AM Fungal Diversity in Selected Medicinal Plants of ... - Springer Link

Indian J Microbiol (July–Sept 2011) 51(3):259–265 DOI 10.1007/s12088-011-0112-7

ORIGINAL ARTICLE

AM Fungal Diversity in Selected Medicinal Plants of Kanyakumari District, Tamil Nadu, India S. K. Sundar • A. Palavesam • B. Parthipan

Received: 2 September 2009 / Accepted: 27 August 2010 / Published online: 1 February 2011 Ó Association of Microbiologists of India 2011

Abstract The association of Arbuscular Mycorrhizal Fungi (AMF) with three medicinally important plants viz., Eclipta prostrata, Indigofera aspalathoides, I. tinctoria collected from three different localities of Kanyakumari District, South India was examined. The study reports the colonization percentage, diversity and species richness of different AM fungi in the rhizosphere of the three medicinal plants and discusses the impact of soil physicochemical characteristics such as soil texture, pH and available macro- and micro nutrient content on AM fungal communities. A total 21 AM fungal species were identified in field conditions of the three plants from three sites. AM fungal species richness, colorization percentage and Shannon index were found to be high in the two Indigofera sp. growing in the hilly areas of Kanyakumari District and were low in E. prostrata collected from the damp regions in the foothills of the three study sites. Five species registered 100% frequency in all the study sites of the three

S. K. Sundar Department of Microbiology, Noorul Islam College of Arts and Science, Kumaracoil, Kanyakumari District 629180, India A. Palavesam Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam, Kanyakumari District 629 502, India B. Parthipan (&) P. G. Department and Research Centre of Botany, S. T. Hindu College, Nagercoil, Kanyakumari District 629002, India e-mail: [email protected]

medicinally important plants with Glomus as the dominant genera. The study states that the mean colonization and diversity patterns were dependant on edaphic factors and type of vegetation. Keywords Arbuscular mycorrhizal fungi Eclipta prostrata Indigofera aspalathoides I. tinctoria Glomus

Introduction Mycorrhizae are non-pathogenic symbiotic soil fungi which invade the root system of plants. This association is not only restricted to the roots of plants, but it is also found in all those organs of plants which are concerned with the absorption of substances from the soil [1]. Among the different types of mycorrhizae, Arbuscular Mycorrhizal Fungi (AMF) have gained much importance in the field of agriculture. The main advantage of mycorrhiza is its greater soil exploration and increasing uptake of P, N, K, Zn, Cu, S, Fe, Mg, Ca and Mn and the supply of these nutrients to the host roots [2, 3]. The occurrence of AM fungi has been reported in many plant communities such as forests [4, 5], grasslands, stepper and praires [6], deserts [7] and mangroves [8]. AM fungi are present in practically all soils and associated with a great variety of plants of different taxonomic groups. Originally medicinal plants in India were reported to be non-mycorrhizal due to the presence of various secondary metabolites. However, AM fungal colonization of medicinal plant roots are now reported [9]. The association of AM fungi with the roots of two of the study plants I. aspalathoies and I. tinctoria has been reported by Sundar et al. [10].

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Kanyakumari District situated in the southern tip of Peninsular India is under strategic geographic location and has rich diversity of medicinal plants scattered over the hills and hillocks of the district. Published data on the AM fungal association in the medicinal plants of Kanyakumari District is not available till date. Hence, the present study was undertaken to survey the AM fungal association in three selected medicinal plants of Kanyakumari District namely E. prostrata L. (Family: Asteraceae), Indigofera aspalathoides, Vahl. ex. Dc and Indigofera tinctoria L. (Family: Leguminosae; Sub family: Papilionaceae (Fabaceae). Fig. 1 Location of the study sites

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Indian J Microbiol (July–Sept 2011) 51(3):259–265

Materials and Methods Description of the Study Sites The three sites selected for the present study were in and around Marunduvalmalai, Munchirai hillocks and Veli hills of Kanyakumari District, Tamil Nadu (Fig. 1). E. prostrata were collected from the bunds of tapioca and plantain fields in the foot hills of Marunduvalmalai (S1), Munchirai plains (S2) and foot hills of Veli hills (S3). The two Indigofera species were collected from Marunduvalmalai hills (S4), Munchirai hillocks (S5) and Veli hills (S6).

0.56 ± 0.03 Each value is an average of six replicates. Values are indicated as mean ± SE

S1 Marunduvalmalai foot hills, S2 plains of Munchirai, S3 Veli foot hills, S4 Marunduvalmalai hills, S5 Munchirai hill rocks, S6 Veli hills

0.61 ± 0.03 0.6 ± 0.016 0.57 ± 0.02 0.59 ± 0.02 0.61 ± 0.02 0.59 ± 0.02 0.61 ± 0.02 Zinc (ppm)

0.71 ± 0.04

5.29 ± 0.03 3.18 ± 0.02 5.43 ± 0.06 3.12 ± 0.06 5.47 ± 0.03 3.14 ± 0.04 5.37 ± 0.06 3.16 ± 0.04 5.37 ± 0.06 3.2 ± 0.01 5.44 ± 0.07 3.1 ± 0.02 5.34 ± 0.02 3.14 ± 0.07 5.51 ± 0.03 3.32 ± 0.06 Iron (ppm) Manganese (ppm)

5.62 ± 0.04 3.41 ± 0.06

1.12 ± 0.01 1.05 ± 0.03 1.2 ± 0.01 1.12 ± 0.03 1.07 ± 0.06 1.19 ± 0.01 1.18 ± 0.01 1.34 ± 0.02 Copper (ppm)

1.45 ± 0.03

152.62 ± 1.34

4.58 ± 0.11 4.81 ± 0.09

161.74 ± 0.92 166.86 ± 0.9

4.14 ± 0.07 4.56 ± 0.14

154.4 ± 1.86 165.05 ± 1.56

4.82 ± 0.07 4.15 ± 0.09 5.04 ± 0.1

152.17 ± 0.95 162.1 ± 1.57 159.56 ± 0.63

5.51 ± 0.1 5.22 ± 0.1

154.41 ± 1.45

Available phosphorus (kg/acre)

Available potassium (kg/acre)

108.58 ± 3.28

0.84 ± 0.02 1.16 ± 0.02 0.9 ± 0.04

109.15 ± 4.17 101.54 ± 1.63 113.6 ± 2.2

0.85 ± 0.04 1.13 ± 0.08 0.88 ± 0.08

101.56 ± 1.32 113.64 ± 2.21 109.41 ± 1.9

0.72 ± 0.02 0.85 ± 0.02 0.75 ± 0.02

111.14 ± 1.8

Organic carbon (%)

Available nitrogen (kg/acre)

119.12 ± 1.4

0.6 ± 0.01 0.51 ± 0.03 0.42 ± 0.06 0.58 ± 0.04 0.56 ± 0.04 0.81 ± 0.04 0.66 ± 0.02 Electrical conductivity (ds m-1) 0.73 ± 0.05

6.3 ± 0.07

Red sandy loam Sandy loam Sandy loam

6.58 ± 0.07 6.7 ± 0.11 6.3 ± 0.09

Red sandy loam Sandy loam Sandy loam

6.4 ± 0.08 6.5 ± 0.1 6.40 ± 0.07

6.2 ± 0.07

Sandy clay loam Sandy clay loam Loam

S3

Soil pH

S5 S4 S5

S6

I. tinctoria

S2

Soil type

Fixed roots were cleared in 2.5% KOH, acidified with 5 N HCl and stained with Tryphan blue (0.05% in lactophenol). Roots that remained dark after clearing were bleached with 3% H2O2 before staining. They were left overnight in Tryphan blue-lactophenol for staining and were examined with a compound microscope (9200–9400) for AM fungal structures. The percentage root length colonization was estimated according to the gridline intersection method [14]. AM fungal spores were isolated by a modification of the wet-sieving and decanting technique [15].

S4

Estimates of AM Fungal Colonization and Isolation of AM Fungal Spores

S1

Soil pH was determined in 1:1 soil:water mixture soon after the soil samples were brought to the laboratory. The available Nitrogen (N), available phosphorus (P) and available potassium (K) were determined according to Clarson [13].

I. aspalathoides

Determination of Soil Characters

E. prostrata

Root and rhizosphere soil samples from each plant species were collected in five replicates at different stages of growth (vegetative and reproductive). Soil samples collected from different individuals of a plant species were mixed to form a composite sample which was used for the isolation of AM fungal spores and for analysis of soil chemistry.

Physicochemical parameters

Sampling

Table 1 Physical and chemical properties of rhizosphere soil of three medically important plants collected from three different localities of Kanyakumari district

The test plants were collected using field and herbarium technique [11] and were identified using the synoptic keys mentioned in flora of Tamil Nadu Carnatic [12]. Voucher specimens of the plants were deposited in the herbarium of P.G and Research Department of Botany, S. T. Hindu College, Nagercoil. E. prostrata L. (Syn, E. alba), belongs to the family Asteraceae and is used as a hepatoprotective agent to treat viral jaundice. I. aspalathoides Vahl. ex. Dc. and I. tinctoria L. belongs to the family Leguminosae; sub family Papilionaceae (Fabaceae). The leaves, flowers and tender shoots of I. aspalathoides are used in the treatment of cancer, tuberculosis and leprosy while the whole plant extract of I. tinctoria is used in the treatment of epilepsy and other nervous disorders and the plant is also used as a natural dye.

S6

Plant Collection and Description

0.46 ± 0.02

261

6.64 ± 0.09


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262


Table 2 Percentage root colonization and AM fungal spore number in the rhizosphere soil of selected medicinal plants from three study sites of Kanyakumari District Species

Study area

Colonization

Spore population (100 g-1 soil)

E. prostrata

Marunduvalmalai foot hills (S1)

67.34

158

I. aspalathoides

I. tinctoria

Plains of Munchirai (S2)

64.07

135

Veli foot hills (S3)

69.95

162


89.81

538


86.36

482

Veli foot hills(S6)

92.72

566

Marunduvalmalai hills (S4) Munchirai hillocks (S5)

75.16 72.55

331 288

Veli hills (S6)

80.94

385

Each value is an average of six replicates. Values are indicated as Mean ± SE

Identification of AM Fungal Spores

Table 3 Frequency distribution of AM fungi in the rhizosphere soil from the three different localities of Kanyakumari district

Intact and crushed spores in polyvinyl alcohol, lactophenol and in Melzer’s reagent were examined and identified according to Schenck and Perez [16] and Mukerji [17]. From the data obtained from soil physico-chemical analysis and spore counts from the rhizosphere soil of the three plants, species richness and Shannon index were computed as per the methods proposed by Magurran [18].

No.

RichnessðRÞ ¼ number of species found in a sample Shannon indexðHÞ ¼ Rpi ln pi Results and Discussion The present study was undertaken to assess the AM fungal symbiosis in three important medicinal plants namely E. prostrata, I. aspalathoides and I. tinctoria. Texture of the rhizosphere soil of E. prostrata in Marunduvalmalai foothills and plains of Munchirai was sandy clay loam, and loamy texture was observed in the rhizosphere soil of E. prostrata plants in Veli foothills (Table 1). The rhizosphere soil of I. aspalathoides and I. tinctoria collected from Marunduvalmalai and Veli hills was sandy loam in texture and in Munchirai hillocks it was red sandy loam. The pH of the rhizosphere soil was slightly acidic to neutral in all the study areas. The macro- and micro-nutrient contents in the rhizosphere soil were higher in Munchirai hillocks followed by the other two sites (Table 1). The three medicinal plants were confirmed to the mycorrhizal, since the roots showed the presence of AM fungal structures in their cortical cells, hyphae ? arbuscules, hyphae ? pelotons or hyphae ? vesicles as described by Lindsay [19]. The percentage AM fungal infection in the three medicinal plants in all three localities were more than 60% (Table 2), but the degrees of colonization in the three plants varied with the soil characteristics and

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AM fungi

E. prostrata

I. aspalathoides

I. tinctoria

S1

S2

S3

S4

S5

S6

S4

S5

S6

1

ADLC

?

?

?

?

?

?

?

?

?

2

AMRW

?

-

-

?

-

?

-

-

-

3

ASCB

-

-

-

-

?

?

-

?

-

4

ABRT

-

-

-

-

-

-

-

?

?

5

GMRG

-

?

?

-

?

?

?

-

?

6

GDCP

-

-

-

-

?

-

-

-

-

7

LAGR

?

?

?

?

?

?

?

?

?

8

LFSC

?

?

?

?

?

?

?

?

?

9

LFLV

-

?

?

?

?

?

?

?

?

10

LGSP

?

?

?

?

?

?

?

?

?

11

LHOI

-

-

-

?

-

?

-

-

-

12

LINR

-

-

-

?

?

?

-

-

?

13

LMCC

?

-

?

-

-

?

?

?

-

14

LMRC

-

-

-

?

?

-

-

-

?

15

LMSS

?

?

?

?

?

?

?

?

?

16 17

LPKS LRBF

-

-

-

?

? -

? -

?

-

?

18

LVSF

?

-

?

-

-

-

-

-

-

19

CCLS

-

-

?

?

-

?

20

CHTG

-

-

?

?

-

?

?

?

-

21

CPLC

?

-

-

-

-

-

-

-

-

ADLC Acaulospora delicata, AMRW A. morrowae, ASCB A. scrobiculata, ABRT A. bireticulata, GMRG Gigaspora margarita, GDCP Gi. decipiens, LAGR Glomus aggregatum, LFSC G. fasciculatum, LFLV G. fulvum, LGSP G. geosporum, LHOI G. hoi, LINR G. Intraradices, LMCC G. macrocarpum, LMRC G. microcarpum, LMSS G. mosseae, LPKS G. pakistanica, LRBF G. rubiforme, LVSF G. versiforme, CCLS Scutellospora calospora, CHTG S. heterogama, CPLC S. pellucida

the nutrient content. The AM spore number of E. prostrata was higher in Veli foot hills compared to that of Marunduvalmalai and Munchirai plains which might be due to the


263

Fig. 2 Photomicrograph of AM fungal spores isolated from the study sites

presence of loamy soil in Veli foot hills and clay loam soil in the two latter sites. In I. aspalathoides and I. tinctoria also the AM colonization and spore count also showed similar results. Rachel et al. [20] reported more AM fungal infection in sandy loam soil followed by other soil types. The two Indigofera sp. showed higher ranges of infection percentage and spore population than E. prostrata. The reason might be that in the present study, the AM infection was studied in E. prostrata plants collected from bunds of Musa paradisica and Manihot utilisima, all of them normally preferring a damp or moist habitat which is not a preferable habitat for AM fungi. The clay content of the soil might also be a reason for the lesser AM fungal association in E. prostrata. Our results were in confirmation with the earlier work by Ragupathy and Mahadevan

[21] who worked on AM fungal association in the soils of Thanjavur district located in the Cauvery delta and reported only moderate AM infection in the plants of the region. Rickerl et al. [22] also reported fewer AM fungal spores in wet and waterlogged soil, which is generally related to reduction or lack of mycorrhizal inoculum. Species diversity of AM fungi was also apparent in the rhizosphere soil of the test plants from the three different study sites. A total of 21 AM fungal species belonging to four known genera were identified in the present study. The present study registered a maximum of 18 AM species in I. aspalathoides followed by 15 species in I. tinctoria and 12 species in E. prostrata. In all the three plants, Glomus was found to be the dominant genus. Vyas [23] reported the dominance of Glomus in the forest and agricultural soils of

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264 Table 4 Species richness, diversity index and species diversity of AM fungi in the rhizosphere soil from the three study sites of Kanyakumari District


Species E. prostrata

I. aspalathoides

I. tinctoria Each value is an average of six replicates. Values are indicated as Mean ± SE

Study area

Shannon


8.0 ± 0.33


7.0 ± 0.21

1.928 ± 0.32


9.0 ± 0.33

2.259 ± 0.26

2.142 ± 0.24


12.0 ± 0.33

2.282 ± 0.34


12.0 ± 0.33

2.288 ± 0.38


15.0 ± 0.47

2.465 ± 0.26

Marunduvalmalai hills (S4)

11 ± 0.21

2.381 ± 0.24

Munchirai hillocks (S5)

10.0 ± 0.33

2.275 ± 0.32

Veli hills (S6)

12.0 ± 0.33

2.395 ± 0.41

Sagar region. Nasim and Bajwa [24] recorded twenty eight species of AM fungi in crop land ecosystem, indicating that about 70% of the AM species composition in made by genus Glomus. In the present study, A. delicata, G. aggregatum, G. fasciculatum, G. geosporum and G. mosseae were identified in the rhizosphere soils of E. prostrata in all the three study sites showing 100 percentage site frequency (Table 3). In I. aspalathoides, A. delicata, G. aggregatum, G. fasciculatum, G. fulvum, G. geosporum, G. intraradices and G. mosseae were detected in the rhizosphere of the three study areas and A. delicata, G. aggregatum, G. fasciculatum, G. fulvum, G. geosporum and G. mosseae showed cent percent frequency in the rhizosphere soil of I. tinctoria in the three study areas. Thus of the 21 species isolated from the rhizosphere soils of the three selected medicinal plants, only five AM fungal species were invariably recorded in all the three plants of the sampled sites. They were A. delicata, G. aggregatum, G. fasciculatum, G. geosporum and G. mosseae (Fig. 2). Marked difference observed in the composition of AM fungi in the rhizosphere soils of the medicinal plants of the three study sites of Kanyakumari district can be attributed to the influence of edaphic factors. I. aspalathoides and I. tinctoria recorded comparatively higher spore number and infection percentage when compared to E. prostrata which may be due to the variation in edaphic factors such as soil texture, soil moisture and the agronomic practices and types of vegetation. In the present study, E. prostrata showed maximum Shannon index in Veli hills followed by Marunduvalmalai foot hills and Munchirai. In I. aspalathoides and I. tinctoria also, the index was high in Veli hills with a species diversity of 2.465 and 2.395, respectively (Table 4). In the present study, the species richness was maximum in Veli region for the three plants. Beena et al. [25] reported a mean species richness of 4.4 on southwest coastal sand dunes of Karnataka and she recorded higher species richness (11) in Ipomoea pes-caprae.

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Species richness

The present data clearly reveals that the sites under investigation are rich in AM fungi. The study also indicates variation in spore number and diversity in the rhizosphere of the three plants surveyed which may be because of soil texture, climate and other soil factors. These finding were in accordance with the study conducted by other authors Muthukumar and Udaiyan [26]. E. prostrata which comes up in damp regions harbours less number of AM fungal spores, infection percentage and diversity when compared to the two Indigofera sp. The study also reveals that, out of the 21 species recorded, five species registered 100% frequency in the three sites with Glomus as the dominant genus. The hilly tracts recorded more spore number and diversity when compared to the foot hills of the three sites. Further trap culture study has to be conducted to reveal any dormant non sporulating species present in the soil [26]. This would also be an initiative to conduct pot trial in the above said plants with these bioinoculants to test their efficacy in improving the plant growth and bioactive principles. Hence the present recording clearly indicates the AM fungal diversity in the rhizosphere of the three medicinally important plants in the study sites situated in the termination of Western Ghats. The motive of this study is also to recommend the mass multiplication of superior strains of the AM fungi and their application along with other rhizosphere microflora to these medicinal plants.

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