SUPPLEMENTARY MATERIAL
Improvement in the yield and quality of kalmegh (Andrographis paniculata Nees) under the
sustainable production system
Rajesh Kumar Vermaa*, Sanjeet Kumar Vermaa, Umesh Pankaja, Anand Guptaa , Khushboo
Khana and Karuna Shankara
a
Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, India
*
Corresponding author. Email: rajeshcimap@rediffmail.com
Abstract
Andrographis paniculata Nees. is an annual erect herb with wide medicinal and pharmacological
applications due to the presence of andrographolide and other active chemical constituents. The
large scale cultivation of the kalmegh is not in practice. The aim of the present study was to
establish sustainable production systems of A. paniculata cv CIM- Megha with the application of
different bioinoculants and chemical fertilizers. A. paniculataherb and andrographolide yield in
the dried leaves was found to be highest (218% and 61.3%, respectively) in treatment T3 (NPK +
Bacillus sp) compared to T1 (control). The soil organic carbon, soil microbial respiration, soil
enzymes activity and available nutrients improved significantly with combined application of
bioinoculants and chemical fertilizers.
Keywords: Kalmegh; andrographolide; bioinoculants; chemical fertilizers; soil organic carbon;
available nutrients
Experimental
Site, climate and soil
The experiment was carried out in 2011-12 cropping year in a fixed barrel (barrel radius 50.0cm)
at the CIMAP research farm, Lucknow, India (26.5˚N, 80.5˚E and at 120m above mean sea
level) during
rainy season with seven treatments combinations and four replication. The
maximum monsoon rains are received in July and August (average annual rain fall 800mm) and
maximum and minimum temperature varied from 25.0 to 43.0˚C and 5.4 to 28.4˚C. The
experimental site is classified as semiarid subtropical zone with hot summer and fairly cool
winters. The soil characteristics of the experiment were soil texture loamy sand, soil pH 8.4,
organic carbon 0.47%, available P18 kg ha-1 and available K 200 kg ha-1.
Multiplication/propagation of bioinoculants/ arbuscular mycorrhizal (AM) fungi
The bacterial cultures (N fixer: Bacillus spp; P solubilizer: Bacillus megaterium and plant growth
promoters: Pseudomonas monteilii) were multiplied in nutrient broth and Jensen’s broth for 24 h
at 210 rpm at 28˚C on an incubator shaker (Singh et al. 2013). The numbers of cells of
bioinoculants were 3.2 x 109, 3.0 x 108 and 3.8 x 108 cfu g-1 carrier, respectively. The compostbased bioinoculants were applied (@ 4 kg ha-1 to kalmegh seedlings by root-dip technique. The
inoculum of AM fungi (Glomus intraradices) was propagated on maize roots (Zea mays L.) for
10 weeks in a 1:1 volume mixture of sterilized sand and soil (5 kg) of low phosphorus content
(7.5 kg ha-1) and subsequently left to dry for 2 weeks. The inoculum potential of AM fungi used
in this study was 4.3 ± 1.3 infecting propagules g-1 of sand–soil mixture following Liu and Luo
(1994).
Treatment and crop culture
The experiment consisted of 7 treatment combinations: T1, an absolute control (soil only) in
which there was no addition of chemical fertilizers; T2, chemical fertilizers only (NPK,
60:40:40); T3, chemical fertilizers along with Bacillus sp. (AZHGF1, NCBI accession no.
JN700924); T4, chemical fertilizers with Bacillus megaterium (BM, ATCC 14581); T5, chemical
fertilizers along with P. monteilii (CRC1, NCBI accession no. HQ 995498); T6, chemical
fertilizers along with arbuscular mycorrhizal fungus (GI, G. intraradices); T7, chemical fertilzers
along with all four bio-inoculants (Bacillus sp., B. megaterium, P. monteilii, G. intraradices)
were applied. For raising crop, 35 days old seedlings (Andrographis paniculata cv.
“CIM_Megha”) were transplanted (two plants in each barrel, month of July). The chemical
fertilizers (NPK) were applied through urea, single super phosphate and muriate of potash at the
rates of 60 kg N, 40 kg P and 40 kg K ha
−1
, respectively. At the time of transplanting, 1\2
nitrogen, full dose of P and K was applied and remaining dose of N was applied after 60 days of
transplanting. Manual weeding and irrigation was done at when required.
Harvesting and drying of herb
Above ground foliage of the kalmegh plant was harvested (after the 120 days of planting) at the
flower initiation, the active compound andrographolide content higher at this stage (Niranjal et
al. 2010). Since the whole plant contain active compound, entire harvested material was shade
dried.
Extraction and estimation of andrographolide
Dried leaves were pulverized to dust. 100 mg of pulverized leaves was extracted three times with
10 ml methanol on water bath for 30 min. All three filtrates were combined with total volume of
30 ml and evaporated to dryness under vacuum. The final volume was made up to 1 ml and pass
through 0.45 µm nylon filter. A 20- µl of this extract was injected into reverse-phase HPLC
system. All analysis were conducted with Waters HPLC system consisting Waters 717 plus auto
sampler, Waters 600 pump; in live solvent degasser, 2996 photodiode array detector. The
separation was carried out using a Waters (X Bridge) C18 column (250 x 4.6mm, 5 µm), having
4.6 x 20 mm guard cartridge. The mobile phase consisted of water and acetonitrile (70:30, v/v)
the flow rate was 0.8 ml/min. The andrographolide content was determined using external
standard (Sigma Chemical, purity 98% ).
Soil analysis
The initial and at post harvest soil of kalmegh crop were collected from 0-15 cm depth in the all
treatments and replication. The collected soil samples were screened, pulverized using a wooden
pestle and mortar, and passed through a 2mm mesh sieve. The chemical and biological properties
of soil were analysed using the methods of (Page et al. 1982).
Statistical analysis
An analysis of variance (ANOVA) was performed by statistical methods (Panse and Sukhatme,
1961). Least significant difference (LSD at P<0.05) and Duncan’s test was used to determine
whether means differ significantly or not.
References
Liu RJ, Luo XS. 1994. A new method to quantify the inoculums potential of Arbuscular
mycorrhizal fungi. New Phytol. 128: 89–92.
Niranjan A, Tiwari SK, Lehri, A. 2010. Biological activites of kalmegh (Andrographis
paniculata Nees) and its active principles-A review.Indian J Nat Prod Res.1:125-135.
Page AL, Miller RH, Keeney DR. 1982. Methods of Soil Analysis. 2nd Edn., American Society
of Agronomy, Madison, WI., USA.
Panse VG, PV Sukhatme. 1961. Statistical methods for agricultural workers. 2nd ed, ICAR, New
Delhi.
Singh R, Soni SK, Patel RP, Kalra A. 2013. Technology for improving essential oil yield of
Ocimum basilicum L. (sweet basil) by application of bioinoculants colonized seeds under organic
field conditions. Indus Crops Prod. 45:335-392.
Table S1. Correlation among yield attributes and soil properties of Andrographis paniculata post harvested soil (Pearson correlation)
Soil/Plant characteristics
L:S ratio Herb yield Andro-
SOC
Soil resp Acid
grap yield
Alkal phos Dehyd Avail P
Avail K
phos
L:S ratio
1.0
Herb yield
0.76**
1.0
Andrograp yield
0. 47*
0.28
1.0
SOC
0.31*
0.63**
0.03
1.0
Soil Resp
0. 53*
0.36
0.55**
0.23
1.0
Acid phos
0.42
0.27
0.25
0.18
0.29
1.0
Alkali phos
0.51*
0.37
0.66**
0.35
0.46*
0.79** 1.0
Dehyd
0.19
0.35
0.37
0.52* 0.25
-.08
0.13
1.0
Avail P
0.69**
0.64**
0.46*
0.08
0.25
0.07
0.23
0.16
1.0
Avail K
0.63**
0.48*
0.40
0.14
0.00
0.26
0.42
-0.21
0.62** 1.0
Note: L: S: leaf stem ratio; Andrograp yield: Andrographolide yield; SOC: soil organic carbon; Soil resp: soil microbial respiration;
Acid phos: soil acid phosphatase enzyme; Alkal phos: soil alkaline phosphatase enzyme; Dehyd: soil dehydrogenase enzyme; Avail
P: available phosphorus; Avail K: available potassium.
**Correlation is significant at 0.01 levels; *Correlation is significant at 0.05 levels.