NODULATION STATUS AND NITROGEN FIXING POTENTIAL OF AKASHMONI ( Acacia auriculiformis ) SEEDLINGS IN TROPICAL MIXED PLANTATIONS

A study was carried out to assess biological nitrogen fixation and observe nodulation status of Acacia auriculiformis (Akashmoni) at mixed plantations in the Seed Research Laboratory and nursery of the Institute of Forestry and Environmental Sciences, Chittagong University (IFESCU), Bangladesh. The plantations consist of one pure planting plot (100% A) and eleven mixed planting plots (1A:1S, 1A:1G, 1A: 1G: 1S, 1A:2S, 1A:2G, 1A:3S, 1A:3G, 2A:1S, 2A:1G, 3A:1S and 3A:1G) of the three common plantation tree species of Bangladesh namely Acacia auriculiformis (A), Swietenia macrophylla (S) and Gmelina arborea (G). In Seed Research Nursery, seedlings of three species were raised in a randomized blocks with three replicates of twelve treatment plots. Nodule number, color, size, shape, form, structure, nodule fresh weight, oven-dry weight and total nitrogen accumulation in the soil of A. auriculiformis seedling were recorded. At the age of 10 month, highest nodulation (143 number) of A. auriculiformis were recorded in the mixed plot (1A:2S) in comparison with pure 100%A plot. Fresh and oven dry weight of roots of the A. auriculiformis seedlings were found significantly (p<0.05) highest in 1A:2S mixed plot. The findings of this study suggest that in comparisons with pure plot, A. auriculiformis fix better nitrogen in mixed plantations.


INTRODUCTION
One of the major management objectives of tropical forests of Bangladesh was to replace the heterogeneous natural forests by the mixed plantations of valuable timber species (Dutta et al. 2014).The management of mixtures of two dominants is more difficult; they can usually only be managed on specific sites and often result in the suppression of one of the species (FAO 1992, Hossain 2008).Nitrogen fixing trees in tropical environments appear to offer both high growth rate and soil enrichment (Binkley and Giardiana 1997).Nitrogen fixing trees may increase the supply of available nitrogen in the soil, benefiting both Nfixing and non-N fixing trees (Binkley et al. 2000).The success of mixed species plantations depends on species attributes and site factors (Forrester et al. 2005) 1 Shourav DUTTA, (corresponding author: shourav.forestry@gmail.com),Institute of Forestry and Environmental Sciences, University of Chittagong, Chittagong-4331, BANGLADESH.Notes: The authors declare that they have no conflicts of interest.Authorship Form signed online.
that affect the balance of positive and negative interactions between the species (Boyden et al. 2005).Nitrogen fixation by NFS is supposed to improve soil N availability (Bouillet et al. 2008, Khanna 1997, Voigtlaender et al. 2012), alleviating N limitation and facilitating the growth of the target species in N 2 limited soils (Forrester et al. 2007).In addition, nitrogen fixing species may enhance phosphorus (P) availability through the rhizosphere acidification due to nitrogen fixation (Hinsinger et al. 2011), increasing the amount of P that cycles in mixed-species plantations compared to monocultures (Forrester et al. 2005).For increasing the effectiveness of the biological nitrogen fixation, studies are needed to carry out on the nodulation and nitrogen fixation in the existing legume flora in different parts of the world (Mahmood and Iqbal 1994).In Bangladesh, information on comparative seedling growth and nodulation of legumes are very scanty (Aryal et al. 1999).There is a wide range of nitrogen fixing plants that have been used in forestry with an objective of raising soil nitrogen levels and subsequently improving the growth of the non-nitrogen fixing forest species (Turvey and Smethrust 1983).But still research on nitrogen-fixing species in general has lagged behind that on food, feed, and forage crops.
Estimates of N 2 -fixation for tropical evergreen forests are extremely rare and highly variable (Cleveland et al. 1999).Over 600 tree species have been reported to fix nitrogen (MacDicken, 1994).In Bangladesh, a total of 98 genera and 332 sub-generic taxa of nitrogen-fixing species, both wild and cultivated have been recorded under the families Caesalpiniaceae (20, 60), Mimosaceae (14, 39) and Fabaceae (64, 233) (Khan et al. 1996).Acacia auriculiformis and Albizia spp.are two most common nitrogen fixing species commonly used in road side and forest plantation, agroforestry, community forestry and homestead plantation programs in Bangladesh.Various exotic nitrogen fixing plants with native nitrogen fixing trees are planted all around the country.A common expectation is that an N 2 -fixing species will enhance nitrogen availability to the other and accelerate nitrogen cycling, so that total growth will be greater.Recently N 2 -fixing tree species such as Acacia auriculiformis, Albizia spp., Leucaena leucocephala etc are planted with non-N 2 fixing tree in the homegarden of Bangladesh (Alam et al. 2005).Acacia auriculiformis, Swietenia macrophylla and Gmelina arborea are the three major plantation tree species proved successful in trials and in large scale plantation programs of Bangladesh (Hossain and Hoque 2013).The present study was aimed to investigate the optimum mixing and benefits of nitrogen fixation of N 2 -fixing A. auriculiformis with nonnitrogen fixing species (S. macrophylla and G. arborea).The present attempt was also made with a view to studying the nodulation potential and nitrogen fixing ability of A. auriculiformis used in the mixed plantations of Bangladesh.

Experiment site
The study was conducted in the nursery seed bed of Seed Research Laboratory of the Institute of Forestry and Environmental Sciences, University of Chittagong (IFESCU) campus, Chittagong, Bangladesh.The experimental design was carried out over a period of ten months from February to November, 2015 in the IFESCU nursery.The experimental site (nursery) lies approximately at the intersection of 91°50´ east longitude and 22°30´ north latitude (Khan et al. 2004) The nursery site enjoys a tropical monsoon climate characterized by hot, humid summer and cool, dry winter (Mahmud et al. 2005).The average monthly mean temperature varied 29.75°C maximum and between 21.14°C minimum (Ahmed, 1990).The annual rainfall in the nursery is 2500-3000 mm which mostly takes place between June and September (Gafur et al. 1979).Relative humidity was generally the lowest (64%) in February and highest (95%) in June-September (Mahmood et al. 2005).
Each plot was 200 cm 60 cm in size with 30 seedlings at a spacing of 20 cm × 20 cm (seedling to seedling distance: 20 cm).Within each mixed plot of 1A:1S or 1A:1G, seedlings of Acacia were planted with Swietenia or Gmelina alternatively.In 2A:1S or 2A:1G plot, two seedlings of Acacia followed by one seedlings of either Swietenia or Gmelina sequentially.Similarly, in 1A:2S or 1A:2G plots one seedling of Acacia followed by two seedlings of Swietenia or Gmelina sequentially.Again, within each mixed plot of 3A:1S or 3A:1G, three seedlings of Acacia followed by one seedlings of Swietenia or Gmelina sequentially.Similarly, in 1A:3S or 1A:3G plot, one seedling of Acacia followed by either three seedlings of Swietenia or Gmelina consequently.

Harvesting and data collection in the seed research laboratory and nursery
Five randomly selected seedlings of each species were harvested carefully from each mix plot and ten seedlings from pure plantation plot at 10 months after germination.The harvested seedlings were washed in tap water and then in distilled water to clean the root regions off all soil particles.Fresh weight of the root of A. auriculformis was measured after removal of all water from the root portion of the washed seedlings.Dry weight of roots was recorded after oven dried at 70°C for 72 hours.

Evaluation of nodulation
For A. auriculiformis nodule numbers per seedling were recorded after measuring the root weight.Then nodules were separated from individual plants and further washed carefully to remove all the soil particles (Solaiman, 1999).The root nodules were counted and their fresh weight was recorded.Nodule status, shape, color and structure were recorded.Nodule numbers in roots were recorded and nodule score was evaluated using the following table (Table 1): Table 1.Classification criteria used to evaluate the nodulation in the roots (Corbin 1977) Soil level Nodule number in roots Nodule score 0 -5 cm 0 Effectiveness of nodules was represented as follows (Peoples, 1989):

Collection of soil samples and determination of nitrogen
In the nursery, soil samples were collected from each plot at different positions from a depth of 30-35 cm near each seedling.Soil samples were then air dried, ground in agate mortar and passed through a 2 mm mesh sieve.The soil samples were analyzed in the chemistry laboratory of institute of forestry and environmental sciences, Chittagong University (Ifescu), Bangladesh to determine total nitrogen.The total nitrogen content was determined by the Kjeldahl method (Bremner, 1965).

Statistical analysis
Analysis of variance (ANOVA) and tests for means (p < 0.05) were run using the means of each variable from each of the three replicate plots.The statistical evaluations of the data obtained as a result of research was carried out though variance analysis according to randomized blocks experimental design.In order to determine the difference between the averages, the Duncan's multiple range test (DMRT) was utilized.All the data collected were analyzed statistically by using spss (Aryal et al. 1999(Aryal et al. , khan et al. 2004)).

RESULTS AND DISCUSSION Nodulation status of A. auriculiformis seedling
Nodules of A. auriculiformis were elongated to ovate with/ without branching and pink/ brown in color.A. auriculiformis nodules were found both in primary and secondary roots of the seedlings (Fig. 2).Nodule number differed significantly among pure and mixed plots (Table 2).Nodules of A. auriculiformis in pure and mixed plots were found pink or brown in color.(Fig. 3).Nodule size varied from 2.6 × 3.3 mm in N5 to 5.2 × 6.3 mm in M5. A. auriculiformis possessed nodules both in single and aggregate forms.Variations in nodule size, form and structure were observed among the treatment plots (Table 3).Maximum number (143) of nodule was recorded in M5 followed by 38 in M3 and 32 in M4.Nodule number was found minimum (6) in N5. A. auriculiformis seedlings showed excellent and good nodulation status in both pure and mixed plots.A. auriculiformis fixed more Nitrogen through nodule formation in their root systems with S. macrophylla than with G. arborea. A. auriculiformis seedlings showed excellent, good or fair nodulation status in mixed plots except N5 plot.Nodulation status found fair in N2 and N4 plots and poor in N5 plot (Fig. 4).At the time of A. auriculiformis seedling harvest, the highest root fresh weight (27.26 g) was recorded in M5 followed by 20.06 g in P1 and 19.07g in M3.Compared with pure plot, the fresh weight of root was significantly higher in mixed plots.The result of variance analysis for root dry weight and nodule fresh weight A. auriculiformis of revealed that the effect of planting pattern was significant (p < 0.05).Mean comparison using Duncan multiple range test (DMRT) showed that the highest root dry weight obtained in mixed plot M5.Compared with pure plot (10.06 g), the dry masses of root were also significantly higher in M5 (19.28g) followed by M2 (10.82 g).The result of analysis of variance revealed that planting patterns had significant effect on the nodule fresh weight of A. auriculiformis (Table 4).Table 4. Comparative root fresh weight (g), nodule fresh weight (g), root dry weight (g) and total nitrogen (%) of ten months old A. auriculiformis seedlings.Highest values of total Nitrogen (0.93%) was measured in mixed M2 plot.Compared with pure plots, the total nitrogen were significantly higher in mixed plots M2 followed by N2 (0.56%) and M3 (0.47%).Among all the plots the sequence of total nitrogen (%) were M2 > N2 > M3 > M4 > M6 > M1 > N3 > N1 > N4 > P1 > M5 > N5 (Fig. 5).

Fig. 5. Percentage of total nitrogen in the soil of twelve experimental plots
The present study reveals a total of 143 nodules per tree for A. auriculiformis in the mixture with S. macrophylla were comparatively more than Aryal et al. (1999) and Khan et al. (2004).Aryal et al. (1999) recorded 55 nodules per tree in a 14 months old plantation and Khan et al. (2004) recorded 61 nodules per tree in a 3 months old plantation for A. auriculiformis respectively.Compared with the findings of several other researchers, the findings of the present study were similar to that of Aryal et al. (1999) and Khan et al. (2004).The present findings also revealed that A. auriculiformis showed excellent nodulation behavior in the mixture with S. macrophylla than with G. arborea.The present investigation also recorded nodule fresh weight (2.01 g) for A. auriculiformis which was greater than Aryal et al. (1999).Aryal et al. (1999) recorded 1.34 g nodule fresh weight for A. auriculiformis.
A. auriculiformis (Akashmoni) is extensively planted in plantations all over the country for its short rotation, wider adaptability and faster growth.S. macrophylla (Mahagony) and G. arborea (Gamar) are two non-nitrogen fixing species extensively planted in forests, marginal lands, institutes, roadsides, railway sites, field borders and homesteads of Bangladesh.From the present investigation, it is evident that the mixed stands possess better effects than the pure stands in improving soil nitrogen.
In this study, the nodulation status of A. auriculiformis seedlings was measured at ten months after germination.In this respect, given the values presented in Table 2, it has been determined according to variance analysis applied that there were statistically significant differences among nodulation of this species in various treatments (p<0.05).According to the results of Duncan Multiple Range test implemented, it has been found that the nodulation behavior of A. auriculiformis in pure plots were lower than in mixed plots.The present study also indicates the increment of nitrogen in the soil through plantation of nitrogen fixing tree species.
Though monoculture of exotic species such as A. auriculiformis has some effect on native biodiversity of Bangladesh, mixed plantation of A. auriculiformis with valuable indigenous tree species particularly G. arborea may show marvelous results.A. auriculiformis may also be planted in degraded hilly areas because it was reported to thrive well on moderately acid, poorly fertile soil and also improves pH, physical and chemical properties of soil.The present investigation indicates three most common plantation tree species (A.auriculiformis, G. arborea and S. macrophylla) of Bangladesh which were similar to Das and Sarkar (2014) conducted in the Bhawal Sal forest, in the central region of Bangladesh.Das and Sarkar (2014) observed significant positive relationship between species growth, diversity and productivity in four mixed species plantations and the subject species were A. auriculiformis, G. arborea, S. macrophylla and A. mangium.
From the present investigation it was also found that nodule size of A. auriculiformis seedlings varied from 2.6 × 3.3 mm in N 5 plot to 5.2 × 6.3 mm in M 5 plot which was greater than Khan et al. (2004).Khan et al. (2004) recorded 3.2 × 6.1 mm nodules in three months old A. auriculiformis seedlings under nursery condition.
Nodule number and nodule mass or nodule weight per unit dry weight of the whole plant or root system are often used in trial comparisons; however, similar information can be obtained by visually scoring nodulation on a 0-5 basis taking into account nodule number, size, pigmentation and distribution.Nodule score is judged by the number of effective nodules in the crown-root zone (regarded as the region 5 cm below the first lateral roots) and elsewhere on the root system (Corbin, 1977).The present study indicates an excellent nodulation status of A. auriculiformis in plantation forests by increasing nodule number below (5 cm) the first lateral roots.
The present study reveals that mixed species plantations of A. auriculiformis with other non-nitrogen fixing species (G.arborea and S. macrophylla) have the potential to improve nodulation, nitrogen fixation and stand productivity over that of Acacia monoculture through catalytic effects on soils by Acacia, which is similar to that of Forrester et al. (2007).A study was conducted by Forrester et al. (2007) reported that nitrogen fixation and productivity were higher in mixed stands of Acacia mearnsii with non-nitrogen fixing Eucalyptus globulus than in A. mearnsii monocultures.
Effectiveness of nodules can generally be gauged by the degree of pink or red coloration of N 2 -fixing bacteroid tissue inside each nodule (Corbin 1977).As a general rule, white or green nodules are inactive and would not be considered when classifying active nodulation (Corbin 1977, Peoples 1989).The present study revealed that nodules of A. auriculiformis in pure and mixed plots were pink or brown in color.So the result indicates all the nodules in the root system were active.

CONCLUSIONS
The results of present investigation showed biological nitrogen fixation and nodulation status of Acacia auriculiformis in mixed plantations in comparison to pure plantations.During the present study, fixation of biological nitrogen in the soil through the root system of A. auriculiformis seedlings was considered as the only way of nitrogen increment in the soil, rejecting all other factors such as rainfall, storm, thunder-bolt, fertilizer treatment etc.The present findings would be so much helpful to carry out further investigation on the growth and development of different nitrogen fixing species to both acidic and alkaline soils before recommending them in mixed plantation programs.These results are based on 10-month old seedlings.So, further researches are necessary to assess the exact performance of A. auriculiformis in fixation of biological nitrogen in the soil.
All the experimental plots in the Seed Research Nursery (a: Two months old seedlings, b: Five months old seedlings).
Biological nitrogen fixation of A. auriculiformis through nodulation (a: Seeds of A. auriculiformis,b: Germination of seeds in seed bed, c: Three months old seedlings under nursery condition, d: Species interactions among A. auriculiformis, S. macrophylla and G. arborea, e:Nodule formation of A. auriculiformis seedlings both in primary and secondary roots).

(
*) Means followed by the same letter(s) in the same column are not significantly different at p < 0.05 (DMRT).

Table 2 .
Nodule number, nodulation status, color, shape, branching status and distribution of nodules of A. auriculiformis in pure and mixed planting plots at 10 months after germination.
(*) Means followed by the same letter(s) in the same column are not significantly different at p< 0.05 (DMRT).

Table 3 .
Nodule size and Structure of ten months old seedlings of A. auriculiformis grown under nursery condition