MATERIALS AND METHODS

Nodules were collected from seven tree legumes growing in and around Karachi under arid environment. Tree species were selected because of their importance in agroforestry and reforestation programs. The tree legumes studied including Albizia lebbeck (L) Benth., Dalbergia sissoo Roxb., Leucaena leucocephala (Lam.) de Wit., Pithecellobium dulce (Rox.) Benth., Prosopis cineraria (L.) Druce, Prosopis glandulosa Torr. and Prosopis juliflora (Swartz.) DC. Rhizobia were isolated from the nodules as described bySomasegaran and Hoben, (1994). Rhizobial isolates from nodules of Vigna mungo (L.) Hepper were also included for comparison. Cultures were maintained on yeast extract mannitol agra medium (YMA) slants as described by Somasegaran and Hoben, (1994). The bacterial isolates were examined for Gram reaction, motility, colony characteristics on YMA medium, YMA with Congo red and YMA with bromothymol blue. They were characterized as rhizobia (Somasegaran and Hoben, 1994). The motility of isolates was determined by hanging drop method (Somasegaran and Hoben, 1994). Cross-inoculation experiments were preformed in chillum jar assemblies developed by Dahiya and Khurana, (1981) which is a modified form of Leonard bottle jar assembly (Somasegaran and Hoben, 1994). The chillum jar unit consisted of a top earthen vessel round at one end and tapering at the other. The lower half (the reservoir) consisted of a glass jar of such dimensions that the chillum vessel sat snugly on its rim and the tapering end of the chillum came to within 2-4 cm of the bottom of the jar. A wick was provided in the tapering end of chillum to help the capillary rise of the liquid from the reservoir to the top of the growth vessel. Coarse river sand, previously acid washed thoroughly, was added to chillum units within 5 cm of the top. Hoagland nitrogen-free nutrient solution (200 mL) was added to each reservoir unit. Top of the growth vessel was covered with glass petri dish and the whole unit was wrapped with thick paper and secured with rubber bands. The units were autoclaved at 121 °C for 2 h. for two alternate days and kept intact until they were brought into use. Undamaged Vigna mungo seeds (courtesy of Dr. Zahoor Aslam, Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan) of uniform size were selected by hand sorting. Seeds were surface sterilized through treating with 0.2% HgCl₂ (acidified with 5 mL/L conc. HCl) for 3 to 5 min. followed by repeated washing with sterile water. The inoculum was applied direct to seed coat by soaking the seeds for half an hour in rhizobial suspension with 10% sucrose solution (4 days old YMA culture). Four inoculated seeds were sown in each assembly immediately after inoculation. In some cases, pregerminated seedlings were inoculated by adding L/mL of suspension of the culture to their bases.

The covering on the top of the assemblies was removed after germination of seeds and sterilized dry gravels were spread over the surface of the sand to check contamination. The assemblies were arranged in randomized blocks using three replicates for each treatment. Uninoculated treatment served as control. Nitrogen free sterile Hoagland solution was supplied to each unit after an interval of 24 h. The assemblies were kept in controlled environment chamber operating at 27 °C with 16 h. illumination. The plants were harvested after six weeks. Nodules were counted on each plant and plants were dried in an oven at 72 °C for 48 h. and weighed. Dried plants were ground and total nitrogen content was determined by Kjeldahl method (Nelson and Sommers, 1980). The data were analyzed statistically and the least significant differences among treatments were evaluated at the 0.05 level of probability.

RESULTS AND DISCUSSION

Rhizobial isolates from tree legumes produced translucent, round and gummy colonies which varied in size between 1.5 and 2.0 mm. The isolates were motile and did not take Congo red stain. They showed varied reaction with bromothymol blue. Rhizobial isolate from Prosopis cineraria and Vigna mungo gave acidic reaction, while Dalbergia sissoo, Leucaena leucocephala, Pithocellobium dulce, Prosopis glandulasa and Prosopis juliflora gave alkaline reactions (Table 1). All the rhizobial isolates from tree legumes except Albizia lebeck formed nodules in Vigna mungo plants. The nodules were globose to be elongated in shape; they were formed on primary as well as secondary roots (Table 2). All the isolates produced moderate nodulation except Vigna mungo which showed abundant nodulation (Table 2).

No nodules were observed in uninoculated control plants. Vigna mungo was successfully cross inoculated with the rhizobial isolates from tree legumes. The number of the formed nodules, dry matter contents and total nitrogen contents of cross infected plants is presented in Table 3. The highest numbers of nodules were produced by isolates from Prosopis glanduloga followed by Dalbergia sissoo which were significantly different (P<0.05) from Vigna mungo and other inoculated treatments. The highest dry weight and nitrogen content were observed in treatment inoculated by Dalbergia sisso followed by Leucaena leucocephala and Prosopis glandulosa which were comparable with Vigna mungo and were significantly different (P<0.05) from other inoculated treatments and uninoculated control treatment.

Early reports of rhizobia associated with tree legumes described them as either of the slow growing type or of the cowpea miscellany type (Basak and Goyal, 1980). There have been reports of alkali producing Rhizobium strains (Hernandez and Focht, 1984) and acid producing Bradyrhizobium strains (Moerira, et al., 1993; Padmanabhan, et al., 1990). Legume trees are infected as much by fast growing rhizobia as by slow growing rhizobia (Moerira, et al., 1998; Trinick, 1980; Turk and Keyser, 1992; Zhang, et al., 1991). Some tree rhizobial strains are host specific, whereas others have a wide host range (Dommergues,

Table 3: The effectiveness of rhizobial isolates from tree legumes on nodulation, dry weight and nitrogen content of Vigna mungo

The means sharing the same letters in each column are not significantly different at P< 0.05

et al., 1984). In the present studies, the isolates from strains effectively nodulate herbaceous legumes root nodules of Albizia lebbeck did not form nodules (Herrera, et al., 1985). Several reports describe on Vigna mungo host. Albizia lebbeck rhizobia also successful experiments where wild rhizobia are more failed to nodulate Vigna radiata host. Duhoux and effective in nitrogen fixation than their compatible Dommergues, (1985) have stated that Albizia lebbeck hosts (Zahran, 2001). Wange, (1989) obtained effective is specific in its rhizobial requirements. It is nodulated symbiosis between Acacia rhizobia and each of only by Bradyrhizobium. Failure of Albizia lebbeck peanut and cowpea. Zhang, et al., (1991) performed rhizobia to infect Vigna mungo and Vigna radiata cross inoculation experiments between rhizobia hosts would need further investigations. One isolated from Acacia and Prosopis and found effective interesting application of rhizobia of wild tree legumes symbiosis with Medicago sativa, Phaeolus vulgaris is their use as inoculum for crop legumes. Many tree and Vicia faba. Iqbal and Mahmood, (1992) tested isolates from seven legumes for their ability to produce root nodules on Leucaena leucocephala host. Isolates from Albizia lebbeck, Pithecellobium dulce and Vigna unguiculata were most effective in nitrogen fixation and induced substantial increase in dry weight and nitrogen contents of the host plant. Zahran, et al., (1999) studied the symbiotic performance of rhizobia isolated from wild tree legumes (Acacia nilotica and Sesbania sesban) and the herbaceous legumes (Alhagi murarum, Melilotus indicus and Trifolium resupinatum) with some grain legumes. About 30% of rhizobial isolates formed effective symbiosis with four legumes (Medicago sativa, Pisum sativum, Vicia faba, Vigna sinensis). Similarly, isolated rhizobia from the tree legume, i.e. Pericopsis moonians, formed nitrogen-fixing nodules in a broad spectrum host, Macroptillium atropurpureum (Lalani Wijesundra, et al., 2000). The high nitrogen-fixing ability of rhizobia associated with Dalbergia sissoo and Leucaena leucocephala in Pakistani soils has been reported by Javid and Fisher, (1989) and Iqbal and Mahmood, (1992), respectively. Higher nitrogenase activity in Leucaena leucocephala has been reported byAryal, et al., (1999) from Indian soils. The present results on the cross-inoculation of Vigna mungo with rhizobia from tree legumes are very encouraging and in conformity with previous studies (Iqbal and Mahmood, 1992; Lalani Wijesundra, et al., 2000; Wange, 1989; Zahran, et al., 1999). The natural rhizobia of wild tree legumes growing in arid zones exhibit higher tolerance to prevailing adverse conditions like salt stress, elevated temperatures and drought (Zahran, 2001). These rhizobia may be used to inoculate wild as well as crop legumes cultivated in reclaimed desert lands. These rhizobia may have specific traits that can be transferred to other rhizobia through genetic engineering tools. However, field trials are needed to prove the effectiveness of these isolates from wild legumes in increasing nitrogen contents of cultivated plants. The cross-infection of agriculturally important legumes with isolates from wild legumes may prove a useful means of increasing nitrogen contents within these plants.

ACKNOWLEDGEMENTS

The funding for this research was provided by NSRDB, Islamabad, Pakistan for the project No. B. Sc. (120)/KU/90. Thanks are due to Dr. Mohammad Qaiser and Dr. Surriya Khatoon from University of Karachi for their help in the identification of some of the plants. Sincere gratitude is expressed to Dr. Zahoor Aslam, Nuclear Institute for Agriculture and Biology, Faisalabad for an providing approved variety of Vigna mungo seeds.

REFERENCES