Journal of Forestry Research (2012) 23(2): 339−344 DOI 10.1007/s11676-012-0262-4
ORIGINAL PAPER
Augmented growth of long pepper in response to arbuscular mycorrhizal inoculation R. K. Singh • P. Gogoi
Received: 2010-11-04;
Accepted: 2011-08-12
© Northeast Forestry University and Springer-Verlag Berlin Heidelberg 2012
Abstract: Arbuscular mycorrhizal (AM) technology is a soil-based fertilization practice for sustainable crop productivity. We evaluated six indigenous Arbuscular mycorrhizal fungi (AMF) strains for their symbiotic response with Piper longum (long pepper), a non-timber forest product holding promise as a commercial crop for its medicinal fruits and roots. Piper saplings were raised in a 10 cm thick sand and soil mix inoculated with various AMF. Under field conditions, plants inoculated with AMF demonstrated better survival (≥80%) than non mycorrhizal plants (58%). Almost all the studied AMF strains increased the plant growth, biomass and nutrient content (N and P) over the uninoculated control. Mycorrhizal inoculation with four AMF species, viz: Glomus fasciculatum, G. clarum, G. etunicatum and G. versiforme greatly enhanced long pepper growth both in the nursery and field conditions. Keywords: Piper longum; Arbuscular mycorrhizal fungi; Survival; Growth; non-timber forest product
Introduction In recent years, sustainable crop production has become a focus in agriculture. One approach to sustainability is cultivation of non-timber forest products such as medicinal plants, aromatic plants, spices, and others. Cultivation of non-timber forest products is advantageous as they are eco-friendly and economically rewarding. Long pepper (Piper longum) holds great promise as a Foundation project: This work was supported by the Department of Biotechnology, New Delhi. The online version is available at http://www.springerlink.com R. K. Singh • P. Gogoi (
)
Department of Botany, Rajiv Gandhi University, Itanagar – 791112, India. E-mail:
[email protected] P. Gogoi Krishi Vigyan Kendra-Sylvan, Hengbung village, Kangpokpi − 795129, India Responsible editor: Yu Lei
commercial crop for its medicinal fruits and roots. It is described in the Ayurvedic and Unani systems of medicine as a valuable drug for the treatments of various kinds of ailments (Sivarajan and Balachandran 1996; Viswanathan 1995). Due to increasing demand at national and global level with the trend of 16.3% increase per annum have added value to this plant species, concurrently entangled in prioritized list by National Medicinal Plant Board of India. It is both cultivated and grows in the wild in Arunachal Pradesh. Its fruits are collected from the forest and sold to local and national pharmaceutical agencies. The rising demand for this species has exhausted its wild stock and necessitated large-scale cultivation. Arunachal Pradesh offers potential for large scale cultivation of P. longum due to its congenial climate and vast land resource. Though the species has been in exploitation by local people for its fruits, no attempt has been made to cultivate it commercially. Hardly, any published literature is available for cultivation of this crop. Therefore, a scientific approach towards its cultivation with the help of Arbuscular mycorrhizal (AM) technology could increase productivity and provide an additional source of income to local people. AM technology is a soil-based fertilization practices for sustainable crop productivity. Several plant species of importance in horticulture and forestry are aided by Arbuscular mycorrhizal fungi (AMF) inoculation in nursery production (Jeffries 1987; Bagyaraj 1992). These soil inhabiting microsymbionts occur widely under various environmental conditions, improve soil structure (Stutz et al. 2000) and have great capacity to increase growth and yield of host plants through efficient nutrient uptake in infertile soils. They also improve water uptake and drought resistance of host plants (Azcón et al. 1991). AM fungi are essential for the survival of many tropical plants (Chang 1994; Strack et al. 2003). Inoculation of trees with AM fungi greatly improves their overall growth in the nursery (Mridha and Xu 2001) and aids early establishment and growth after transplanting. This is due to reduced transplantation shock and better nutrient and water uptake through the mycorrhizal hyphal link available in the inoculated seedlings. The objectives of this study were to evaluate the effectiveness of six indigenous AMF species on growth and development of P.
340 longum in green house conditions, and to evaluate their effectiveness in reducing the transplantation shock and increasing survival and growth in field conditions.
Materials and methods Study site and sampling The study was conducted in the green house and botanic garden of Rajiv Gandhi University, Arunachal Pradesh, India. The soil was classified as sandy loam. The pH value of the soil was 5.50±0.71, and it contained 1.54±0.14 (%) soil organic carbon and 3.98 ± 0.65 (µg⋅g-1) available phosphorous. AMF species used in the study were obtained from soil samples of field sites of Arunachal Pradesh. The trap cultures were prepared by using soil samples of each site and Zephyranthes ajex as host plant following the procedure of Morton et al. (1993). The AMF spores were isolated by wet sieving and decanting (Gerdemann and Nicolson 1963) from the trap culture and identified on the basis of standard keys, identification manuals by Schenck and Perez (1990) and current species descriptions at International Culture Collection of AM Fungi[http://invam.caf.wvu.edu/Myc-Info/Taxonomy/species.ht ml] and [http://www.amf-phylogeny.com/]. The pure culture of identified species was prepared in sterilized earthen pots (15 cm in diameter) filled to the top with sterile sand: soil mix (1:1) using Zephyranthes ajex as host plant. Modified Hogland solution (100 mL) with low P (Talukdar and Germida 1993) was added to the potting mixture once in a fortnight and watering to the pots was done to the field capacity every week. After four months, plants were harvested, and soil was gently mixed with chopped root fragments and air dried for five days inside a room and used as inoculum. The inoculum potential (IP) of each culture was estimated adopting the most probable number (MPN) method (Porter 1979). For each AMF species, inoculation beds were prepared separately in the green house. The beds were filled with a mixture of sand and soil (1:1) and the rooting medium was inoculated into the bed with culture of individual AMF species by uniformly distributing soil-based AM inoculum @ 40 g⋅kg−1 soil. Beds were 10 cm deep with sand and soil (1:1), where inocula were placed by preparing a single layer on top of the bed, so that growing root of Long pepper cuttings can pass through this inoculum layer, then the remaining soil was topped over the inoculum layer, leveled and pressed down. One inoculation bed without any inoculum was also prepared. Large numbers of pepper cuttings of uniform size (approximately 5−6 cm height and 3 mm diameter on average) were planted temporarily in each bed and kept for 30 days before transplanting. Nursery experiment The experimental design was completely randomized with ten replicates of each treatment. Treatments consisted of saplings without AMF (control) and pre-infected saplings with any one of
Journal of Forestry Research (2012) 23(2): 339−344 the six AMF species. Saplings along with rhizosphere soil were removed (without disturbing the root zone) from respective nursery beds and planted by making a core in the centre of pots containing 200 g autoclaved sand and soil mixture. The experimental pots were maintained in the green house at a temperature of 22±1°C, with 12 h fluorescent illumination with 8,000 lx light intensity, and water was supplied daily to maintain the soil moisture level close to field capacity. A basal dose of nutrients in the form of Urea (N=46.67%), Single super phosphate (P=16%) and Murate of potash was incorporated @ 66, 110, and 30 mg⋅kg-1 soil, respectively. Harvesting was carried out 90 days after planting (DAP) and plant height was recorded. Shoot and root dry weights were determined after drying the plant samples at 60oC to a constant weight in a hot air oven. The phosphorus content of the shoot and root was determined by Hanson’s method (1950). For determination of total nitrogen, the microkjeldhal method of Bremner & Mulvaney (1982) was followed. Chlorophyll content of leaf was measured by using Arnon’s method (1949). Field experiment The experiment was also done in a completely randomized block design with seven treatments and ten replicates each. The treatments were AMF inoculated P. longum saplings, and another one was without inoculation of AMF (control). The experimental site was a slope area in the botanic garden. The size of the experimental plot was 4.25 m × 6.5 m. The soil of the plot was ploughed well and the farmyard manure @ 10 ton⋅ha-1 and single super phosphate @ 125 kg⋅ha-1 was incorporated. The compost amended soil mixtures were wetted with an appropriate amount of water and used to form raised beds (0.2 m height). Ten saplings were transferred randomly in separate rows to the field from each of the respective AMF inoculation beds. The growth performance and survival rate of the saplings were recorded 6 months after planting in the field. Statistical analyses The data were subjected to statistical analyses using Analysis of Variance (ANOVA) and the means were separated by Duncan’s multiple range test (DMRT). Statistical significance was set at α = 0.05). Data were analyzed using computer statistics programme SPSS (2000) Version 10.1.0.
Results Isolation A total of 23 AM fungal species were isolated after trap and pure culturing and identified on the basis of morphological characters. Based on the number of infective propagules present in the pure culture of each AM species as estimated by the most probable number (MPN) method, six AMF species were selected for the efficiency test both in field and green house conditions. Glomus
341
Journal of Forestry Research (2012) 23(2): 339−344
Maximum shoot length (31.0 cm) and maximum percent increment of shoot fresh weight and shoot biomass production (965.38% and 671.43% respectively) over controls was observed in plants inoculated with G. fasciculatum. Only three species (Glomus sp 2, G. fasciculatum and G. clarum) showed a significant increase (p < 0.05) over uninoculated controls in respect to root fresh weight and root dry weight as well as total biomass. A significant increase (p < 0.05) in chlorophyll content over the control was observed in plants inoculated with G. fasciculatum, G. clarum, G. versiforme and G. etunicatum only (Table 1).
fasciculatum produced the highest number (5,400) of infective propagules followed by G. mosseae (390), Glomus sp. 2 (240), G. clarum (240), G. etunicatum (200), and G. versiforme (140). Nursery experiment The effect of inoculation of different AM fungi on growth performance of P. longum plants was evaluated in pot conditions. AM inoculation had a positive effect on growth of the plant in terms of shoot length and fresh and dry weights of shoot and root (Table 1).
Table 1. Effect of AM fungal inoculation on plant growth parameters of P. longum saplings. AMF species
Shoot length
Shoot fresh weight
Shoot dry weight
Root fresh weight
Root dry weight
Chlorophyll content
(cm)
(g)
(g)
(g)
(g)
(mg⋅g-1)
Control Glomus mosseae
07.3
a
20.7
c
0.26 2.77
16.0
G. versiforme Glomus sp. 2
22.8
c
21.0
c
2.09
G. etunicatum
0.54
cd
1.03
0.35
b
1.12
b
1.72
0.20
0.53a
b
0.98b
b
1.16b
a
0.89b
b
0.55a
a
1.40b
0.18
c
0.29
b
0.36
0.39a
a
0.24
0.77
b
0.17
a
0.30
c
b
0.38
1.78
ab
1.46
b
b
a
d
0.47
1.90
16.5
0.66
c
bc
bc
b
0.62
0.37
d
b
a
b
1.82
31.0
G. clarum
0.07
b
d
G. fasciculatum
a
0.76
0.17
AMF = Arbuscular mycorrhizal fungi, Values with the same letter within same row for each parameter are not significantly different at p ≤ 0.05 levels by Duncan’s Multiple Range Test with respect to species main effect.
G
Fig. 1
Fig. 2
AMF species
Shoot P and N concentration in P. longum plant inoculated
with different AMF species in green house condition. Columns for each parameter with same alphabet are not significantly different at p
