Indian Journal of Biotechnology
Vol 12, January 2013, pp 13-19
Microbial inoculants to support tea industry in India
Anita Pandey*, Shipra Singh and Lok Man S Palni
G B Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora 263 643, India
A long term study has been carried out for over a decade across various tea gardens, located in different parts of Indian
Himalayan Region (IHR), with a focus on rhizosphere microbiology of tea. Occurrence of ‘negative rhizosphere effect’
exerted by the established tea bushes, in contrast to the normal stimulatory effect exhibited by the plants in general as well
as young tea bushes, is the first and foremost feature associated with tea plants. Colonization by large populations of
antagonists and lowering of the soil pH are the other important characteristics associated with tea rhizosphere. With a view
of scaling down the use of chemical fertilizers, experiments were conducted on isolation, characterization and formulation of
suitable microbial inoculants for use in tea plantations across hilly regions. Based on a number of attributes related to plant
growth promotion and disease control, selected bacterial and mycorrhizal species have been developed in suitable
formulations for application in tea plantations. The post-application observations indicated the benefits of microbial
inoculations on seed, cutting and tissue culture raised tea plants. The present paper is an attempt to review the basic and
applied research work carried out on rhizosphere microbiology of tea, particularly in the last two decades, with reference to
possible application in the tea industry.
Keywords: Indian Himalayan Region (IHR), microbial inoculants, rhizosphere effect, tea plantations
Introduction
Lorenz Hiltner (1904)1 was the first to recognize
the importance of microbial activity associated
with the root system in respect of plant nutrition. The
term “rhizosphere” was used to describe the zone of
intense microbial activity around roots, and two major
research areas of potential value related to the
rhizosphere effect–plant nutrition and biocontrol were
identified. Every plant species provides an individual
and specific site of microbial activity in the form of a
“rhizosphere”. Tea [Camellia sinensis (L.) O. Kuntze]
is an economically important cash crop of India.
Besides its economic importance as a beverage, tea
bushes also provide an excellent “rhizosphere” site for
studying plant-microbe interactions, under a given set
of environmental conditions. This is on account of
various features associated with tea plantations,
namely, (1) in nature, tea is a small to medium sized
tree (Fig. 1A), which is maintained as a shrub through
cultural practices, i.e., by regular cycles of pruning
(Fig. 1B); (2) it is essentially grown as monoculture,
with some shade trees, over large areas; (3) tea plants
prefer acidic soils with pH between 4.5 to 5.6; (4)
it is grown in more than 50 countries around the
world from Georgia (43°N latitude; USA) to Nelson
(42°S latitude; South Island, New Zealand), and from
sea level to 2300 m above mean sea level under
varied environmental conditions and temperature
ranging from –8° to 35°C with precipitation from about
1,150 to 8,000 mm per year2-6
.
In order to understand the microbial diversity of tea
rhizosphere, first of all basic studies were carried out
on asymbiotic (free-living bacteria and fungi) as well
as symbiotic (arbuscular mycorrhizal fungi) associates
of tea rhizosphere. The investigations were conducted
on the rhizosphere of young as well as established
(4 to 123-yr-old) tea bushes, represented by both
chinery and assamica types. The study sites included
bushes in both well managed and abandoned tea
plantations, representing monsoonal, subtropical and
temperate locations. The sites of tea plantations
used for this study in India included: (1) Banuri Tea
Experimental Garden, Palampur, Himachal Pradesh,
(2) Mansambal Tea Estate, Palampur, Himachal
Pradesh, (3) Temi Tea Estate, Temi, Sikkim, (4) Singell
Tea Estate, Kurseong, West Bengal, (5) Pant Tea Estate,
Bhowali, Nainital, Uttarakhand, and (6) Kausani Tea
Plantations, Kausani, Bageshwar, Uttarakhand.
Tea Rhizosphere: Characteristic Features
Negative Rhizosphere Effect
The rhizosphere of tea has been found to possess
many characteristic features. Initial experiments
conducted at the Banuri Tea Experimental Garden and
————————
*Author for correspondence:
E-mail: [email protected]
INDIAN J BIOTECHNOL, JANUARY 2013
14
Mansambal Tea Estate in District Kangra (Himachal
Pradesh) gave interesting results and important leads
necessitating further investigations. The first and
foremost feature associated with the established tea
bushes was found to be the occurrence of a “negative
rhizosphere effect”, as reflected in terms of R:S
(rhizosphere:soil) ratios below one (Table 1). The
number of microorganisms in the rhizosphere soil of
established tea bushes, contrary to the young tea
bushes, was found to be lower in comparison to the
bulk soil. It is also in contrast to the normal
stimulatory effect exerted by the roots of other plants
on soil microorganisms. While bacteria appeared to
be the most suppressed group in the rhizosphere of
established tea bushes, actinomycetes and fungal
populations were also suppressed but to a lesser
extent. Further, the negative rhizosphere effect
seemed to be more pronounced in tea bushes of
assamica type in comparison to the chinery type. The
effect was much greater in the case of well maintained
tea gardens vis-à-vis abandoned gardens7-9
.
Factors Causing Negative Rhizosphere Effect
The major factor influencing the biological activity in and around the rhizosphere is the amount and nature of root exudates. The experiments conducted on microbial analyses of tea rhizosphere indicated that the tea roots, after attaining a certain age, also start secreting exudates that contain antimicrobial
metabolites to which bacteria are the most susceptible group. This was demonstrated through bioassays using the bacterial isolates dominant in the tea rhizosphere. Supplementing the culture medium with
rhizosphere soil extract (aqueous) resulted in a marked reduction in the number of bacterial colonies
7.
This also indicated that at least some of the antimicrobial
metabolites accumulate in the rhizosphere soil, and that the same are water soluble and heat tolerant.
Colonization by larger population of antagonists,
known for various categories of antagonism, such as,
antibiosis, competition, parasitism, and predation, was
observed to be the second important factor
responsible for the overall reduction in the number of
microorganisms in rhizosphere. Results relating to
relative inhibition observed in soil dilution plates
prepared from the established tea rhizosphere, and the
inverse relationship seen under ‘in situ’ conditions
between the bacterial and fungal populations in the
soil, support the occurrence of an intense microbe-
microbe interaction in the rhizosphere of established
tea bushes10
,11
. Lowering of the soil pH with age was
found to be the third important factor resulting in
suppression of microbial communities in the tea
rhizosphere12
. Lowering of pH by tea plantlets under
‘in vitro’ conditions also supports these observations13
.
Characteristics of Microbial Diversity of Tea
Rhizosphere
Free-living Microbes
The rhizosphere of established tea bushes under the
influence of negative rhizosphere effect was observed
as a remarkable example of natural phenomenon.
Species of Bacillus dominated the bacterial
population, B. subtilis being the most abundant
(Fig. 2A). B. mycoides, B. licheniformis, B. cereus,
Fig. 1—(A & B)—Some study locations: A. Old and abandoned tea plants at Ranidhara, Almora (Uttarakhand); B. Tea plantations at
Banuri Tea Experimental Garden, Himachal Pradesh (courtesy: Madhu Sharma).
PANDEY et al: MICROBES IN SUPPORT OF TEA INDUSTRY
15
and B. megaterium were other important species12
.
This would seem to be related to the endospore
forming ability of the genus Bacillus and survival
under adverse conditions14
, including the factors
related to the ‘negative rhizosphere effect’ seen in
case of tea. It was interesting to observe that
Pseudomonas, a common soil bacterium, that was
isolated frequently from the soil samples collected
from the rhizosphere of young tea plants, was not
prevalent in soil samples taken from the established
tea rhizosphere. In view of the known importance of
genera Bacillus and Pseudomonas, B. subtilis in
particular, the species were tested for its antagonistic
properties (Figs 2B & C) as well as their survival at
lower temperatures15,16
. Species of Penicillium and
Trichoderma dominated the list of fungi in the
rhizosphere of established tea bushes. The dominant
species of Penicillium and Trichoderma isolated
from different locations included P. erythromellis,
P. janthinellum, P. lanosum, P. raistrickii, T. konengii,
and T. pseudokonengii11,17
. A large number of
actinomycetes forming brown to gray hard pustule
like colonies with brown to black secretion that was
diffusible in the agar were also isolated. Based on
their colony and cell morphology and biochemical
characters, these were grouped under the genus
Streptomyces18
. More recent studies conducted on the
microbial diversity of tea rhizosphere with focus on
functional aspects, antagonism in particular, using both
conventional and modern methods have shown
conformity with the earlier findings19-22
. The latest
study of Zhao et al,23
conducted on determination of
bacterial communities of tea soils using the nested
PCR-DGGE method, has been found to be in line
with the earlier findings of Pandey and Palni7,12
that
were based on traditional culture dependent methods.
Colonization of Arbuscular Mycorrhizal (AM) Fungi
The AM fungi are well known for their symbiotic
association with plant roots and for the support they
Table 1—R:S ratios for 3 groups of microorganisms at 3 depths during a 12 months cycle (Pandey and Palni7)
Months Bacteria Actinomycetes Fungi
Soil depth (cm)
0-15 15-30 30-45 0-15 15-30 30-45 0-15 15-30 30-45
Nov 0.29 0.08 0.04 0.63 0.46 0.30 0.51 0.44 0.40
Dec 0.59 0.57 0.42 11.2 0.08 0.16 0.80 2.00 -
Jan 0.91 0.93 0.74 1.29 0.13 1.10 0.26 0.79 1.00
Feb 0.98 0.86 0.84 0.39 0.66 0.47 1.24 2.33 -
Mar 0.44 0.36 1.01 0.24 1.76 0.57 1.55 0.89 -
Apr 0.51 0.51 0.44 0.85 0.92 1.04 0.86 0.82 0.22
May 0.48 0.57 0.09 0.80 0.56 0.23 0.81 0.38 -
Jun 0.64 0.50 0.09 0.65 0.62 0.22 0.65 0.26 -
Jul 0.65 0.16 0.73 0.93 1.16 0.48 0.82 0.83 0.48
Aug 0.52 0.56 0.92 0.52 0.50 0.56 0.66 1.17 0.38
Sep 0.16 0.26 0.31 0.34 0.56 0.25 1.23 1.23 0.77
Oct 0.80 0.90 1.06 0.73 0.30 0.06 0.76 0.94 0.76
R:S = Rhizosphere:Soil; - = not detected
Fig. 2—(A-C)—A. Dominance of B. subtilis on a soil dilution plate (a heavy snow fall on the previous day of sampling), B & C.
Production of antimicrobial substances by B. subtilis against B. mycoides (B) and Botryodiplodia theobromae (C).
INDIAN J BIOTECHNOL, JANUARY 2013
16
provide in mineral solubilization. With a view of
understanding the diversity and colonization of AM
fungi, the root and rhizosphere soil samples collected
from very old (well established) but abandoned tea
plants (Ranidhara, Almora, Uttarakhand) as well as
recently planted and well maintained tea bushes
(Kausani, Bageshwar, Uttarakhand) in the Kumaun
region of Uttarakhand have been investigated. Trap
cultures of AM fungi were developed using an
appropriate amount of soil as inoculums, and Eleusine
coracana and Zea mays were used as hosts for
reconfirmation of the identity of AM fungi. Tea roots
were found to be heavily colonized by AM fungi
(approx. 70-90%) in both the cases, with an average
spore density of 162 and 185 spores 25 g-1
soil in the
established (abandoned) and young (well maintained)
tea bushes, respectively. General AM colonization
events in tea roots have been shown in Fig. 3.
The occurrence of four genera, viz., Acaulospora,
Gigaspora, Glomus, and Scutellospora was recorded
from the established tea plants from Ranidhara (likely
to be of seed origin), while species of the genus
Glomus along with a few spores of Acaulospora
species were recorded in samples taken from the
young tea bushes (Table 2)24
. It may be important to
note that the established but very old and abandoned
tea plants at Ranidhara were growing under natural
conditions. Based on the local information these
plants were >100-yr-old and had neither received any
chemical fertilizer nor subjected to any pruning
cycles, at least for the last 20 yr. On the other hand,
tea plantations at Kausani were young (6 to 7-yr-old),
cutting raised, grown as monoculture and were
maintained under prescribed pruning cycles, as well
as received appropriate amounts of chemical
fertilizers at regular intervals. Detection of only two
genera of AM fungi from young, well maintained tea
bushes against a total of four genera found in the
samples taken from naturally growing old and
abandoned tea plants is indicative of the likely
negative effect of the use of chemicals on the
diversity of AM fungi. A recent report on lack of
AM colonization in tea plants cultivated in Northern
Iran25
is interesting, and may be attributed to various
edaphic and climatic conditions, as well as cultural
practices. In addition, the composition of AM fungi
is also likely to be strongly affected by individual
host species. Comparative assessments on diversity of
AM fungi, associated with rhododendrons and
Taxus baccata from Uttarakhand, have also been
performed26,27
. Microbial Inoculants for Tea Plantations
Natural rhizosphere is an ideal site for the isolation
of “beneficial” microorganisms. With a view to
develop promising bioinoculants for use in tea
plantations located in hilly regions across the country,
considerable efforts have been put to explore
the below ground microbial diversity of tea plants
growing at various locations. Following a series
of screening tests, the bacterial isolates, namely,
B. subtilis and P. corrugata, were selected with
Fig. 3—(A-D)—Colonization of tea roots by AM fungi: (A)
Appresorium formation at the root surface just before entry; (B)
Intra and inter cellular growth of hyphae; (C) Formation of vesicles;
& (D) Formation of arbuscules inside root cortical cells (×400).
Table 2—General characteristics of colonization by AM fungi in natural and cultivated tea sites during periods
of active growth and dormancy
Natural site Cultivated site AM fungal
colonization features Active growth Dormancy Active growth Dormancy
Colonization (%) 77.6±4.40 97.3±0.78 86.4±3.02 98.1±0.79
Spore density 162±30.29 189±16.85 185±12.68 202±9.78
No. of genera 04 04 02 02
No. of morphotypes 35 33 27 23
Diversity index 1.80±0.12 2.05±0.10 1.75±0.13 1.94±0.10
Simpson index 0.20±0.04 0.20±0.05 0.21±0.05 0.17±0.04
PANDEY et al: MICROBES IN SUPPORT OF TEA INDUSTRY
17
potential to be developed as bioinoculants for use in
tea plantations. Most of the commercially developed
plant growth promoting rhizobacteria (PGPRs)
belong to the genus Bacillus and among the genus,
strains of B. subtilis are particularly well known as
PGPRs. This is mainly because of their antagonistic
ability (Figs 2B & C) and tolerance toward
environmental stress16,28,29
. P. corrugata was found to
produce siderophores and antimicrobial substances,
causing the inhibition of fungal growth, and possessed
nitrogenase and phosphate solubilizing activity. The
isolate appeared to be a psychrotroph and could grow
between 4 to 35°C8,30
. The significance of the ability
to fix nitrogen combined with root colonization in
diazotrophic pseudomonads has been recognized for
developing bacterial inoculants for temperate regions
in the Canadian High Arctic31
. These two bacterial
cultures, B. subtilis and P. corrugata, have been
accessioned by the Agricultural Research Culture
Collection, International Depository Authority, Illinois,
USA (Acc. nos. NRRL B-30408 & NRRL B-30409,
respectively).
Under field conditions, the success of microbe
based “fertilizer” technology depends on the availability
of suitable inoculants in appropriate formulations in
easy to ‘store and use form’. For commercialization,
viability of the bioinoculant(s) in prescribed formulation
with preservation of strain characteristics over a
defined period in storage is of utmost importance32,33
.
Therefore, the selected bioinoculants have been tested
in suitable formulations (broth, charcoal and alginate
beads) using bioassays. And the viability of bacterial
formulations was evaluated following storage at 4oC
and room temperature upto 3 yr34,35
. While broth or
charcoal based formulations were found to be suitable
for applications, bacteria entrapped in alginate beads
were suitable for long term storage and/or transport.
In case of seed raised plants of tea, cracked seeds
were coated with the bacterial inoculant(s) using a
charcoal based formulation. This resulted in improved
growth of treated seedling plants in comparison to
untreated control plants. The treatment also proved
useful in respect of tissue culture raised (TCR) plants
of tea. The TCR plants were transferred to autoclaved
soil and then inoculated with a liquid formulation of
bacterial species during laboratory to land transfer.
The bacterial inoculants were the primary colonizers
and provided the first line of defense to TCR plants of
tea, resulting in near 100% survival against 45-50%
survival observed in the control plants. The analyses
of rhizoplane and rhizosphere soil (of treated and
control plants) indicated that the major cause of
mortality during laboratory to land transfer of TCR
plants (control) of tea was fungal attack, mainly by
Fusarium oxysporum36
. With a view of introducing
bioinoculant(s) in tea plantations, suspension of
selected bacterial strains was inoculated in the
rhizosphere region of young seedlings and cutting-
raised plants of tea under net house conditions. It was
found that the introduced bacteria successfully
colonized the rhizosphere and stimulated the general
microflora. The root colonization efficiency of
the bacterial inoculants was also worked out using
antibiotic resistance markers37
. The work of
Chakraborty and coworkers38-40
has also indicated the
importance of several tea rhizosphere associated
bacteria as growth promoters and biocontrol agents.
Soil based AM fungal consortia have also been
developed both from the natural and cultivated tea
rhizospheres. Following inoculation with the consortium
(of two different origin) under greenhouse conditions,
plant growth promotion was observed in the bioassays
conducted using maize and wheat as test plants. In both
the cases significant increase in the growth of maize and
wheat plants, al beit to different degrees, was recorded.
In subsequent bioassays conducted on tea, both AM
fungal consortia were found to promote the growth of
tea plants significantly in non-sterilized acidic soil. It
was noted that while both the consortia promoted
growth of tea plants, AM fungal consortia developed
from natural tea rhizosphere was found to perform
better41
. The inoculation also resulted in significant
increase in a number of quality parameters, such as,
amino acids, total protein and polyphenols, caffeine, and
sugar content of tea leaves42
.
Organic Tea in India: The Scope and Possibilities While the production of organic tea has received
attention in recent years, it needs to be made cost-
effective to be competitive with other forms of teas.
In the Indian context, a report published in ENVIS
Newsletter43
showed that the price differential
between the organic and so called non-organic teas
varied from 200-300% in early 1990s, which had
come down to about 50% by the year 2000. The
premium for organic tea compared to conventional tea
in the international market ranged between 25-30% in
the year 200644
. These data indicate that it is likely
that the price for organic tea will continue to decrease
in relation to conventional grown tea in future. As a
INDIAN J BIOTECHNOL, JANUARY 2013
18
result, the transition to consumption of organic tea
would become commercially acceptable at a large
scale. Moreover, diversification into other products,
such as, herbal tea, green tea and other commodities
may also help in increased acceptability of organic
farming of tea.
In a country like India, where large labour force is
available at a relatively cheaper rates, organic farming
is likely to be seen as a good and cost-effective solution
in the long term, keeping in view of the increasing
costs involved in chemical farming with associated ill
effects. Currently, most organic planters are in the
transition phase and, hence, have to bear the burden of
high productivity costs. As planters continue to practice
organic tea cultivation, the production costs are expected
to reduce, making India as one of the important
producers of organic tea. Besides, the availability of
good marketing channels for securing a good premium
for the organic produce is equally important. The real
boost for the promotion of organic tea cultivation will
depend on support offered by the big tea companies,
showing preference for the organic teas.
Conclusion
Many reports regarding the so called “failure” of
microbial fertilizers are available in the literature. The
main cause behind such reports, in general, can be
ascribed to the use of microbes that were isolated from
one environment and introduced in a completely
“alien” or different environment. Microbes, being
rhizosphere as well as environment specific, often fail
to survive in the alien environments. Documentation of
microbial diversity in various ecological niches of
Indian Himalayan Region (IHR) including tea
plantations has been under consideration in recent
times45-47
. In the studies reported in the present review,
due consideration was given to the understanding of
biodiversity associated with the tea rhizosphere as
well as the ecological competence of the isolated
microbes. The selected microbes were isolated from
the rhizosphere of tea growing in temperate locations.
The approach adopted in these studies does provide a
model for developing microbial inoculants for specific
plants growing under specific set of environmental
conditions. The future studies are expected to use
native bioinoculants to provide protection against a
range of minor and major pathogens, and also to
support and enhance the growth of tea bushes by way
of improved nutrition. The approach, thus, described
should subsequently result in reducing the use of
chemical fertilizers. This is particularly important for
the hilly states of India that have embraced the slogan
of being green and organic.
Acknowledgement
Professor N K Jain is thanked for support and
guidance on the possibility of using microbes as
inoculants and biofertilizers in tea gardens located in the
mountain regions. The Department of Biotechnology
and Ministry of Environment and Forests, Government
of India, New Delhi are thanked for financial support
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