Microbial inoculants to support tea industry in India

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  • 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 effectplant 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 (43N latitude; USA) to Nelson

    (42S 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 35C 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: anita@gbpihed.nic.in

  • 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--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 1R: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 2General 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 (%) 77.64.40 97.30.78 86.43.02 98.10.79

    Spore density 16230.29 18916.85 18512.68 2029.78

    No. of genera 04 04 02 02

    No. of morphotypes 35 33 27 23

    Diversity index 1.800.12 2.050.10 1.750.13 1.940.10

    Simpson index 0.200.04 0.200.05 0.210.05 0.170.04

  • 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 35C8,30

    . 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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