*Rao. M.V.1. V.K. Garg.2. R.K. Dixon3 and W.D. Kellev.3 "Department of Plant Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 024, TN, India. 2Biomass Research Center, National Botanical Research Institute, Lucknow 226 001, UP, INDIA. 3School of Forestry, Auburn University, Auburn, AL 36849, USA

GROWTH AND SYMBIOSIS OF LEUCAENA LEUCOCEPHALA (LAM.) DE WIT SEEDLINGS INOCULATED WITH SPECIFIC ECTO- AND ENDOMYCORRHIZAL FUNGI

It is now recognized that most terrestrial plants have a mycorrhizal association (Dixon and Marx,1987). If the mycorrhizal association is well-developed the host plant may benefit through improved water absorption (Duddrige et al, 1980), uptake of nutrients (Harley and Smith, 1983), resistance to soil pathogens (Bagyaraj, 1983; Marx, 1972) and tolerance to environmental stress (Dixon and Marx, 1987). Many leguminous trees including Leucaena species are colonized by mycorrhizal fungi. A limited number of field observations suggests that leucaena is primarily endomycorrhizal but some species or varieties may also form ectomycorrizae.

Preliminary observations reveal that inoculation of Leucaena species with vesicular-arbuscular mycorrhizal (VAM) fungi improves juvenile growth. Dual inoculation with VAM and Rhizobium improves growth and nodulation in several legumes, including leucaena (Salinas et al, 1985). Although mycorrhizal fungi are beneficial to leucaena, most preliminary studies have been conducted in non-sterile soil or inoculation procedures included introduction of several symbiotic fungi (Manjunath et al, 1984). The results of these experiments make it difficult to determine which symbiotic fungi are most beneficial to the host. The objective of this experiment was to evaluate the growth and nodulation of Leucaena leucocephala (Lam.) de Wit var K8 inoculated with specific VAM and ectomycorrhizal fungi in sterile soil.

Materials and Methods. The experiment was implemented in a randomized complete block design. Seedlings were inoculated with two ectomycorrhizal fungi Pisolithus tinctorius (Pers.) Coker and Couch, and Laccaria laccata (Scop, ex Fr.) Bk. and Br. and four endomycorrhizal fungi Glomus deserticola Trappe, Bloss and Menge, Glomus intradices Trappe, Bloss and Menge, Gigaspora margarita Beck, and Hall and Glomus etunicatum Beck, and Gerd. Nonmycorrhizal plants (NM) were maintained as a control. Each treatment was replicated five times and both mycorrhizal and nonmycorrhizal plants were inoculated with Rhizobium.

Leucaena leucocephala seed were scarified, mixed with a peat based culture of Rhizobium inoculum and sown in 2.5 liter pots containing a fumigated sandy loam soil (Manjunath et al, 1984). Ectomycorrhizal (15cc) and endomycorrhizal (2,5cc) fungal inoculum were placed below the seeds in pots of the respective treatments. The mycorrhizal inoculum was produced previously using standard methods (Schenck, 1982). During the study the photoperiod was approximately 14h daily and seedling potting medium was watered on alternate days to field capacity. All seedlings received 100ml of full strength Hoagland’s nutrient solution at weekly intervals (Hoagland and Amon, 1950).

After three months all seedlings were harvested. Mycorrhizal colonization of the seedlings was measured as ratio of the number of mycorrhizal lateral roots/total number of lateral roots. Endomycorrhizal colonization was determined after clearing the roots with KOH and staining with trypan blue (Phillips and Hayman, 1970). Ectomycorrhizal colonization was evaluated using methods described by Marx (1972). Component dry weights of the roots, shoots, and leaves were obtained after oven drying at 70°C to constant weight. Seedling leaf area was measured by using LiCOR leaf area meter. The data were subjected to analysis of variance and Duncan’s multiple range test.

Bethenfalvay, G.J. and J.F. Yoder. 1981. The Gfyine-Glomus-Rhizobium symbiosis. Physiol. Plant. 52:141-145.

Dixon, R.K. and D.H. Marx. 1987. Mycorrhizae. In: Cell and Tissue Culture Methods in Forestry (J. Bonga and D. Durzan, eds.) Martinus Nijhoff Publishers, Boston, MA, USA, pp. 336-350.

Duddridge, J.A, A. Malibari and D.J. Read. 1980. Structure and function of mycorrhizal rhizomorphs with special reference to their role in water transport. Nature 287:834-836.

Harley, J.L. and S.E. Smith. 1983. Mycorrhizal symbiosis. Academic Press, New York, NY, USAHoadland, D.R. and D.I. Arnon. 1950. The water culture method for growing plants without soil.

Calif. Agr. Exp. Sta. Cir. 347.Levisohn, I. 1956. Growth stimulation of forest tree seedlings by the activity of free living

mycorrhizal mycelia. Forestry 29:53-59.Linderman, R.G. and C.A. Call. 1979. Enhanced rooting of woody plant cuttings by mycorrhizal

fungi. J. Am. Soc. Hort. Sci. 102:629-632.Manjunath, A, D.J. Baguaraj and H.S. Gopala Gowda. 1984. Dual inoculation with VA

mycorrhizal and Rhizobium is beneficial to Leucaena. Plant and Soil 78:445-448.Marks, G.C. and R.C. Foster. 1973. Structure, morphogenesis, and ultrastructure of

ectomycorrhizae. In: Ectomycorrhizae, Their Ecology and Physiology. Eds. Marks, G.C. and T.T. Kozlowski. Academic Press, New York. Pp. 1-41.

Marx, D.H. 1972. Ectomycorrhizae as biological deterrents to pathogenic root infections. Ann. Rev. Phytopathol. 10:429-454.

Nalini, P.A., M.S.B. Reddy, and D.J. Bagyaraj. 1986. Selection of an efficient VA mycorrhizal fungus for Leucaena, a preliminary report. Leucaena Research Reports 7:61-65.

Phillips, J.M. and D.S. Hayman. 1970. Improved for procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Sec. 55. 158-161.

Salinas, J.G., J.I. Sanz, and E. Sieverding. 1985. Importance of VA mycorrhizae for phosphorus supply to pasture plants in tropical Oxisols. Plant and Soil 84:347-360.

Schenek, N.C. 1982. Methods and principles of mycorrhizal research. American Phytopathological Society, St. Paul, MN, USA

South, W.T. and M. Habte. 1985. The influence of starter nitrogen and mycorrhizal inoculation on the post-transplanting. LRR 6:95-96.

Taufigul, A. and M. Habte. 1985. Interaction of Leucaena with Glomus fasciculatum in a typical oxisol. LRR 6:97-98.

Results. Leucaena leucocephala seedlings inoculated with Glomus deserticola, Glomus intradices, Glomus etunicatum and Gigaspora margarita developed abundant endomycorrhizae with typical vesicles and arbuscules (Table 1). The abundance of chlamydospores collected from the seedling rhizosphere soil suggests that these fungi were reproducing. In contrast, seedlings inoculated with Pisolithus and Laccaria did not develop typical ectomycorrhizae. Hyphae of the ectomycorrhizal fungi were intimately linked to seedling roots but complete fungal mantles and Hartig nets were not observed.

Table 1. Leaf area, nodule dry weight and mycorrhizal colonization of Leucaena leucocephala

Treatment Mycorrhizalcolonization

(%)

Noduledryweight(mg)

Leafarea

(cm2)Laccarialaccata

Od 266a 632a

Pisolithustinctorius

Od lOlab 550a

Glomusdeserticola

45c 262a 650a

Glomusintradices

91a 217a 398a

Glomusetunicatum

82ab 208a 508a

Gigasporamargarita

66b 204a 569ab

Nonmycorrhizal Od 170ab 650a

= 0.05).

Nodule dry weight of seedlings inoculated with Laccaria laccata, Glomus deserticola, Glomus intradices, Glomus etunicatum and Gigaspora margarita were significantly larger than the nonmycorrhizal plants or those treated with Pisolithus tinctorius (Table 2). Plants with abundant nodules and endomy-corrhizae were generally larger than nonmycorrhizal seedlings. Inoculation with Laccaria laccata stimulated a significant increase in the shoot growth of leucaena.

Discussion. Previous reports indicate that many legumes are highly dependent on the synergistic tripartite symbiosis of the host, mycorrhizal fungi and rhizobia (Salinas et aL, 1985). Our results suggest that L. leucocephala also significantly benefits from this tipartite symbiosis. Mycorrhizal colonization and growth of leucaena varied significantly with the Glomus and Gigaspora isolate (Nalini, 1986; Taufigul and Habte, 1985). The efficiency of mycorrhizal symbiosis in leucaena and other tree species may be significantly influenced by host-symbiont genetics (Dixon, 1989; Manjunath et aL, 1984).

Examination of older root systems in the field suggests leucaena develops ectendomycorrhizal or ectomycorrhizal relationships. In this study juvenile leucaena did not develop ectomycorrhizae with Pisolithus tinctorius or Laccaria laccata. In contrast, other nitrogen-fixing trees such as Alnus and Casuarina form ectomycorrhizae with specific fungi in field and laboratory environments. Given the biological significance of mycorrhizae to leucaena this matter deserves further investigation.

Table 2. Height, root collar diameter and shoot, root, leaf dry weight of Leucaena leucocephala __________seedlings inoculated with ecto- and endomycorrhizal fungi. ____________________Treatment ^height ■■ '•'•Moot' Snoot Riiot*' ■ :L*a£ . ;

collar dry dry drydiameter weight weight weight

(mm) (mm) (g) _____ (g)Laccaria 495a 6.8ab 3.4a 5.lab 3.6alaccataPisolithus 428ab 6.9ab 3.0ab 4.4b 2.8atinctoriusGlomus 493a 6.3b 2.9ab 5.9ab 3.2adeserticolaGlomus 410ab 7.6a 2.9ab 5.3ab 2.6aintradicesGlomus 394b 7. lab 2.6b 5.6ab 3.0aetunicatumGigaspora 384b 6.6b 2.9ab 5.6ab 3.5amargaritaNonmycorrhizal 390b 6.7b 2.6b 4.8ab 3.3aMeans followed by common letter are not significantly different by Duncan’s multiple range test (P = 0.05).

Shoot and root growth of leucaena was significantly stimulated by inoculation with Laccaria laccata and depressed by Pisolithus tinctorius. Previous reports revealed that ectomycorrhizal fungi could stimulate root (Linderman and Call, 1979) and shoot (Levisohn, 1956) growth of endomycorrhizal tree seedlings without colonization. The differential response of leucaena to inoculation with two ectomycorrhizal symbionts suggests a complex biological relationship between the host and fungi (Marks and Foster, 1973).

Earlier reports indicate that mycorrhizal colonization significantly improves host phosphorus and micronutrient nutrition, tolerance to adverse soil pH, or drought and pathogen resistance (Dixon and Marx, 1987). The processes of legume nodulation and nitrogen fixation have been shown to directly benefit from the presence of physiologically active mycorrhizal fungi (Bethenfalvay, 1981). Differences in leucaena growth due to mycorrhizal colonization were relatively small in the fertile, well-watered soils used in our brief study. However, inoculation of leucaena with an efficient mycorrhizal fungus in phosphorus deficient soils, or high stress sites, may result in dramatic increases in juvenile growth (Salinas, 1985; South and Habte, 1985).

Acknowledgement. This research was supported by the U.S. Agency for International Development and the Winrock International Institute for Agricultural Development under the Forestry/Fuelwood Research and Development Project.

References:

Bagyaraj, D.J. 1983. Biological interaction with VA mycorrhizal fungi. In: VA mycorrhiza. Eds. C. LI. Powell and D.J. Bagyaraj, CRC. Press, Boca Raton, USA, pp. 131-153.