Communications in Soil Science and Plant Analysis

ISSN: 0010-3624 (Print) 1532-2416 (Online) Journal homepage: http://www.tandfonline.com/loi/lcss20

Bio-inoculants enhance growth, nutrient uptake,

and buddability of citrus plants under protected
nursery conditions

Amandeep Singh, Anirudh Thakur, Sandeep Sharma, P.P.S. Gill & Anu Kalia

To cite this article: Amandeep Singh, Anirudh Thakur, Sandeep Sharma, P.P.S. Gill & Anu
Kalia (2018): Bio-inoculants enhance growth, nutrient uptake, and buddability of citrus plants
under protected nursery conditions, Communications in Soil Science and Plant Analysis, DOI:
10.1080/00103624.2018.1526946

To link to this article: https://doi.org/10.1080/00103624.2018.1526946

Published online: 12 Oct 2018.

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COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS
https://doi.org/10.1080/00103624.2018.1526946

Bio-inoculants enhance growth, nutrient uptake, and buddability

of citrus plants under protected nursery conditions
Amandeep Singha, Anirudh Thakura, Sandeep Sharmab, P.P.S. Gilla, and Anu Kaliab
a
Department of Fruit Science, Punjab Agricultural University, Ludhiana India; bDepartment of Soil Science, Punjab
Agricultural University, Ludhiana India

ABSTRACT ARTICLE HISTORY

Bio-inoculants have been used for enhancing plant growth in horticultural Received 9 May 2018
crops even in nutrient-limited soils. The present research evaluated the effect Accepted 22 August 2018
of inoculation of two growing media with six biofertilizer consortia on the
KEYWORDS
growth and buddability of ‘Rough lemon’ rootstock subsequently budded with
Azospirillum; PSB; AM fungi;
‘Kinnow’ mandarin variety. Soil + FYM + Cocopeat (SFC) with Azospirillum growing media; rough
(Azo) + AM fungi consortium improved seed germination, seedling growth, lemon; Kinnow mandarin
chlorophyll, P, K, root growth, root epidermis, and cortical region thickness;
xylem and phloem diameter in rough lemon; budding success and growth in
Kinnow mandarin saplings. It also increased OC, P, and K levels in growing
media. AM spore count and AM root colonization showed a positive correla-
tion with budding success, root P and soil P content. SFC fortified with
Azo + AM fungi resulted in better growth and buddability of rough lemon
seedlings along with better growth of Kinnow mandarin saplings.

Introduction
Worldwide, citrus fruits are grown over an area of 8.9 million ha with 137.8 million tons production.
China (34.9 m tons), Brazil (19.1 m tons), India (11.15 m tons), USA (8.5 m tons), Mexico (7.8 m tons),
Spain (7.1 m tons) and Egypt (4.4 m tons) are the major producers of Citrus in the world. India is the
third largest citrus producing country in the world with an area of 1.02 m ha and annual production of
11.15 m tonnes (FAOSTAT 2015). In India, citrus is the third largest fruit industry after banana and
mango. Kinnow mandarin a cross between Citrus nobilis Lour × C. deliciosa Tenora developed by H. B.
Frost at University of California, Riverside, USA, was introduced by Dr. J.C. Bakshi in the year 1954 at
the Punjab Agricultural University, Regional Fruit Research Station, Abohar, Punjab. Kinnow has
confined to the semiarid and arid regions of northern India (Panigrahi et al. 2014)
Rough lemon (Citrus jambhiri Lush.) is being used as a rootstock for Kinnow mandarin as it is
vigorous in growth, impart large fruit size and early maturity, deep root system; and tolerant to drought,
citrus tristeza virus (CTV), citrus exocortis viroid (CEV), and citrus xyloporosis viroid. Rough lemon also
has tolerance to alkaline soils and moderate tolerance to salinity. The huge demand for pedigreed and
healthy citrus plants has made citrus nursery production a specialized commercial industry. The
production of healthy and vigorous seedlings of rough lemon is important for producing large qualities
of planting material of Kinnow mandarin by commercial nurseries. The soils of North Indian plains are
high in pH resulting in the poor availability of nutrients especially phosphorus. In order to achieve good
growth of plants under nursery conditions, the nutrient status, organic matter content, and water-
holding capacity are the important properties of the growing media. Citrus has sparse root system
without root hairs as a result, early season nutrient and water supply are very essential.

CONTACT Anirudh Thakur anirudhthakur@pau.edu Department of Fruit Science, Punjab Agricultural University, Ludhiana
141 004, India
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lcss.
© 2018 Taylor & Francis
2 A. SINGH ET AL.

Organic matter (OM) and water holding capacities can be favorably amended with the fortification of
different organic materials. The selection of an organic growing media is very critical for using mycor-
rhizal fungi (Ustuner et al. 2009). In our earlier studies, better seedling growth of rough lemon and
Kinnow mandarin saplings was observed in growing media comprising soil + farmyard manure
(FYM) + cocopeat (2:1:1 v/v) in comparison to soil + FYM (2: 1 v/v) which is widely used in the
commercial nurseries (Bhagat, Thakur, and Dhaliwal 2013). Use of biofertilizers like phosphate solubi-
lizing bacteria (PSB), non-symbiotic N2-fixing bacteria and Arbuscular mycorrhizal (AM) fungi for
enhancing plant growth is used in horticultural crops due to higher cost and hazardous effects of
chemical fertilizers (Wu, Zou, and Wang 2011). AM fungi colonize roots of plants and help in nutrient
uptake (Jansa, Weimken, and Frossard 2006) and maintenance of soil aggregate structure (Rilling 2004).
Mycorrhizal inoculation improved plantlet growth and phosphorus uptake in banana (Ortas et al. 2017).
Inoculation of nursery saplings with beneficial microorganisms can be an efficient way of spreading
biofertilizers in the rhizosphere of fruit orchards. Pre-inoculation of pomegranate nursery seedlings with
selected nitrogen-fixing bacteria and Arbuscular mycorrhiza fungi alone or in combination may help in
the production of vigorous plants capable to survive and thrive in stress soils (Mir et al. 2012). Further,
mycorrhiza-inoculated seedlings have a significant impact on newly established citrus orchards (Ortas
2012). Mycorrhizal inoculation increased plant growth parameters such as shoot height, diameter and
shoot and root dry matter in sour orange (Ortas and Ustuner 2014). Inoculation of sour orange (Citrus
aurantium L.) seedlings with commercial AM fungi inoculum stimulated the growth of sour orange
seedlings regardless of phosphorus level (Al-Karaki 2013). The citrus nursery plant is a composite plant
hence, unlike the previous studies, there is a need to study the effect of biofertilizers on the growth of
rootstock as well as the budded plant. It was hypothesized that supplementation of growing media with
biofertilizers, viz.. AM fungi, phosphate solubilizing bacteria, and Azospirillum may promote growth and
buddability of rough lemon seedlings and subsequent Kinnow mandarin plants. The present studies were
conducted with the objective to investigate the effect of biofertilizers and growing media on growth of
rough lemon seedlings and Kinnow mandarin plants.

Materials and methods

Experimental site and material
The present studies were carried out in the Citrus Nursery, Department of Fruit Science, Punjab
Agricultural University, Ludhiana, India, located at 29.3° N latitude and 76.5° E longitude, 270 m amsl
from 2014 to 2016. The experimental site falls in sub-tropical semi-arid (steppe) region (BSh) as per
the Koppen‒Geiger climate classification (BSh) region (Peel, Finlayson, and McMahon 2007). The two
growing media [Soil + FYM (2:1 v/v) and Soil + FYM + Cocopeat (2:1:1 v/v)] were prepared during
the month of May, 2014 on a concrete floor and spread in 30 cm thick layers and treated with
Basamid® Granular (Dazomet 98%) a soil fumigant @ 50 gm−2. The heaps were covered with a
transparent plastic sheet for 21 days for fumigation and solarization to eliminate soil-borne pathogens.
After two turnings of the media at weekly intervals, the media were fortified with biofertilizer
formulations alone or in combinations after 3 weeks of removal of plastic sheets as mentioned in
Figure 1 and filled in black polythene bags (30 × 15 cm size, 62.5 µ thickness). Rough lemon seeds
collected from a single tree of rough lemon from a rough lemon block (3, 750 m2) propagated with
scion buds from a single rough lemon tree were sown in black polythene bags four weeks after filling
the bags in the month of August. These polythene bags were kept under protected condition under a
green shade net house (50% shade). Seeds germinated in around 3 weeks with more than one seedling
per seed due to nucellar embryony. The uniform-sized seedlings were retained to keep only one
seedling per polybag. The rough lemon seedlings were ‘Inverted T–budded’ at 23 cm above soil level by
taking scion buds from Kinnow mandarin mother plants in foundation block of Kinnow mandarin
maintained in 40 mesh stainless steel screen houses. The soil used for the preparation of growing
media was TypicUstochrept, sandy loam in texture (131 g clay, 148 g silt, and 721 g sand kg−1) with
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 3

Seed germination Number of leaves

100 150

Leaf number
80 110
60
70
Per cent

40
20 30
0 -10
Control
AZO
PSB

Control
AMF

AZO+AMF

AZO
PSB
AMF

AZO+AMF
AZO+PSB+AMF

AZO+PSB+AMF
AZO+PSB

AZO+PSB

AZO+PSB
AZO+PSB
Control
AZO
PSB
AMF

AZO+AMF

Control
AZO+PSB+AMF

AZO
PSB
AMF

AZO+AMF
AZO+PSB+AMF
Soil + FYM Soil + FYM + Cocopeat Soil + FYM Soil + FYM + Cocopeat

Leaf area
Seedling height
80 30
60 20

cm2
mm

40 10
20 0

AZO
PSB
Control
AZO
PSB
AMF

AZO+AMF

Control
AZO+PSB+AMF

AMF

AZO+AMF
AZO+PSB

AZO+PSB+AMF
AZO+PSB
0
AZO+PSB+A…
AZO+PSB+A…

PSB
PSB

AZO+AMF
Control
AZO

AMF

AZO+AMF

Control

AMF
AZO

AZO+PSB
AZO+PSB

Soil + FYM Soil + FYM + Cocopeat Soil + FYM Soil + FYM + Cocopeat

Seedling girth Chlorophyll content

7 60
Spad units

6.5 40
mm

6 20
5.5 0
AZO+PSB+A…
AZO+PSB+A…

AZO+PSB+A…

AZO+PSB+A…
PSB

PSB
AMF

AZO+AMF
Control

AMF

AZO+AMF

Control
AZO

AZO

AZO+PSB
AZO+PSB

Control

Control
AZO
PSB
AMF

AZO+AMF

AZO
PSB
AMF

AZO+AMF
AZO+PSB

AZO+PSB

Soil + FYM Soil + FYM + Cocopeat Soil + FYM Soil + FYM + Cocopeat

Figure 1. Effect of growing media and biofertilizers on seed germination and shoot growth of rough lemon. The vertical bars
represent standard error and are not visible if the values are very small.

alkaline pH (8.1) and free from salinity (0.36 dSm−1). The soil had less quantity of nutrients, which
include medium in organic carbon (0.42%), medium in available phosphorus (25.5 kg ha−1), and high
in potash (311.2 kg ha−1). The experimental soil was collected from an area that was fallow for more
than ten years. The organic amendments viz. farmyard manure (FYM) had 20.5% organic carbon, 0.4%
phosphorus, 0.4% potassium; and cocopeat had 34.4% organic carbon, 0.7% phosphorus (P) and 1.0%
potassium (K). Uniform cultural operations viz. irrigation, hoeing, weeding, and plant protection
measures were followed uniformly for all the plants during the entire period of experimentation.

Treatments
Rough lemon seedlings were grown in two growing media Soil + FYM (2:1 v/v) and Soil + FYM + Cocopeat
(2:1:1 v/v) fortified with biofertilizers alone or in combinations, viz.. Azospirillum (AZO), phosphorus
4 A. SINGH ET AL.

solubilizing bacteria (PSB), Arbuscular Mycorrhizal fungi (AM Fungi), AZO + PSB, AZO + AM fungi, and
AZO + PSB + AM fungi and tested for their effect on growth and buddability of rough lemon seedlings.
Azospirillum and phosphate solubilizing bacteria (104 CFU/g) were applied at the rate of 50 g culture per Kg
of potting media. The AM Fungi consortium [Funneliformis mosseae (Nicol. & Gerd.) Gerd.& Trappe,
Gigaspora margarita Becker & Hall, and Acaulospora laevis Gerd.& Trappe] isolated from the rhizosphere
of Indian gooseberry (Emblica officinalis Gartner) was cultured in maize plants and propagules comprised
of infected root bits and spores. The AM Fungi inoculum was applied 5 cm below the seeds prior to sowing
at the rate of 10 g per pot as a thin layer. The Azospirillum and phosphate solubilizing bacteria cultures were
having Azospirillum sp. and Pseudomonas sp. respectively.

Observations recorded
The observations on seed germination of rough lemon were recorded six weeks after sowing and
percent germination was calculated. The observations on rough lemon seedling height, stem dia-
meter, leaf area, shoot dry weight, root dry weight, length, root diameter, and number of secondary
roots were recorded before the budding operation one year after the sowing. Third, the fourth and
fifth leaf from the terminal end from each seedling were collected for plant nutrient analysis. Total P
and K concentrations were determined in triple-acid (nitric acid: sulphuric acid: perchloric acid
(HNO3:H2SO4:HClO4) 10:3:1) digests using the ammonium molybdate method for P (Nelson and
Sommers 1996) and flame photometery method for K (Westerman 1990).
The observation on budding success on the basis of the number of sprouted buds was measured 60 days
after budding. The growth character of budding viz. sprout length, sprout diameter, number of leaves per
plant, and leaf area and leaf nutrient content recorded after nine months of budding. The leaf chlorophyll
content was measured by a non-destructive method using SPAD-502 plus chlorophyll meter (Konica
Minolta, INC, Japan). Twenty-five leaves were chosen randomly in each replication and the chlorophyll
content readings (−9.9 to 199.9) were recorded and expressed as spad units. Length of the taproot, the
diameter of taproot, and the number of secondary roots were studied under root pattern. Available P in the
media was extracted by shaking with 0.5 M NaHCO3 adjusted to pH 8.5, for half an hour (Olsen et al. 1954).
Potassium was extracted by shaking the sample with 1N NH4OAc (pH 7) solution (1:5 soil solution ratio)
for 5 min. The suspension was then filtered using Whatman No. 1 filter paper. The filtrate was analyzed for
available K with the help of flame photometer (Knudsen, Peterson and Pratt 1982). The nutrient levels were
expressed on a percent dry weight basis. The soil moisture was recorded in polybags with Kinnow
mandarin plants at 15 cm depth during the period of peak moisture demand by Soil Moisture Meter
(BST-SM400), Bionics Scientific Technologies (P) Ltd, India after 48 h of irrigation (200 ml water per
polybag).
The root colonization was assessed by the method published by Phillips and Hayman (1970) and
the root colonization percentage was estimated. The spore density was assessed in the rhizosphere
soil by wet sieving and decantation technique (Gerdemann and Nicolson 1963). Nutrient agar media
was used for bacterial count, potato dextrose agar for fungal count, and actinomycetes isolation agar
for actinomycetes count by serial dilution method. The petriplates were rotated to distribute the soil
inoculum throughout the medium. The agar was allowed to solidify, after solidification the plates
were inverted and incubated at 28°C for 7 days. At the end of the incubation period, when the plates
were showing at least 10 and more than 100 colonies per plate they were used for counting. The
average colony count was calculated per gram of soil as per the formula, population per gram of dry
soil = (population per gram of wet soil × 100)/90.

Scanning electron microscopy

The Scanning Electron Microscopy (SEM) of the citrus seedling roots was performed to decipher the
variation in the total girth, tissue packaging and dimensions of the stele tissue, and gross morphology
in control and culture inoculated plants. The tap roots from the respective treatments were uprooted,
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 5

washed with running tap water to rinse off the adhering dirt and soil particles. The roots were later
placed in 20% alcoholic solution to clear the tightly adhering soil and debris from the root surface.
The transverse sections of the roots were obtained with tissue thickness of 0.5 to 1.0 mm using a
sharp surgical blade from the root-shoot junction region. The diced tissues were fixed in 2.5%
glutaraldehyde (Bozzolla and Russel, 1999), washed with buffer, and post-fixed in osmium tetra-
oxide. The tissue was again rinsed with buffer to get rid of the excess secondary fixative followed by
dehydration by treatment with graded ascending ethanol series (from 30 to 100%). The dehydrated
samples were dried overnight in vacuum dessicator, and sputter coated with 10–20 nm gold layer in
Hitachi E-1010 sputter coater before imaging in SEM (Hitachi S-3400N) @ 15.0 KV.

Statistical analysis
The experiment was laid as Completely Randomized Design with two factors, two growing media
and seven biofertilizer combinations with n = 30. The data were analyzed using statistical software
SAS 9.3 (SAS Institute, Cary, NC). The mean separation was done using (LSD) the least significant
difference at p ≤ 0.05 following significant ANOVA. The correlation analysis was done using
statistical software Statistix 8.1 (Statistix, Tallahassee FL).

Results
Seed germination and shoot growth of rough lemon
The combined application of biofertilizers had a greater effect on seed germination than their individual
application (Figure 1). Application of Azospirillum + AM fungi resulted in the highest increase (12%) in the
seed germination (97.33%) over control which did not differ significantly from the combined application of
Azospirillum + phosphate solubilizing bacteria + AM fungi. The highest germination (98.0%) was recorded
in growing medium soil + FYM + cocopeat fortified with biofertilizer combination Azospirillum + AM
fungi. It did not differ significantly from the seed germination recorded in growing media soil + FYM
having biofertilizer combination Azospirillum + PSB; Azospirillum + AM fungi; or
Azospirillum + phosphate solubilizing bacteria + AM fungi. Irrespective of the growing media, the highest
average seedling height (63.60 cm), stem diameter (6.69 mm), number of leaves (122.83), leaf area
(27.33 cm2) and chlorophyll units (62.46 spad units) of rough lemon seedlings was recorded in
Azospirillum + AM fungi which did not differ significantly from the values recorded with the application
of Azospirillum + PSB + AM fungi.

Rough lemon root growth

Irrespective of the biofertilizers, higher root length (27.85 cm) and the number of secondary roots
(44.23) were recorded in growing media Soil + FYM + Cocopeat in comparison to Soil + FYM
(Figure 2). The highest average root length (31.50 cm) was recorded with Azospirillum + AM fungi
followed by Azospirillum + phosphate solubilizing bacteria + AM fungi (28.66 cm). Similarly, higher
root diameter (7.12 mm) and the number of secondary roots (49.83) were recorded with
Azospirillum + AM fungi. The root diameter in Azospirillum + AM fungi did not differ significantly
from the root diameter recorded in all the other biofertilizer treatments except Azospirillum and
control. Whereas, the number of secondary roots recorded in Azospirillum + AM fungi did not differ
significantly from the number of roots in Azospirillum + phosphate solubilizing bacteria.

Nutrient uptake
The highest nitrogen content (3.33%) was found in media Soil + FYM + Cocopeat fortified with
biofertilizer combinations Azospirillum + phosphate solubilizing bacteria + AM fungi and minimum
6 A. SINGH ET AL.

Root length
40
30

cm
20
10
0

Control

AZO+PSB+AMF
AZO

AMF

AZO+PSB+AMF
Control

AMF
AZO

AZO+AMF
PSB

AZO+PSB
AZO+AMF

PSB

AZO+PSB
Soil + FYM Soil + FYM + Cocopeat

Root diameter
9
6
mm

3
0

AMF
Control

AMF
AZO

AZO+PSB
AZO+AMF
AZO+PSB+AMF
Control
AZO

AZO+PSB
AZO+AMF
AZO+PSB+AMF
PSB

PSB

Soil + FYM Soil + FYM + Cocopeat

Number of secondary roots

60
Number/ plant

40
20
0
Control

AMF

AZO+PSB+AMF
Control

AMF
AZO

AZO+PSB
AZO+AMF

AZO

AZO+PSB
AZO+AMF
AZO+PSB+AMF
PSB

PSB

Soil + FYM Soil + FYM + Cocopeat

Figure 2. Effect of growing media and biofertilizers on root growth of rough lemon seedlings. The vertical bars represent standard
error and are not visible if the values are very small.

(2.79%) in Soil + FYM without any biofertilizer (Figure 3). Highest mean phosphorus content
(0.27%) was observed in Azospirillum + AM fungi which did not differ statistically with phosphorus
levels in biofertilizer treatment Azospirillum + phosphate solubilizing bacteria + AM fungi. The
maximum phosphorus content (0.29%) found in media Soil + FYM + Cocopeat fortified with
Azospirillum + AM fungi and the minimum (0.21%) was found in control without any biofertilizer.
Among all the treatments maximum mean potassium content were observed in
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 7

Leaf nitrogen
4
3
2

Per cent
1
0

Control

AMF

AZO+PSB+AMF
Control

AZO+PSB+AMF
AZO

AZO

AMF
PSB

AZO+PSB
AZO+AMF

PSB

AZO+PSB
AZO+AMF
Soil + FYM Soil + FYM + Cocopeat

Leaf phosphorus
0.4

0.2
Per cent

0
Control

AMF

AMF
AZO

AZO+PSB
AZO+AMF
AZO+PSB+AMF
Control
AZO

AZO+PSB
AZO+AMF
AZO+PSB+AMF
PSB

PSB

Soil + FYM Soil + FYM + Cocopeat

Leaf potassium
1.2
0.9
Per cent

0.6
0.3
0
AZO

AMF

AMF
AZO+PSB
AZO+AMF
AZO+PSB+AMF
Control

AZO+PSB
AZO+AMF
AZO+PSB+AMF
Control
AZO
PSB

PSB

Soil + FYM Soil + FYM + Cocopeat

Figure 3. Effect of growing media and biofertilizers on leaf nitrogen, phosphorus and potassium of rough lemon seedlings. The
vertical bars represent standard error and are not visible if the values are very small.

Azospirillum + phosphate solubilizing bacteria (0.73%) treatment which did not differ statistically
with all the remaining treatments except control (0.65%). The highest potassium content (0.90%)
was recorded in media Soil + FYM + Cocopeat with Azospirillum + AM fungi.
8 A. SINGH ET AL.

Budding Success Buddling girth

120 8
80 6
Per cent

mm
4
40
2
0 0
PSB
Control

AZO+AMF

PSB
AZO

AMF

Control
AZO

AMF

AZO+AMF
AZO+PSB+AMF

AZO+PSB+AMF
AZO+PSB

AZO+PSB

PSB
Control

PSB
AZO

AMF

Control

AMF
AZO+AMF

AZO

AZO+AMF
AZO+PSB

AZO+PSB+AMF

AZO+PSB+AMF
AZO+PSB
Soil + FYM Soil + FYM + Cocopeat Soil + FYM Soil + FYM + Cocopeat

Buddling length
40 Number of leaves
30 25

20
cm

15
10 No./ plant
5
0

PSB
Control
AZO

AMF

AZO+AMF
AZO+PSB+AMF
Control
AZO
PSB
AMF

AZO+AMF
AZO+PSB

AZO+PSB+AMF
AZO+PSB
Control

Control
AZO
PSB

AZO
PSB
AMF

AZO+AMF

AMF

AZO+AMF
AZO+PSB+AMF

AZO+PSB+AMF
AZO+PSB

AZO+PSB

-5

Soil + FYM Soil + FYM + Cocopeat Soil + FYM Soil + FYM + Cocopeat

Buddling leaf area Media moisture content

35 20
25 10
cm2

Per cent

15 0
Control

Control

AMF
AZO

AMF
AZO+PSB
AZO+AMF
AZO+PSB+AMF

AZO

AZO+PSB
AZO+AMF
AZO+PSB+AMF
PSB

PSB

5
-5
Control

Control
AZO
PSB

AZO
PSB

AZO+AMF
AMF

AZO+AMF

AMF
AZO+PSB

AZO+PSB
AZO+PSB+AMF

AZO+PSB+AMF

Soil + FYM Soil + FYM +

Soil + FYM Soil + FYM + Cocopeat Cocopeat

Figure 4. Effect of growing media and biofertilizers on budding success of rough lemon, the growth of Kinnow mandarin
buddlings, and media moisture content (%). The vertical bars represent standard error and are not visible if the values are very
small.

Budding success
Irrespective of the biofertilizers, higher budding success (83.09%) was recorded in growing media
Soil + FYM + Cocopeat (Figure 4) in comparison to Soil + FYM (80.66%). The highest budding
success (91.67%) was observed in Soil + FYM + Cocopeat fortified with biofertilizers
Azospirillum + AM fungi. It was followed by budding success in Soil + FYM + cocopeat with
Azospirillum + PSB + AM fungi which did not differ significantly from budding success recorded in
Soil + FYM + cocopeat with AM fungi.
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 9

Growth of Kinnow mandarin buddlings

Higher buddling length (24.14 cm), number of leaves (16.95), leaf area (22.00 cm2) and buddling
diameter (5.79 mm) were recorded in Kinnow mandarin saplings (Figure 4) in growing media
Soil + FYM + Cocopeat in comparison Soil + FYM, irrespective of the biofertilizers treatment.
Among the six biofertilizer treatments, the highest average sprout length (27.50 cm), leaf area
(23.33 cm2), and sprout diameter (6.19 mm) were found with the application of
Azospirillum + AM fungi. The highest sprout length (29.00 cm) and the number of leaves (21.33)
were recorded in Soil + FYM + Cocopeat fortified with Azospirillum + PSB + AM fungi. However,
the highest sprout diameter (6.20 mm) was recorded in media Soil + FYM + Cocopeat fortified with
Azospirillum + AM fungi.

Organic carbon, media phosphorus, media potassium, root phosphorus, and media moisture
Irrespective of the potting mixture, Azospirillum + AM fungi (Figure 5) resulted in the highest
organic carbon (4.33%), media phosphorus (0.39%), media potassium (459.33 ppm), and root
phosphorus (0.13%). The biofertilizer treatment Azospirillum + AM fungi did not differ from
Azospirillum + PSB + AM fungi; and Azospirillum + PSB and AM fungi for the organic carbon
levels. The highest organic carbon (6.06%), media phosphorus (0.41%), media potassium
(607.00 ppm) and root phosphorus (0.14%) was recorded in Soil + FYM + Cocopeat fortified with
biofertilizer Azospirillum + AM fungi.

Arbuscular mycorrhiza spore count and root colonization

Likewise, the growing media, Soil + FYM + Cocopeat (Figure 5) resulted in significantly higher AM
fungi spore count (26.42 spores 10 g−1 media) and root colonization (29.76%) in comparison to
Soil + FYM (21.71 spores 10 g−1 media and 24.09%), irrespective of the biofertilizers. The highest
AM fungi spore count (39.66 spores 10 g−1 media) and root colonization (39.67%) was also seen in
Soil + FYM + Cocopeat fortified with biofertilizer Azospirillum + AM fungi. It was followed by
Soil + FYM having Azospirillum + AM fungi in which AM fungi spore count and root colonization
of 35.0 spores per 10 g media and 36%, respectively, were recorded. AM spore count and AM root
colonization showed positive correlation (Table 1) with budding success (0.73 and 0.70, respectively),
root phosphorus content (0.73 and 0.66, respectively) and soil phosphorus content (0.68 and 0.45,
respectively).

Colony count
Irrespective of the biofertilizer treatments, higher counts were recorded for bacteria, fungi, and actinomy-
cetes colonies in the potting media Soil + FYM + Cocopeat over Soil + FYM at 120 days after inoculation
(Figure 6a–c). The microbial counts significantly increased from day zero to day 120, irrespective to the
growing media and biofertilizer treatments. All the biofertilizer treatments significantly influenced the mean
microbial counts. After 120 days of inoculation, the highest bacterial counts (98.9 106CFU/g) were recorded
with Azospirillum+ AM fungi in Soil + FYM + Cocopeat and it closely followed by bacterial counts (96.7
106CFU/g) with Azospirillum+ PSB + AM fungi in the same growing media. Similarly, higher fungal counts
(30.44 106CFU/g) were recorded in Soil + FYM + Cocopeat in comparison to Soil + FYM (27.90 106CFU/g).
The highest fungal count (56.1 CFU/106) was observed with the application of phosphate solubilizing
bacteria in Soil + FYM + Cocopeat which was followed by 53.9 106CFU/g counts in Azospirillum + PSB in
Soil + FYM. The biofertilizer treatments Azospirillum; AM fungi; and Azospirillum + PSB + AM fungi in
Soil + FYM + Cocopeat did not differ significantly for fungal counts (52.22 106CFU/g). The biofertilizer
treatments also significantly increased the actinomycetes counts with maximum count (96.1 106CFU/g) was
recorded with the application of Azospirillum + PSB followed by Azospirillum+ AM fungi (88.9 106CFU/g)
and AM fungi (88.33 106CFU/g) AM fungi in Soil + FYM + Cocopeat.
10 A. SINGH ET AL.

Root Phosphorus AM root colonization

Per cent
0.2 50

Per cent
30
0.1
10
0
PSB

PSB
Control
AZO

AMF

AZO+AMF

Control

AMF

AZO+AMF
AZO
AZO+PSB+AMF

AZO+PSB+AMF
AZO+PSB

AZO+PSB

Control
AZO
PSB

Control
AMF

AZO+AMF

AZO
PSB
AMF

AZO+AMF
AZO+PSB+AMF

AZO+PSB+AMF
AZO+PSB

AZO+PSB
-10

Soil + FYM Soil + FYM + Cocopeat Soil + FYM Soil + FYM + Cocopeat
Media Phosphorus Organic carbon
0.5 8
Per cent

0.4 6

Per cent
0.3
0.2 4
0.1 2
0 0
PSB

PSB
Control

AMF

AZO+AMF
AZO

AMF

AZO+AMF

Control
AZO

AZO+PSB+AMF
AZO+PSB+AMF
AZO+PSB

AZO+PSB

PSB
Control
AZO
PSB
AMF

AZO+AMF

Control
AZO

AMF

AZO+AMF
AZO+PSB+AMF
AZO+PSB+AMF

AZO+PSB
AZO+PSB
Soil + FYM Soil + FYM + Cocopeat Soil + FYM Soil + FYM + Cocopeat
AM Spore count
Media potassium
750
10 g soil

40
500
ppm

20
250
0
0
PSB

PSB
Control
AZO

AMF

AZO+AMF

Control

AMF

AZO+AMF
AZO
AZO+PSB

AZO+PSB+AMF

AZO+PSB

AZO+PSB+AMF

Control

Control

PSB
AZO
PSB
AMF

AZO+AMF

AZO

AMF

AZO+AMF
AZO+PSB+AMF

AZO+PSB

AZO+PSB+AMF
AZO+PSB

Soil + FYM Soil + FYM + Cocopeat

Soil + FYM Soil + FYM + Cocopeat

Figure 5. Effect of growing media and biofertilizers on root phosphorus, AM fungi root colonization, media phosphorus, organic
carbon, AM fungi spore count, and media potassium. The vertical bars represent standard error and are not visible if the values are
very small.

Discussion
Rough lemon shoot and root growth
The growing media Soil + FYM + Cocopeat (2:1:1 v/v) resulted in better growth of rough lemon seedlings
and Kinnow mandarin saplings over Soil + FYM (2:1 v/v) as reflected by the various growth parameters.
Higher rough lemon rootstock height, diameter, number of leaves, leaf area, root length, and number of
secondary roots were recorded in rough lemon seedlings in media Soil + FYM + Cocopeat in comparison
to Soil + FYM (Figures 1–2). Addition of cocopeat improved seedling height of rough lemon seedlings.
The results are in conformity with the earlier results of Bhagat, Thakur, and Dhaliwal (2013), that
addition of cocopeat improved plant growth. The enhancement of seed germination following the
Azospirillum + AM fungi application might be attributed to the role of bio-inoculants in enhancing
the availability of nitrogen and phosphorus in the soil to the germinating seeds which might have resulted
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 11

Table 1. Pearson correlation coefficients of sapling growth and soil parameters.

Sprout Budding Buddling Root Soil AM spore AM root
length success girth Phosphorus Phosphorus count colonization
Sprout length 1
Budding success 0.466** 1
Buddling girth 0.753** 0.408** 1
Root Phosphorus 0.509** 0.685** 0.444** 1
Soil Phosphorus 0.653** 0.503** 0.526** 0.724** 1
AM spore count 0.598** 0.734** 0.462** 0.726** 0.685** 1
AM root colonization 0.457** 0.702** 0.654** 0.663** 0.452** 0.731** 1
*Correlation is significant at the 0.05 level.
**Correlation is significant at the 0.01 level.

in consequent enhancement in the metabolic activity resulting in higher germination (Jain, Kumar, and
Panwar 2007). The results clearly indicated that the application of Azospirillum along with AM fungi is
beneficial in improving the growth of rough lemon seedling. In the present studies, the inclusion of
cocopeat and bio-inoculant combination of Azospirillum + AM fungi increased media organic carbon,
media P, media K and root P in the media (Figure 5). Application of Azospirillum along with PSB or AM
fungi or PSB + AM fungi resulted in more beneficial effect on rough lemon growth characteristics over
their individual application. Azospirillum + AM fungi did not differ significantly for growth parameters
of rough lemon with the application of Azospirillum + PSB + AM fungi. The improvement of rootstock
seedling growth in these media may be due to the improvement of organic carbon, media P, media K, and
media moisture levels (Figures 3–5). The higher levels of leaf N, P, and K, and root P (Figures 3–4) also
suggest higher nutrient uptake by the citrus plants in the media and bio-inoculants combination
resulting in better growth of rough rootstock and Kinnow mandarin plants. The SEM images of the
transverse section of rough lemon roots showed rough lemon plants grown in Soil + FYM + Cocopeat
had thinner epidermis and cortical region in comparison to the one in Soil + FYM + Cocopeat fortified
with Azospirillum + AM fungi consortia samples. Besides, the vascular bundles of the rough lemon
seedlings grown in Soil + FYM + Cocopeat had smaller diameter of the xylem/phloem which indicate the
better water and nutrient conduction potential of the consortia (Azospirillum + AM fungi) treated plants
(Plates 1–2). The Azospirillum + AM fungi also resulted in the highest root length, root diameter, and
number of secondary roots (Figure 2). The improvement of plant growth in AM fungus treated plants
may be due to larger root systems which enhance higher water and nutrient uptake. AM fungus increased
shoot biomass and lateral roots in trifoliate orange (Chen et al. 2017). In the present investigations,
significant improvement in organic carbon, P, K, and root P levels were recorded in growing media
soil + FYM + cocopeat (2:1:1) and Azospirillum + AM fungi. AM fungi contribute to increased nutrient
status in the mycorrhizosphere by decomposing of organic amendments or organic N compounds but,
plants may have an additional benefit through additional nitrogen provided through N2 fixation. Felker
et al. (2005) noticed enhanced growth and branching in Prosopis sp. following Azospirillum inoculation.

Nutrient uptake
The application of biofertilizers increased the N, P, and K contents in rough lemon leaves (Figure 3). In
the rough lemon seedlings grown in growing media Soil + FYM + Cocopeat, the average leaf potassium
level increased by 25.9% in comparison to the leaf potassium levels in Soil + FYM. Similarly, in
Soil + FYM + Cocopeat, the average leaf P and N levels were increased by 17.4 and 7.2%, respectively,
over in rough lemon seedlings grown in Soil + FYM. Addition of cocopeat in the growing media also
increased organic carbon in media by 126.2%, media phosphorus (21. 4%), media potassium (50.1%),
root phosphorus (37.5%), AM fungi spore count (21.7%) and AM root colonization (23.5%). The
boinoculant treatment Azospirillum + phosphate solubilizing bacteria + AM fungi resulted in the
highest increase (13.2%) in leaf nitrogen content of rough lemon over control. Whereas,
Azospirillum + AM fungi resulted in the highest increase of 17.4and 9.2% in leaf P and K over control.
12 A. SINGH ET AL.

The increase in nutrient concentration by biofertilizers may be due to increase in root length and
phosphorus uptake by roots. Mycorrhizal inoculation increased growth, root infection, and P uptake in
banana (Ortas et al. 2017). In the present study also, the improvement of rough lemon seedling growth
might be due to the solubilization and mobilization of P from the soil to the plants. Azospirillum + AM
fungi also showed the highest increase in media organic carbon (144.6%), root P (85.7%), media P
(105.3%), media K (580%), AM fungi spore count (131%) and root colonization (65.7%) over unin-
oculated control. Bacteria may improve plant nutrient uptake by solubilizing insoluble phosphorous
and releasing potassium from silicate in soil. AM fungi have been found to increase nutrient uptake in
plants especially the immobile nutrients like P, Zn, and Cu and ammonium-N (Tanaka and Yano 2005;
Ortas 2012). AM fungi absorb and transfer the major, macro-, and micronutrients necessary for plant
growth (Lester 2009). Highest N and P uptake has been observed in dual inoculation with biofertilizers
may be due to improved symbiotic nitrogen fixation and phosphatase activity.

Budding success and Kinnow mandarin sapling growth

The higher budding success and growth of Kinnow mandarin saplings in terms of Kinnow buddling
length, number of leaves, leaf area, and buddling girth in media Soil + FYM + Cocopeat over Soil + FYM
may also be due to improvement of soil physical and chemical properties following the addition of
cocopeat as discussed earlier. All the biofertilizer treatments improved the budding success and growth of
Kinnow mandarin budlings. However, Azospirillum + AM fungi resulted in highest 89.8% budding
success which was 18.5% higher than control (75.8%). This biofertilizer combination resulted in the
highest increase sprout length (37.5%), leaf area (41.4%), number of leaves, and sprout girth. In citrus, the
root hairs are present in some species but, they are relatively shorter than other tree species. Addition of
biofertilizers improved the growth of sour orange, Volkamer lemon, and Cleoptra mandarin (Wang and
Qui 2006). The colonization with AM fungi can significantly increase leaf number and dry mass by
improving the supply of water and nutrients to the citrus plants (Wu and Zou 2009). Wu, Zou, and Wang
(2011) reported that AM fungi association significantly increased stem diameter as well as shoot and root
biomass in trifoliate orange. The increase in plant growth with the application of biofertilizers might be
due to the growth-promoting effect of Azotobacter and AM fungi that improved N, P and K availability
(Figure 3). High total chlorophyll content and more accumulation of various metabolites were attributed
to the enhanced plant growth and biomass production (Kohler et al. 2007). The variation in the
effectiveness among AM fungal species is due to the differences in the spore population and percent
root colonization.

Microbial colony count

All the biofertilizer treatments significantly influenced the microbial counts of bacteria, fungi, and
actinomycetes in growing media (Figure 6). Higher microbial colony count was recorded in medium
Soil + FYM + Cocopeat in comparison to Soil + FYM. Cocopeat improves physical properties of soil; serves
as a buffer against temporary drought stress; and lowers the chances of decline in plant growth during the
growing season. The improvement in soil moisture (Figure 4), organic carbon, media P, and media K levels
(Figure 5) may be responsible for higher microbial counts in Soil + FYM + Cocopeat in comparison to
Soil + FYM. The higher bacterial counts in combined application of Azospirillum+ AM fungi and
Azospirillum + phosphate solubilizing bacteria + AM fungi suggests that plant growth promoting rhizo-
bacteria act as a helping tool for AM fungi for better establishment and functioning of symbiosis (Kohler
et al. 2007). Plant growth-promoting rhizobacteria (PGPR) may colonize the rhizosphere, the root surface
or even superficial intercellular spaces of plants (McCully 2001). There is a positive synergism between
bacterial and AM fungi communities (Berg 2009), one of the two dominates the colonization of the
rhizosphere from time to time due to its invasive capacity and effectivity for rapid establishment of the
symbiotic relationship (Rigamonte, Pylro, and Duarte 2010). The highest arbuscular mycorrhiza spore
count and root colonization percentage were also observed with the application Azospirillum + AM fungi
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 13

A. 0 days 120 days

90
60
30
0

AZO+PSB+A…

AZO+PSB+A…
Control

AMF

Control

AMF
AZO

AZO
PSB

AZO+PSB

AZO+AMF

PSB

AZO+PSB

AZO+AMF
Soil + FYM Soil + FYM + Cocopeat

B. 0 days 120 days

60

40

20

0
AZO

PSB

AZO

PSB
AZO+AMF

AZO+AMF
Control

AMF

Control

AMF

AZO+PSB+AMF
AZO+PSB+AMF
AZO+PSB

AZO+PSB
Soil + FYM Soil + FYM + Cocopeat

C 0 days 120 days

.
90
60
30
0
PSB

PSB
Control

AZO

AMF

AZO+AMF

AZO+PSB+AMF

Control

AZO

AMF
AZO+PSB

AZO+AMF

AZO+PSB+AMF
AZO+PSB

Soil + FYM Soil + FYM + Cocopeat

Figure 6. Effect of growing media and biofertilizers on colony count (106CFU*/g) of bacteria (A), fungi (B) and actenomycetes (C) in
growing media. *Colony forming unit. The vertical bars represent standard error and are not visible if the values are very small.

Plate 2. SEM images of the transverse sections of rough lemon seedling roots grown in Soil + FYM + Cocopeat fortified with
Azospirillum + AM fungi.
14 A. SINGH ET AL.

Plate 1. SEM images of transverse sections of rough lemon seedling roots grown in Soil + FYM + Cocopeat.

(Figure 5). This finding clearly indicates that the co-inoculation of AM fungi with biofertilizers is beneficial
in enhancing the AM fungal root colonization and spore load in the rhizosphere of rough lemon seedlings.
The mechanism by which bacteria stimulate AM colonization and spore proliferation are not properly
understood. Metabolic activities viz. production of essential metabolites, inductions of cell wall degrading
enzymes, increased root cell permeability, and changes in hormonal balance may be the possible factors for
stimulation of AM fungal colonization (Barea, Azcón-Aguilar, and Azcón 1997).

Conclusion
In conclusion, the inclusion of cocopeat and bio-inoculants in the standard growing media of
soil + FYM improved the growth characteristics and buddability of rough lemon. In comparison
to individual application, the combined application of Azospirillum along with PSB/AM fungi/
PSB + AM fungi was more beneficial in improving the seed germination and growth parameters
of rough lemon, and growth of Kinnow mandarin buddlings. The highest seedling height, stem
diameter, number of leaves, leaf area, chlorophyll units, and budding success of rough lemon
seedlings were recorded in Azospirillum + AM fungi which did not differ significantly from
Azospirillum + PSB + AM fungi. Addition of Azospirillum + AM fungi also resulted in highest
sprout length, leaf area, number of leaves, and sprout girth of Kinnow mandarin buddlings. The
roots of rough lemon plants grown in Soil + FYM + Cocopeat fortified with Azospirillum + AM
fungi consortia had thicker epidermis and cortical region; and xylem and phloem vessels with a
higher diameter in comparison to the ones in Soil + FYM + Cocopeat.

Acknowledgments
The authors are thankful to the Punjab Agricultural University, Ludhiana, India for providing the necessary financial
assistance for the investigations.

Notes on contributors
Amandeep Singh, M.Sc. (Fruit Science). Research interest use of biofertilizers to improve plant growth and nutrient
uptake of citrus nursery plants.;

Anirudh Thakur, Ph.D. (Fruit Science), Assistant Professor of Horticulture. He has undergone Post-Doctoral Research
training at Curtin University, Perth, Australia for a period of one year. At present, he is associated with the research
group working on crop improvement, standardization of production and post-harvest handling technologies for low
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 15

chill peach, plum, apple and other temperate fruits. He is also associated with the Hi-Tech containerised nursery
production of virus free citrus plants as per the internationally accepted techniques under protected conditions. He has
been associated with 6 externally aided projects as Principal Investigator or Co- Principal Investigator. He was
associated with writing and editing two books “Temperate Fruit Crop Breeding: domestication to cultivar develop-
ment" by S N Ghosh, M K Verma and Anirudh Thakur, and "Underutilized Fruit Crops: importance and cultivation"
Ghosh S N, Singh A and Thakur Anirudh. He has published 54 research publications, 30 presentations in International
and National Symposia/ Seminars and 16 extension articles. He has done 12 TV and Radio talks which include one
hour life phone-in programmes. He was awarded University Gold Medals for academic excellence in Bachelor’s and
Master’s programmes. He also attained first position in Ph.D. Horticulture programme of Punjab Agricultural
University, Ludhiana. He has been awarded with Senior Research Fellowship of Indian Council of Agricultural
Research, New Delhi, India during Ph. D. He has been awarded eight prizes for best presentations at International
and National Seminars and Symposia. He has guided four Masters and one Ph. D. student.

Sandeep Sharma, Ph. D. (Soil Science), Assistant Soil Microbiologist. Major Research interests are soil microbial and
biochemical transformations under tillage and residues management practices; and Soil microbiological indices under
different land use cropping systems.

P.P.S. Gill, Ph. D. (Fruit Science), Senior Horticulturist, working on nutrient management in fruit plants, tree
architectural engineering for improved fruit quality and yield and post-harvest management of fruits.

Anu Kalia, Ph. D. (Microbiology), Assistant Professor (Nano Science). Major research interests are green synthesis and
characterization of nanomaterials to discern nanomaterial-microbe interactions; synthesis of nanoscale plant nutrients
and development of novel nanofertilizers; and development of degradable polymer based nanocomposite packing
films.

References
Al-Karaki, G. N. 2013. The effect of arbuscular mycorrhizal fungi on the establishment of sour orange (Citrus aurantium)
under different levels of phosphorus. Acta Horticulturae 984:103–08. doi:10.17660/ActaHortic.2013.984.8.
Barea, J. M., C. Azcón-Aguilar, and R. Azcón. 1997. Interactions between mycorrhizal fungi and rhizosphere
microorganisms within the context of sustainable soil-plant systems. In Multitrophic Interactions in Terrestrial
Systems, ed. A. C. Gange and V. K. Brown, 65–77. Oxford: Blackwell Science.
Berg, G. 2009. Plant microbe interactions promoting plant growth and health perspectives for controlled use of
microorganisms in agriculture. Appied Microbiology Biotechnology 84:11–18. doi:10.1007/s00253-009-2092-7.
Bhagat, S., A. Thakur, and H. S. Dhaliwal. 2013. Organic amendment influence growth, buddability success in rough
lemon. Biological Agriculture and Horticulture 29:46–57. doi:10.1080/01448765.2013.759782.
Bozzola, J. J., and L. D. Rusell. 1999. Electron Microscopy: Principles and techniques for biologists. 2nd Edition, Boston:
Jones and Bartlett. 670 p.
Chen, W., J. Li, H. Zhu, P. Xu, J. Chen, and Q. Yao. 2017. The differential and interactive effects of arbuscular
mycorrhizal fungus and phosphorus on the lateral root formation in Poncirus trifoliata (L.). Scientia Horticulturae
217:258–65. doi:10.1016/j.scienta.2017.02.008.
FAOSTAT. 2015. Food and agriculture organization corporate statistical database. http://www.fao.org/faostat/en
Felker, P., D. Medina, C. Soulier, G. Velicce, M. Velarde, and M. Gonzalez. 2005. A survey of environmental and
biological factors (Azospirillum sp, Agrobacterium rhizogenes, Pseudomonas aurantiaca) for their influence in
rooting cuttings of Prosopis alba clones. Journal of Arid Environment 61:227–47. doi:10.1016/j.jaridenv.2004.09.010.
Gerdemann, J. W., and T. H. Nicolson. 1963. Spores of mycorrhizal Endogone species extracted from soil by wet sieving
and decanting. Transactions of the British Mycological Society 46:235–44. doi:10.1016/S0007-1536(63)80079-0.
Jain, A. K., S. Kumar, and J. D. S. Panwar. 2007. Role of Rhizobium, phosphorus and micronutrients on growth and
nodulation of green gram (Vigna radiata (L.) Wilczek). Advance in Plant Sciences 20:337–39.
Jansa, J., A. Weimken, and E. Frossard. 2006. The effects of agricultural practices on arbuscular mycorrhizal fungi. In
Function of Soils for Human Societies and the Environment, ed. E. Frossard, W. Blum, and B. Warkentin, 89–115.
London: Geological Society.
Knudsen, D., G. A. Peterson, and P. F. Pratt. 1982. Lithium, sodium and potassium. In Methods of Soil Analysis Part2.
Chemical and Microbiological Properties A, ed. L. Page, R. H. Miller, and D. R. Keeney, 225–46. Madison, WI, US
App: Soil Science Society of America.
Kohler, J., F. Caravaca, L. Carrasco, and A. Rolden. 2007. Interactions between a plant growth-promoting rhizobacter-
ium, an AM fungus and phosphate-solublizing fungus in the rhizosphere of Lactuca sativa. Applied Soil Ecology
35:480–87. doi:10.1016/j.apsoil.2006.10.006.
Lester, D. 2009. Buying and applying mycorrhizal fungi. Max Yield USA 48:126–31.
16 A. SINGH ET AL.

McCully, M. E. 2001. Niches for bacterial endophytes in crop plants a plant biologist’s: A review. Australian Journal of
Plant Physiology 28:983–90.
Mir, M., G. I. Hassan, K. Sheikh, and S. K. Sharma. 2012. Impact of biofertilizers on growth, nutrient uptake, yield,
metabolism and rhizospere activities of Pomegranate. Applied Biological Research 14:168–75.
Nelson, D. W., and L. E. Sommers. 1996. Total carbon, organic carbon and organic matter. In Methods of Soil Analysis.
Part 3: Chemical Methods, ed. D. L. Sparks, A. L. Page, P. A. Helmke, R. H. Loeppert, P. N. Soltanpour, M. A.
Tabatabai, C. T. Johnston, and M. E. Sumner, 961–1010. No. 5. Madison, WI, USA: Soil Science Society of America,
Book Series.
Olsen, S. R., C. V. Cole, F. S. Watanabe, and L. A. Dean. 1954. Estimation of Available Phosphorus in Soils by
Extraction with Sodium Bicarbonate. USDA. Circular 939. Washington, D.C.: USDA.
Ortas, I. 2012. Mycorrhiza in citrus growth and nutrition. In Advanced in Citrus Nutrition, ed. A. K. Srivastava, 469–
84. The Neitherland: Springer-Verlag.
Ortas, I., M. Rafiquea, C. Akpinarc, and Y. A. Kacard. 2017. Growth media and mycorrhizal species effect on
acclimatization and nutrient uptake of banana plantlets. Scientia Horticulturae 217:55–60. doi:10.1016/j.
scienta.2017.01.025.
Ortas, I., and O. Ustuner. 2014. Determination of different growth media and various mycorrhiza species on citrus
growth and nutrient uptake. Scientia Horticulturae 166:84–90. doi:10.1016/j.scienta.2013.12.014.
Panigrahi, P., R. K. Sharma, M. Hasan, and S. S. Parihar. 2014. Deficit irrigation scheduling and yield prediction of
‘Kinnow’ mandarin (Citrus reticulate Blanco) in a semiarid region. Agricultural Water Management 140:48–60.
doi:10.1016/j.agwat.2014.03.018.
Peel, M. C., B. L. Finlayson, and T. A. McMahon. 2007. Updated world map of the Koppen-Geiger climate
classification. Hydrology and Earth System Sciences 4:439–73. doi:10.5194/hessd-4-439-2007.
Phillips, J. M., and D. S. Hayman. 1970. Improved procedures for clearing and staining parasitic and vesicular
arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycological Society
13:31–32.
Rigamonte, A. E., V. S. Pylro, and G. F. Duarte. 2010. The role of mycorrhization helper in the establishment and
action of ectomycorrhiazae associations. Brazilian Journal of Microbiology 41:832–40. doi:10.1590/S1517-
83822010000400002.
Rilling, C. M. 2004. Arbuscular Mycorrhizae and terrestrial ecosystem process. Ecology Letters 7:740–54. doi:10.1111/
j.1461-0248.2004.00620.x.
Tanaka, Y., and K. Yano. 2005. Nitrogen delivery to maize via mycorrhizal hyphae depends on the form of N supplied.
Plant Cell and Environment 28:1247–54. doi:10.1111/pce.2005.28.issue-10.
Ustuner, O., S. Wininger, V. Gadkar, H. Badani, M. Raviv, N. Dudai, S. Medina, and Y. Kapulnik. 2009. Evaluation of
different compost amendment with AM fungal inoculums for optimal growth of chives. Compost Science and
Utilization 17:257–65. doi:10.1080/1065657X.2009.10702432.
Wang, B., and Y. L. Qui. 2006. Phylogenetic distribution and evolution of mycorrhizas in land plants. Mycorrhiza
16:299–363. doi:10.1007/s00572-005-0033-6.
Westerman, R. L. 1990. Soil Testing and Plant Analysis. 3rd ed. Madison,WI, USA: SSSA.
Wu, Q. S., and Y. N. Zou. 2009. The effect of dual application of arbuscular mycorrhizal fungi and polyamines upon
growth and nutrient uptake on trifoliate orange seedlings. Notulae Botanicae Horti Agrobotanici Cluj-Napoca
37:95–98.
Wu, Q. S., Y. N. Zou, and G. N. Wang. 2011. Arbuscular mycorrhizal fungi and acclimatization of micropropagated
citrus. Communications in Soil Science and Plant Analysis 42:1825–32. doi:10.1080/00103624.2011.587570.