Diversity of Arbuscular Mycorrhizal Fungi in Disturbed and Undisturbed Forests of Karbi Anglong Hill District of Assam

Conversion of tropical forests to agricultural lands is one of the leading causes of biodiversity loss. To assess the impact of such forest conversion on microbial diversity, the present study investigated the diversity of Arbuscular Mycorrhizal Fungi (AMF) in undisturbed forests (UF), Slash-and-Burn Fields (SBF) and Monoculture Forests (MF). The study sites are located adjacently on a hilly slope. Arbuscular Mycorrhizal (AM) fungal spores were extracted by wet-sieving and decanting technique from the three sites. A total of 12 AM fungal taxa belonging to 4 different genera were extracted and identified. Glomus was the dominant genus in all three sites. Spore density was highest in UF, lower in SBF, and lowest in MF. The high diversity of AM fungi in undisturbed forest with naturally higher plant species diversity suggested that disturbance affect the abundance and richness of arbuscular mycorrhizal spores. Our results show that the forests of Karbi Anglong Hill District of Assam contained a high AM fungal diversity and species richness. The AMF diversity is significantly affected by the land use practices practiced by the people and no step has been initiated to restore this important group of microorganisms by forest management practices.

species richness can influence plant community structure and an increase in plant productivity and diversity 7,8 .Because of the beneficial effects on plant as well as soil health, the importance of AMF in restoration and re-establishment of fragile and degraded ecosystem is well recognized [9][10][11] .This explains the need to enumerate and identify the indigenous AMF population which can be successfully applied in restoration practices.
Conversion of tropical forests to agricultural lands is one of the leading causes of biodiversity loss 12 .The destruction of these forests is taking place at an alarming rate in tropics and the forests of Karbi Anglong Hill Disrict of Assam are also no exception to this.The major threats to the rich biodiversity of the region are expansion of agricultural activities, shifting cultivation, encroachment etc, 13 .The studies on the impact of these vegetation disturbances on microbial flora in general, and AMF in particular, is almost nil in this part of Indo-Burma biodiversity hotspot region.Therefore, to assess the impact of such forest destruction on microbial diversity, the present study investigated the diversity of AMF in undisturbed forests (UF), slash-and-burn fields (SBF) and monoculture forests (MF).The study also aimed to catalogue the Arbuscular Mycorrhizal (AM) fungal species so that they can be used in future for restoration and regeneration of degraded forests and maintenance of sustainable forestry.

Study site
The study site is located in Karbi Anglong Hill District (92 0 45' and 93 0 54' East Longitude and 25 0 45' and 26 0 35' North Latitude) of Assam, India.The three sites-UF, SBF and MF are located adjacently on a hilly slope at an altitude of 232 MSL.Annual mean air temperature was 24.4 0 C Total annual precipitation was 1052 mm, and the rainy season lied between May to September (730 mm).The soil was classified as sandy loam.
The undisturbed forest is a moist semievergreen forest 14  Reserve forest areas subjected to Jhum or shifting cultivation are artificially regenerated by Forest Departments under jhum area rehabilitation programme to improve and restore the fragile ecosystem of the hills.Our study site MF is about 20 years old and consists of artificially regenerated fast growing species of teak (Tectona grandis Linn.).

Sampling procedure
Sixteen soil samples from the rhizosphere region of plants were arbitrarily collected in June-July 2009, at sampling points 20 m apart on the middle line transect of each site.Each soil sample was 200 g to a depth of 15 cm.The soil was placed in sealed plastic bags and stored at 4 0 C until analysis.

Spore isolation and identification of AM fungi
One hundred grams of soil from each field sample was used for spore isolation, using wet sieving and decanting method of Gerdemann and Nicolson 15 .All healthy AMF spores were counted using stereo-zoom microscope.Intact AM spores were transferred onto glass slides containing polyvinylalcohol-lactophenol with or without Melzer's reagent and identified under a compound microscope at upto 400x magnification.AM fungi were identified using keys of Schenck and Perez 16 and culture database established by the International collection of Vesicular and Mycorrhizal Fungi (http://invam.caf.wvu.edu).Permanent slides are stored at Microbial Ecology Laboratory, Dept of Botany, Gauhati University, Assam, India.

Statistical analysis
AM fungal composition in field samples was evaluated based on spore isolation frequency (IF), density, relative abundance (RA), Shannon-Wiener index of diversity (H'), Simpson diversity index (D) and Sorenson's similarity coefficients (Cs).

AM fungal composition
A total of 12 AM fungal taxa were isolated from forty eight soil samples collected at three different sites.Five of these (41.6%) were identified to species level, and seven (58.3%) were identified to genus.Seven of them belonged to the genus Glomus, three to Acaulospora, one to Ambispora and one to Gigaspora.All the 12 AM fungal taxa were found in UF, whereas SBF and MF harboured only 7 and 4 taxa respectively.The Shannon-Wiener index of diversity (H') was higher in the UF (2.08) than in the SBF (1.66) or MF (1.17).The Sorenson's similarity coefficients (Cs) of AM fungal community composition was higher between UF and SBF (0.73) than between UF and MF (0.5).Similarly, Simpson's diversity index (D) was highest in UF (0.84), slightly lower in SBF (0.78) and lowest in MF (0.65).The three diversity indexes are tabulated in Table 1.

Isolation frequency and Relative abundance of AMF
Glomus was the dominant genus in all three sites followed by Acaulospora (Table 2) The three species, Glomus manihotis, Glomus sp4, and Acaulospora sp1 were common in all three sites with IF > 50%.The relative abundance of dominant genus Glomus was highest in SBF (73.3%) than in UF (65.7%) and MF (64%).Acaulospora, the second most frequented genus showed the highest relative abundance in MF (35.8%) than in UF (32.2%) and SBF (24.7%).The dominant genus Glomus manihotis has the highest relative abundance in all the three sites, and was much higher in MF (46.6%) than in SBF (32.1%) and UF (24.1%).

Spore density of AMF
There was a significant difference in the total spore density of AM fungi among the UF (879±19.5 spores per 100g soil), SBF (174±5.3spores per 100g soil) and MF (103±4.4spores per 100g soil).The dominant genus Glomus had the highest spore density in UF (581±23 spores per 100g soil) than in SBF (128±7 spores per 100g soil) and MF (66±6.4spores per 100g soil), and followed by second dominant genus Acaulospora, which had 285±43.2spores per 100g soil in UF, 43±11.8spores per 100g soil in SBF and 37±8.2 spores per 100g soil in MF.Spore density of four AM fungus genera isolated from 3 sites is given in Figure 1.

DISCUSSIONS
Since spores are highly resistant to adverse environmental conditions 17 , we used spore quantification to enumerate the diversity of AMF in disturbed and undisturbed forests.The three sites i.e.UF, SBF and MF were selected in such a way that they were located adjacently to each other on a hilly slope and with similar climatic conditions.The results of the present investigation indicated a significant influence of disturbance on total spore density of AM fungi.Spore density was highest in UF, lower in SBF, and lowest in MF.The high spore density of AM fungi in undisturbed forests with naturally higher plant species diversity suggested that disturbance affect the abundance of arbuscular mycorrhizal spores.A similar result was obtained by Su and Guo 18 , who reported that the mean spore density of AMF was significantly decreased in overgrazed as compared to non-grazed plots in the Inner Mongolia steppe.Reduction in spore density due to agricultural practices has also been described by Li et al., 19 , who found that the highest spore density occurred in never-cultivated field, slightly lower in old field and lowest in cultivated field in hot and arid ecosystem of Southwest China.Reduced spore density in slash-and-burn fields and monoculture forests can be explained by the fact that disturbance disrupts the soil fungi by removing above ground biomass on which these obligate symbionts depend for their carbon source and by breaking the hyphal network leading to a reduction in mycorrhizal colonization 20 .Moreover, clear-cutting of forests combined with fire exposes soils to desiccation, high temperature (>500 o C), and rain erosion 21 and these parameters can be attributed to the lower AM fungal spore counts in slash-andburn fields immediately after their conversion.However, other studies have documented an equal or higher AM fungal spore counts and diversity in pastures, and other deforested stands compared to adjacent natural forests 22,23 .
These studies, however, attributed that the improved population may be a consequence of after land conversion which provided ample time for natural re-colonization of these sites by annual grasses and herbaceous plant species, serving as host to AMF, and thereby, is associated with higher AM fungal spore production.Although, monoculture forests site, in our study, was about 20 years old and artificially regenerated with Tectona grandis Linn., the forest floor was barren and covered with fallen twigs and leaves of the plant itself with very little or no herbaceous plant cover.Forest management factors like site preparation, shrubclearing etc, along with high erosion rate of the sandy loam soil in sloppy tract; have resulted in the forest floor removal.Such and other reasons (like physical properties of the soil which has not been considered here) may account for the minimal AM fungal spore counts in monoculture forests.A total of 12 AM fungal species belonging to four different genera were extracted and identified directly from the field soil samples of three study sites.All the twelve AM fungal species were isolated from undisturbed forests compared to seven from slash-and-burn fields and four from monoculture forest.The Shannon-Wiener Index of diversity was higher in undisturbed forests due to the evenness in relative abundance of the greater number of AM fungal species compared to slash-and-burn forests and monoculture forests.Sorenson's coefficients showed a greater similarity between undisturbed forests and slash-and-burn fields due to prevalence of more number of common AM fungal species in both sites.
The prevalence of Glomus manihotis and Acaulospora sp 1 in all the three study sites along with a high IF, spore density and RA indicates that these species are more tolerant to soil disturbances.Further study is essential to establish their usefulness and role in maintaining the fragile ecosystem of this hill district.
Since in this study the samples were collected during June-July at the middle of the rainy season, and AMF being seasonal in their sporulation pattern 24 , we were not sure whether all AM fungus species could be isolated, and therefore, with longerterm sampling AMF diversity in this area would definitely increase.We conclude that the forests of this hill district contained a high AM fungal diversity.The AMF diversity is significantly affected by the land use practices practiced by the people and no step has been initiated to restore this important group of microorganisms by the forest management practices.

Fig. 1 :
Fig. 1: Spore density of four AM fungus genera isolated from different study sites (UF, SBF and MF)

Table 1 : Diversity indices of AM fungi in different study sites (undisturbed forests
, UF; slash-and-burn fields, SBF and monoculture forests, MF)