Response of Leucaena esculenta to Endomycorrhizae and RMzobium inoculation M. Valdbs,* F. Reza-AlemBn and V. Furlan Dual inoculations on Leucaenna esculenta plants of either Glomus versifome and Rbizobium loti NGR 8 or Glomus sp. and R. loti ENCB 31, gave higher growth and phosphorus accumulation compared with treatments with a single micro-symbiont. The above combinations could be used in a re-forestation programme with L. esculenta in the highlands of Oaxaca State, Mexico. The use of endomycorrhized plants is recommended to alleviate the planting shock-phase, particularly in arid zones, and also the risk of root colonization by possibly less-efficient indigenous fungi. Key words: Endomycorrhizae,
Glomus versifome,
Leucaena esculenta, R&&um.
Leucaena esculenta, whose common name is red huaje (red leucaena), is an important tropical tree legume indigenous to the State of Oaxaca, Mexico, a region highly affected by erosion and desertification. This agro-forestry plant is used extensively for human consumption (leaves, flowers, pods and seeds) and firewood, and it also has a high potential as fodder. Red leucaena is also used in land reclamation, re-forestation, erosion control, for hedges, and as a shade crop (National Research Council 1984; Cruz-Cisneros 1989). This plant depends on its rhizobial nodulation for the supply of N, particularly in the N-deficient soils which are very common in tropical regions. In addition to Rhizabium, endomycorrhizal fungi usually colonize the fine roots of leguminous plants. These fungi help leucaena to grow in soils low in minerals such as phosphorus. In several cases, mycorrhizal fungi and rhizobia are synergistic. This has led workers from different parts of the world to exploit the potential of the double symbiosis to increase the productivity of many leguminous crops (Bagyaraj
1984).
The present study was designed to evaluate, for the first time, the beneficial effects of dual endomycorrhizal fungi-rhizobia inoculation on red leucaena.
M. Vald&s and F. Aeza-AlemAn
are with the Escuela
National
de Cienaas
Biol6gicas, lnstituto PoliUcnico N&on& Apartado Postal 63-246, MBxico, D.F 02800. Mexico. V. Furlan is with the Stalion de Recherches. Agriculture Canada, 2560 Boulevard Hochelaga, Sainte-Foy, QuBbec, GlV 2J3, Canada. ‘Corresponding author. @ 1993 Rapid Communications
of Oxford
Ltd
Materials
and Methods
Soil The soil used in Oaxaca at 5 to 40
this study was collected on the highlands of cm depth. The soil, a loamy sand, had a pH of 6.2, 1.5% (w/w) organic matter, 0.079% (w/w) N, and 3.86 &g extractable P (Bray I). The soil was sieved at 2 mm, mixed with sand (2:1, w/w) and the non-sterilized mixture used to fill pots, 15 cm in diameter.
Mcroorganisms
The endomycorrhizal fungi used were Glamus versiforme (Karst.) Berth and Clamus sp., a native fungus. For G. vers@rme inoculation, chopped, colonized onion roots were spread at the rate of 2 g per pot, 2 cm below soil surface. For the GLom~s sp., 40 spores were
inoculated per plant. Three different strains of Rhizobium were used: R. loti ENCB 31, TAL 1145 and NGR 8. Two ml of the liquid culture, containing 3 x 10’ cells/ml, were poured in the pot after seedling transplantation. Seedsand Treatments Leucaena seeds were scarified with cone H2S04 for 20 min to ensure a high germination; at the same time, this treatment kil!s fungi and other pathogens on the seed. The seeds were then washed 10 times with sterile distilled water. The scarified seeds were germinated at 28°C in Petri dishes containing moist sterilized sand. When the radicle was .I cm long, two seedlings were
transplanted per pot. There were 15 treatments as follows: N; P; N and P; each of the three RkizubiMm strains; each of the two endomycorrhizal fungi (alone and control (unfertilized
combined with each Rkizobitrm and uninoculated). N, as urea,
strain); and a and P, as triple
superphosphate, were each applied at the equivalent rate of 6 g/m’.
M. Valdk, F. RemAle&n
and V. Furlan
The experiment was conducted in a greenhouse and pots were arranged in a randomized block design with four replicates per treatment. After 26 weeks of growth, plants were harvested and the following characteristics recorded: shoot and nodule dry mass; root colonization by the endomycorrhizal fungus, using the technique of Giovanetti & Mosse (1980); nodule nitrogenase activity (acetylene reduction), measured by the method of Somasegaran & Hoben (1985); dry leaf N and P content, measured by chemical analysis. The analysis of variance (ANOVA) was used to test the results.
Results
(1985). Inoculations of Glomtrs versiforme with R. loti NGR 8 or Glomtrs sp. with R. lofi ENCB 31 produced the highest growth increases and phosphorus accumulations observed. Habte & Manjunath (1987) demonstrated that the improved growth response of endomycorrhizal .!,. leucocephala was closely related to improved P uptake. Inoculations of Glomus DersifOrme with either R. loti ENCB 31 or TAL 114.5 produced a significantly higher tissue N content compared with all other treatments. There was no significant difference in nodule dry mass between the plants inoculated with Rhizobium only and plants dually inoculated. This may be explained by the fact that measurements were taken only at the end of the experiment. It is well known that, with time, container volume becomes a limiting factor for root system development and possibly for nodulation. Manjunath & Habte (1988) did sequential monitoring on mycorrhizal and non-mycorrhizal leucaena plants and observed different patterns of the variables measured during the experiment. Nitrogenase activity was significantly higher in plants inoculated with Glomus sp. and either R. lofi TAL 1145 or NGR 8 compared with other dual inoculation treatments and even with those plants inoculated with Rhizobium only. It is possible that the nitrogenase activity resulting from these two Rhizobiwn strains was particularly stimulated in the presence of Glomtls sp. but more likely it was time-delayed in the other treatments. An experiment performed by Smith & Daft (1977) showed that phosphate uptake, nodulation and nitrogenase activity of mycorrhizal
and Discussion
Endomycorrhizal leucaena plants had a dry mass not significantly different from that of the control plants (Table I). Surprisingly though, their tissue N content was over three times that of non-mycorrhizal plants. A higher N content in mycorrhizal plants has already been observed by other researchers and is discussed by Cooper (1984). Plants inoculated with Rhizobim strains produced a significantly larger dry biomass than the endomycorrhizal ones and had a similar N content. There were no differences in P content for plants inoculated with Glomtts or with Rhizobiwn. Among the different dual inoculations with endomycorrhizal fungi and rhizobia, some treatments showed a significant plant dry mass or N and P tissue content enhancement when compared with either fungal or rhizobial inoculation alone (Table I). Many similar cases of synergistic effects are reported by Bagyaraj (1984) and Huang et al.
Table 1. Effect of nltrogenase actlvlty,
N P fertlllzatlon, root colonization
Treatment
Shoot dry mass (9)
1.49-
Control N P N and
P
98
in a column
World ]ouml
followed
and
Nodule dry mass (mg)
Rhlzobium
2.03’ 1.64a 2.97b
2018
3.20b
203’
2.44a 1.94”
3.31b 3.51b 2.42b 3.94c
2128 185a 155a
same
of Microbiology and Biotechnology. Vol 9, 1993
letter
on
red
leucaena
growth,
Root w-1
0.90=
significantly
40”
3.20b 3.03b
673’ 646” 927a
67b 408 39”
3.34b 2.4fY 4.72’
a37a 920= 9278
37a 3oa
4.W 3.15b 3.35b
1033b 1242’ 1306’
65b 70b 61b 42a
3.61b 2.91b
1048b 1105b
46’ 57b
1 .05a 1.25’ 2.97b
5.25b not
805’ 705’ 9248
1.33”
3.46’ 3.11a 2.798 5.62b
are
nodulatlon,
colonlzatlon
1.54a 2.31’
193a 1588 197a 214a superscript
strains
Nltrogenase actlvlty (pmol C2H,I p1ant.h) 0 0 0 0 0 0
4.3v 2.92b 2.70b by the
fungi P content.
0 0 0 0 0 0
1.47a 1.7v 2.45b
Glomus versiforme Glomus sp. Rhizobium loti ENCB 31 Rhizobium loti TAL 1145 Rhizobium loti NGR 8 G. versiforme + R. loti ENCB 31 G. versiforme + R. loti TAL 1145 G. versiforme + R. loti NGR 8 Glomus sp. + R. loti ENCB 31 Glomus sp. + R. loti TAL 1145 Glomus sp. + R. loti NGR 8 l Means test).
and endomycorrhizal and tissue N and
different
(PI
0.05;
Duncan’s
34a 33a 41’
multiple-range
Endomycorrhizae the growth period and that plants varied during enhancement of these parameters due to the Endomycorrhizae preceded enhancement of growth. Even if the control plants have an important root colonization, resulting from endomycorrhization by indigenous species, the shoot dry mass of the controls was significantly less than that of plants inoculated with Glomus sp. and R. loti ENCB 31 under the same conditions (Table I). This indicates that any indigenous species were less efficient at promoting growth enhancement compared with the inoculated species. Daft & Nicolson (1966) reported that high colonization is not the only factor involved when considering the mycorrhizal response. The effects of mycorrhiza on plant growth depend, to some degree, on the balance between the development of root colonization and the concentration and availability of phosphate and perhaps other nutrients. Furlan & Bemier-Cardou (1989) emphasized the necessity of having the optimal ratio of available nutrients in the soil if the host plant-endomycorrhizal fungus system was to be at its most efficient. Finally, we suggest the use of ready-endomycorrhized seedlings in field culture to diminish the risk of root colonization by possibly less-efficient indigenous fungi. This technique is particularly recommended in arid regions to survival. It is well known that increase seedling endomycorrhizae can improve drought-tolerance and reduce transplant injury of plants (Cooper 1984).
Acknowledgements The authors wish to thank Dr D. PrCvost, Agriculture Canada Research Station, Sainte-Foy, for her valuable comments and suggestions in reviewing this manuscript and Mrs C. Tremblay for typing the manuscript.
and Rhizobium
on Leucaena
References Bagyaraj, J. 1984 Biological interactions with VA mycorrhizal fungi. In VA Mycorrhiza, eds Powell, CL. & Bagyaraj, DJ. pp. 131-153. Boca Raton, Florida: CRC Press. Cooper, K.M. 1984 Physiology of VA mycorrhizal associations. In VA Mycorrhiza, eds Powell, CL. & Bagyaraj, D.J. pp. 153-186. Boca Raton, Florida: CRC Press. Cruz-Cisneros, R. 1989 Produccibn de forraje con especies no convencionales en terrenos degradados de1 estado de Morelos. MSc Thesis, Universidld Autbnoma de Mexico, Mexico City, Mexico. Daft, M.J. & Nicolson, T.H. 1966 Effect of En&one mycorrhiza on plant growth. New Phyfologisf 65, 343-350. Furlan, V. & Bemier-Cardou, M. 1989 Effects of N, P, and K on formation of vesicular-arbuscular mycorrhiza, growth and mineral content of onion. Phznf und Soil 113, 167-174, Giovannetti, M. & Mosse, B. 1980 An evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. Nezu Phyfologisf
84,
489-500.
Habte, M. & Manjunath, A. 1987 Soil solution phosphorus status and mycorrhizal dependency in Leucaena leucocephala. Applied Environmental
Microbiology
53, 797-801.
Huang, R.S., Smith, W.K. & Yost, R.S. 1985 Influence of vesicular-arbuscular mycorrhiza on growth, water relations, and leaf orientation in Leucaena leucocephalu (Lam.) de Wit. NEW Phyfologist
99, 229-243.
Manjunath, A. & Habte, M. 1988 Development of vesiculararbuscular mycorrhizal infection and the uptake of immobile nutrients in Leucaena leucocephala. Plant and Soil 106, 97-103. National Research Council 1984 Lerrcuena: Promising Forage and Tree Crop for the Tropics, 2nd edn. Washington DC: National Academy Press. Smith, SE. & Daft, M.J. 1977 Interactions between growth, phosphate content and nitrogen fixation in mycorrhizal and non-mycorrhizal Medicago sativa. Australian ]ournal of Physiology 4,403-413. Somasegaran, R. & Hoben, HJ. 1985 Methods in LegumeRhizobium Technology. University of Hawaii: NIFTAL Project and MIRCEN.
(Received in revised
form 14 ]uly
1992;
accepted
17 July
World normal of Microbiology and Biotechnology. Vol 9. 1993
1992)
99
Glomus versifome,
Leucaena esculenta, R&&um.
Leucaena esculenta, whose common name is red huaje (red leucaena), is an important tropical tree legume indigenous to the State of Oaxaca, Mexico, a region highly affected by erosion and desertification. This agro-forestry plant is used extensively for human consumption (leaves, flowers, pods and seeds) and firewood, and it also has a high potential as fodder. Red leucaena is also used in land reclamation, re-forestation, erosion control, for hedges, and as a shade crop (National Research Council 1984; Cruz-Cisneros 1989). This plant depends on its rhizobial nodulation for the supply of N, particularly in the N-deficient soils which are very common in tropical regions. In addition to Rhizabium, endomycorrhizal fungi usually colonize the fine roots of leguminous plants. These fungi help leucaena to grow in soils low in minerals such as phosphorus. In several cases, mycorrhizal fungi and rhizobia are synergistic. This has led workers from different parts of the world to exploit the potential of the double symbiosis to increase the productivity of many leguminous crops (Bagyaraj
1984).
The present study was designed to evaluate, for the first time, the beneficial effects of dual endomycorrhizal fungi-rhizobia inoculation on red leucaena.
M. Vald&s and F. Aeza-AlemAn
are with the Escuela
National
de Cienaas
Biol6gicas, lnstituto PoliUcnico N&on& Apartado Postal 63-246, MBxico, D.F 02800. Mexico. V. Furlan is with the Stalion de Recherches. Agriculture Canada, 2560 Boulevard Hochelaga, Sainte-Foy, QuBbec, GlV 2J3, Canada. ‘Corresponding author. @ 1993 Rapid Communications
of Oxford
Ltd
Materials
and Methods
Soil The soil used in Oaxaca at 5 to 40
this study was collected on the highlands of cm depth. The soil, a loamy sand, had a pH of 6.2, 1.5% (w/w) organic matter, 0.079% (w/w) N, and 3.86 &g extractable P (Bray I). The soil was sieved at 2 mm, mixed with sand (2:1, w/w) and the non-sterilized mixture used to fill pots, 15 cm in diameter.
Mcroorganisms
The endomycorrhizal fungi used were Glamus versiforme (Karst.) Berth and Clamus sp., a native fungus. For G. vers@rme inoculation, chopped, colonized onion roots were spread at the rate of 2 g per pot, 2 cm below soil surface. For the GLom~s sp., 40 spores were
inoculated per plant. Three different strains of Rhizobium were used: R. loti ENCB 31, TAL 1145 and NGR 8. Two ml of the liquid culture, containing 3 x 10’ cells/ml, were poured in the pot after seedling transplantation. Seedsand Treatments Leucaena seeds were scarified with cone H2S04 for 20 min to ensure a high germination; at the same time, this treatment kil!s fungi and other pathogens on the seed. The seeds were then washed 10 times with sterile distilled water. The scarified seeds were germinated at 28°C in Petri dishes containing moist sterilized sand. When the radicle was .I cm long, two seedlings were
transplanted per pot. There were 15 treatments as follows: N; P; N and P; each of the three RkizubiMm strains; each of the two endomycorrhizal fungi (alone and control (unfertilized
combined with each Rkizobitrm and uninoculated). N, as urea,
strain); and a and P, as triple
superphosphate, were each applied at the equivalent rate of 6 g/m’.
M. Valdk, F. RemAle&n
and V. Furlan
The experiment was conducted in a greenhouse and pots were arranged in a randomized block design with four replicates per treatment. After 26 weeks of growth, plants were harvested and the following characteristics recorded: shoot and nodule dry mass; root colonization by the endomycorrhizal fungus, using the technique of Giovanetti & Mosse (1980); nodule nitrogenase activity (acetylene reduction), measured by the method of Somasegaran & Hoben (1985); dry leaf N and P content, measured by chemical analysis. The analysis of variance (ANOVA) was used to test the results.
Results
(1985). Inoculations of Glomtrs versiforme with R. loti NGR 8 or Glomtrs sp. with R. lofi ENCB 31 produced the highest growth increases and phosphorus accumulations observed. Habte & Manjunath (1987) demonstrated that the improved growth response of endomycorrhizal .!,. leucocephala was closely related to improved P uptake. Inoculations of Glomus DersifOrme with either R. loti ENCB 31 or TAL 114.5 produced a significantly higher tissue N content compared with all other treatments. There was no significant difference in nodule dry mass between the plants inoculated with Rhizobium only and plants dually inoculated. This may be explained by the fact that measurements were taken only at the end of the experiment. It is well known that, with time, container volume becomes a limiting factor for root system development and possibly for nodulation. Manjunath & Habte (1988) did sequential monitoring on mycorrhizal and non-mycorrhizal leucaena plants and observed different patterns of the variables measured during the experiment. Nitrogenase activity was significantly higher in plants inoculated with Glomus sp. and either R. lofi TAL 1145 or NGR 8 compared with other dual inoculation treatments and even with those plants inoculated with Rhizobium only. It is possible that the nitrogenase activity resulting from these two Rhizobiwn strains was particularly stimulated in the presence of Glomtls sp. but more likely it was time-delayed in the other treatments. An experiment performed by Smith & Daft (1977) showed that phosphate uptake, nodulation and nitrogenase activity of mycorrhizal
and Discussion
Endomycorrhizal leucaena plants had a dry mass not significantly different from that of the control plants (Table I). Surprisingly though, their tissue N content was over three times that of non-mycorrhizal plants. A higher N content in mycorrhizal plants has already been observed by other researchers and is discussed by Cooper (1984). Plants inoculated with Rhizobim strains produced a significantly larger dry biomass than the endomycorrhizal ones and had a similar N content. There were no differences in P content for plants inoculated with Glomtts or with Rhizobiwn. Among the different dual inoculations with endomycorrhizal fungi and rhizobia, some treatments showed a significant plant dry mass or N and P tissue content enhancement when compared with either fungal or rhizobial inoculation alone (Table I). Many similar cases of synergistic effects are reported by Bagyaraj (1984) and Huang et al.
Table 1. Effect of nltrogenase actlvlty,
N P fertlllzatlon, root colonization
Treatment
Shoot dry mass (9)
1.49-
Control N P N and
P
98
in a column
World ]ouml
followed
and
Nodule dry mass (mg)
Rhlzobium
2.03’ 1.64a 2.97b
2018
3.20b
203’
2.44a 1.94”
3.31b 3.51b 2.42b 3.94c
2128 185a 155a
same
of Microbiology and Biotechnology. Vol 9, 1993
letter
on
red
leucaena
growth,
Root w-1
0.90=
significantly
40”
3.20b 3.03b
673’ 646” 927a
67b 408 39”
3.34b 2.4fY 4.72’
a37a 920= 9278
37a 3oa
4.W 3.15b 3.35b
1033b 1242’ 1306’
65b 70b 61b 42a
3.61b 2.91b
1048b 1105b
46’ 57b
1 .05a 1.25’ 2.97b
5.25b not
805’ 705’ 9248
1.33”
3.46’ 3.11a 2.798 5.62b
are
nodulatlon,
colonlzatlon
1.54a 2.31’
193a 1588 197a 214a superscript
strains
Nltrogenase actlvlty (pmol C2H,I p1ant.h) 0 0 0 0 0 0
4.3v 2.92b 2.70b by the
fungi P content.
0 0 0 0 0 0
1.47a 1.7v 2.45b
Glomus versiforme Glomus sp. Rhizobium loti ENCB 31 Rhizobium loti TAL 1145 Rhizobium loti NGR 8 G. versiforme + R. loti ENCB 31 G. versiforme + R. loti TAL 1145 G. versiforme + R. loti NGR 8 Glomus sp. + R. loti ENCB 31 Glomus sp. + R. loti TAL 1145 Glomus sp. + R. loti NGR 8 l Means test).
and endomycorrhizal and tissue N and
different
(PI
0.05;
Duncan’s
34a 33a 41’
multiple-range
Endomycorrhizae the growth period and that plants varied during enhancement of these parameters due to the Endomycorrhizae preceded enhancement of growth. Even if the control plants have an important root colonization, resulting from endomycorrhization by indigenous species, the shoot dry mass of the controls was significantly less than that of plants inoculated with Glomus sp. and R. loti ENCB 31 under the same conditions (Table I). This indicates that any indigenous species were less efficient at promoting growth enhancement compared with the inoculated species. Daft & Nicolson (1966) reported that high colonization is not the only factor involved when considering the mycorrhizal response. The effects of mycorrhiza on plant growth depend, to some degree, on the balance between the development of root colonization and the concentration and availability of phosphate and perhaps other nutrients. Furlan & Bemier-Cardou (1989) emphasized the necessity of having the optimal ratio of available nutrients in the soil if the host plant-endomycorrhizal fungus system was to be at its most efficient. Finally, we suggest the use of ready-endomycorrhized seedlings in field culture to diminish the risk of root colonization by possibly less-efficient indigenous fungi. This technique is particularly recommended in arid regions to survival. It is well known that increase seedling endomycorrhizae can improve drought-tolerance and reduce transplant injury of plants (Cooper 1984).
Acknowledgements The authors wish to thank Dr D. PrCvost, Agriculture Canada Research Station, Sainte-Foy, for her valuable comments and suggestions in reviewing this manuscript and Mrs C. Tremblay for typing the manuscript.
and Rhizobium
on Leucaena
References Bagyaraj, J. 1984 Biological interactions with VA mycorrhizal fungi. In VA Mycorrhiza, eds Powell, CL. & Bagyaraj, DJ. pp. 131-153. Boca Raton, Florida: CRC Press. Cooper, K.M. 1984 Physiology of VA mycorrhizal associations. In VA Mycorrhiza, eds Powell, CL. & Bagyaraj, D.J. pp. 153-186. Boca Raton, Florida: CRC Press. Cruz-Cisneros, R. 1989 Produccibn de forraje con especies no convencionales en terrenos degradados de1 estado de Morelos. MSc Thesis, Universidld Autbnoma de Mexico, Mexico City, Mexico. Daft, M.J. & Nicolson, T.H. 1966 Effect of En&one mycorrhiza on plant growth. New Phyfologisf 65, 343-350. Furlan, V. & Bemier-Cardou, M. 1989 Effects of N, P, and K on formation of vesicular-arbuscular mycorrhiza, growth and mineral content of onion. Phznf und Soil 113, 167-174, Giovannetti, M. & Mosse, B. 1980 An evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. Nezu Phyfologisf
84,
489-500.
Habte, M. & Manjunath, A. 1987 Soil solution phosphorus status and mycorrhizal dependency in Leucaena leucocephala. Applied Environmental
Microbiology
53, 797-801.
Huang, R.S., Smith, W.K. & Yost, R.S. 1985 Influence of vesicular-arbuscular mycorrhiza on growth, water relations, and leaf orientation in Leucaena leucocephalu (Lam.) de Wit. NEW Phyfologist
99, 229-243.
Manjunath, A. & Habte, M. 1988 Development of vesiculararbuscular mycorrhizal infection and the uptake of immobile nutrients in Leucaena leucocephala. Plant and Soil 106, 97-103. National Research Council 1984 Lerrcuena: Promising Forage and Tree Crop for the Tropics, 2nd edn. Washington DC: National Academy Press. Smith, SE. & Daft, M.J. 1977 Interactions between growth, phosphate content and nitrogen fixation in mycorrhizal and non-mycorrhizal Medicago sativa. Australian ]ournal of Physiology 4,403-413. Somasegaran, R. & Hoben, HJ. 1985 Methods in LegumeRhizobium Technology. University of Hawaii: NIFTAL Project and MIRCEN.
(Received in revised
form 14 ]uly
1992;
accepted
17 July
World normal of Microbiology and Biotechnology. Vol 9. 1993
1992)
99
