998), few fungal parasites of nematode have been reported from Pakistan (Munir et al., 1988; Zaki and Maqbool, 1993; Zaki, 1999a; Khan et al., 1994). A survey was therefore carried out to isolate fungi parasitic on root-knot nematodes (Meloidogyne spp.).
Materials and Methods
A survey of the cultivated fields of Karachi University campus, Shah Faisal colony, Korangi, Memon Goth, Linkroad, Gharo, Mirpur Sakro, Thatta and Kathore area was carried out during January, 1999 to September, 1999. A total number of 150 plant specimens belonging to 13 plant species showing symptoms of root knot disease were collected. Soil sample 250 g at 10 cm depth collected in a polythene bags.
Isolation and identification of fungal parasites: Egg masses of root knot nematodes were hand picked with the help of a sterilized forcep, surface sterilized in 1% Ca(OCl)2 for 1-2 minutes, rinsed twice in sterile distilled water and plated onto 0.8 percent water agar supplemented with penicillin (100,000 units/L.) and streptomycin (0.2 g/L.).
Eggs of root knot nematodes were extracted by shaking the infected roots in 2 percent sodium hypochlorite solution, collected on a 400 mesh sieve and 1ml of the suspension was evenly spread onto water agar plates.
Meloidogyne females were obtained by teasing gall root tissues with the help of a sterilized needle under a stereoscope dissecting microscope. After surface sterilization in 2 percent sodium hypochlorite solution females were washed thoroughly with sterile distilled water and plated onto water agar plates as described earlier.
Meloidogyne eggs were extracted by sodium hypochlorite solution and washed thoroughly in sterile distilled water and kept for hatching at 25°C. For predacious or trapping fungi, one gram soil sample was sprinkled onto water agar plates and freshly hatched juveniles suspension containing 100-200 juveniles/ml were pipetted onto water agar surface.
There were three replicates of each samples and dishes were incubated at room temperature (25-30oC) for 3-5 days. As soon as some fungal colonies appeared, hyphal fragment was transferred onto PDA plates and fungi were identified after reference to Booth (1971), Nelson et al. (1983), Domsch et al. (1980) and Thom and Raper (1945).
Results and Discussion
From 150 root and soil samples infested with root knot nematodes collected from 9 localities of vegetable crops, total of 16 genera comprising 28 species of fungi were isolated (Table 1).
| Table 1: | Fungal parasites associated with root-knot nematodes |
| *New report **New record from Pakistan | |
| Fig. 1(a-c): | Conidiophore emerging from females of Meloidogyne spp., a) Aspergillus nidulans, b) A. terreus and c) Paecilomyces lilacinus |
Of these Acremonium butyri, Alternaria alternata, Catenpria sp. Cephalosporium sp., Cladosporium cladosporoides, Cladosporium sp., Cunninghamella elegance, Curvularia lunata, Penecillium sp., Rhizoctonia solani and sterile fungi were appeared to be new record on root knot nematode, Meloidogyne spp., in Pakistan. Aspergillus spp. were found most frequent, parasitizing almost all the stages of root knot nematodes. Of these A. flavus and A. fumigatus have been earlier recorded on root knot and cyst nematodes (Khan and Sexana, 1995) whereas A. nidulans (Fig. 1a), A. tamarri and A. terreus (Fig. 1b) were found associated with females, eggs and egg masses and appeared to be new reports on Meloidogyne spp. Fusarium species appeared second major fungal group found associated with different forms of root knot nematodes. F. exosporium, F. pallidoroseum, F. solani and Fusarium sp. have been reported on Meloidogyne spp. Ma et al., (1991) but F. anthophillum on eggs appeared to be new report. Paecilomyces lilacinus (Jatala et al., 1979) and Verticillium chlamydosporium (Morgan-Jones et al., 1981) are well known nematophagous fungi isolated from eggs, egg masses and females of root knot nematodes. Both of these fungi have been extensively studied under green house and field trials (Ehteshamul-Haque et al., 1994; Zaki, 1999b; Parveen et al., 1998). Culture of P. lilacinus used in the previous studies was supplied by Dr. P. Jatala of Peru and is deposited in Karachi University Culture Collection as KUCC-244. Natural infection of P. lilacinus is recorded first time recorded from Pakistan (Fig. 1c). A zoosporic fungus Catenaria sp. and trapping fungus Arthrobotrys sp. isolated from nematode suppressive soil during the present survey. Both of these fungi were associated with Meloidogyne larvae. Catenaria sp. has been reported by Zaki (1999a) but Arthrobotrys sp. appeared to be new record from Pakistan. During the present study, Ulocladium atrum was found associated with eggs of Meloidogyne root knot nematode. The fungus has been reported from cyst of Globodera rostochiensis (Munir et al., 1988). There is need to test the efficacy of these fungi in vitro and in vivo for the control of plant parasitic nematodes.
Acknowledgment
This work, was carried out under University Grant Commissions grant which is sincerely acknowledged.