Study on Antagonistic and Growth Promotion Potential of Certain Exo and Endophytic Bacteria of Mulberry (Morus SPP)

CONTENTS

1. INTRODUCTION

2. REVIEW OF LITERATURE

3. METERIALS AND METHODS

4. RESULTS

5. DISCUSSION

6. SUMMARY AND CONCLUSION

7 BIBLIOGRAPHY

DECLARATION

We hereby declare that this investigation entitled ‘ Study on antagonistic and growth promotion potential of certain exo and endophytic bacteria of mulberry (Morus spp.) ’ submitted by us to the oxford college of Science Post Graduated Center affiliated to Bangalore University in partial fulfillment of the requirement for the award of the degree of Master of Science in Microbiology, is a bonafide work carried out by us under the guidance of Dr. Pratheesh Kumar P. M., Scientist-D, Mulberry Pathology & Microbiology Laboratory, Central Sericulture Research & Training Institute, Central Silk Board, Ministry of Textiles, Govt. of India, Mysore during 2017-2018.

We further declare that the results presented here have not been previously submitted for any degree or diploma, either in this or in any other university.

Akanksha Varun, Shwetha G & Yashaswini R

Place: Mysuru

Date: 8.06.2018

ACKNOWLEDGEMENT

We offer humblest salutation at the feet of God, and our parents for their kind blessing throughout our life. We are extremely happy to make this opportunity to acknowledge my gratitude to those who are associated in the preparation of this dissertation. Words fail to express our profound regards from innermost recess of our heart to Dr. Pratheesh Kumar. P.M. Scientist-D, Mulberry Pathology & Microbiology Laboratory, CSR & TI, Mysore for the valuable help, constant guidance and wise counseling extend right from the selection of topic to the successful completion of the dissertation work , his scholarly insight shaped the content of this work.

Our special thanks to Dr.Bharathi. S Head of the Department of Microbiology, Bangalore for her co-operation and encouragement during the course of this study. My special heartfelt thanks to our guide Vienna fernandes for their guidance.

Our special thanks to Mr. T. Balakrish, Technical Assistant and Mr. Kumar and other staff members of Mulberry Pathology Laboratory. CSR&TI, I&TI for various assistances extended during the course of this study.

We wish to express our respect to God, our parents, our brothers, sisters and friends for their love and affection and for giving confidence to me without which it would not have been possible for us to attain this task.

Akanksha Varun, Shwetha.G & Yashaswini R

Place: Mysore

Date 08.06.2018

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LIST OF TABLES

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LIST OF FIGURES

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INTRODUCTION

In India, sericulture has started thousands of years ago and over the years silk has become an inseparable part of Indian culture and tradition. It is also one of the highest employment providers by combining both agriculture and industry. India is the second largest producer in silk in the world and process the distinction of producer of all the 4 varieties of commercial silk namely, mulberry silk (Bombyx mori), eri silk (Samia ricini), tasar silk (Antheraea mylitta) and muga silk (Antheraea assamensis). Among these 4 varieties of silk produced, mulberry accounts for 75%, tasar 7%, eri 17% and muga 1% of total raw silk production. The major silk producing states in India are Karnataka, Andhra Pradesh, Tamil Nadu, West Bengal and Jammu & Kashmir. The raw silk production of mulberry silkworm shows increasing trend during the last decade due to development of high yielding mulberry varieties and silkworm breeds coupled with technological innovation in cultivation and rearing practices. Natural silk is unique with its own importance, elegance and luster, natural silk is comfortable to wear and good for health. Silk is resistance against too high temperature. The elegant feature of silk thread is due to its fine luster and soft touch.

Historical evidences reveal that China is the first country where sericulture was practiced in the Chan-tong province secretly for more than 3000 years. Slowly in 1200 BC this industry spread over to Korea and then to Japan during third century BC itself. In 19th century sericulture industry flourished in France and epidemic of pebrine disease broke out and totally wiped out the industry not only in France but also in Middle East and Europe. Then, sericulture spread to Tibet and India about 140 BC. Mulberry cultivation and silk industry first began in the areas flanking the rivers Brahmaputra and Ganges. When British came to India, they found a flashing silk trade in India. Murshidabad district of West Bengal became one of the famous centers of silk industry. The East India Company decided to modernized the rearing and realign techniques. In Jammu and Kashmir, Mysore and West Bengal this industry flourished well. Later on West Bengal declined in the production of silk. World war-II saved the Indian silk industry for a total disaster with the outbreak of war the demand for silk is increased tremendously. In 1948 the country was divided in to India’s and Pakistan and as a result some of the silk producing areas passed over to Pakistan and Bengal In 1948 big strike was taken for rehabilitate the silk industry.

Sericulture is the care of little insect that produce silk thread it is very delicate work that require attention and patience throughout the life cycle of silk moth from egg to cocoon. Sericulture also includes activity of raising the food plants (mulberry for rearing silkworm). Mulberry is the only food for silkworm (Bombyx mori) and is grown under varied climatic conditions ranging from temperate topics mulberry leaf is major economic component in sericulture since the quality and quality leaf produced per unit area has direct bearing on cocoons harvested. Mulberry is a hard plant capable of thriving under the variety of agro climatic conditions. Mulberry can be grown as bush, high bush, medium trees or long trees bush type is widely grown in south India which grows heights and last for about 15 years and it is perennial plant. It needs attention throughout the year agronomical practices such as pruning, intercultural operations and application of fertilizers as well as crop protection.

Quality of mulberry leaves in addition to proper maintenance of temperature and humidity place an important role in healthy silkworm, leaf quality plays important role in production of quality of cocoon. The leaves during its feeding time should not contain dust particles or water droplets on surface of leaf. Disease attacked and poor quality leaves should not to be fed to the silkworms as these have direct impact on health of silkworm and in turn results poor quality of silk produced.

Mulberry is prone to many diseases caused by various pathogens. Powdery mildew caused by Phyllacativa corylea, leaf spot caused by Cercospora moricola. Root rot caused by a group of pathogens such as Fusarium solani, Rhizoctonia bataticola, Fusarium oxysporum and Botryodiplodia theobromae as well as root knot caused by Meloideogyna incognita are some of the major diseases prevalent in mulberry crops in India. Since disease deteriorates the quality of mulberry leaves these qualitatively inferior leaves lead to production of poor quality cocoon and hence farmers get less income by selling such as cocoons further by feeding the disease affected mulberry leaves to silkworm larvae become weak and hence are susceptible to various diseases leading to larval mortality and large scale silkworm crop loss. Therefore control of mulberry disease is a vital concern among sericulture farmers.

In the recent past, various methods are development for control of mulberry diseases, this includes cultural and biological methods and least preference was given to chemical control measures due to adverse effect of plant protection chemicals on soil, plants and also to human beings. The chemicals generally used for control of diseases accumulate in water bodies, pollute the air, and in some cases have other tremendous effect o environment. Cultural control could not address to control the disease fully especially a crop like mulberry has to follow the recommended agronomical practices for better yield. This is mainly because when the agronomical practices are developed, focus was only given to the yield improvement and not for control of diseases. Also, agronomical practices could not contain severe soil borne disease like root rot. Though biological control agents such as Trichoderma are recommended to control soil borne diseases, the effectiveness of these organisms depends on various microclimatic factors to get desired results. Therefore a uniform effectiveness has not been attained from many biocontrol agents especially soil applied ones.

Identification of antagonistic microorganisms that are attached to rhizosphere, phylloplane or living as endophytes of mulberry are therefore vital so that there may not be a problem for establishment of these microbes and hence easily be utilized for disease control. These organisms provide multiple benefits such as promotion of plant growth and yield and suppression of phytopathogens. These organisms are studied widely for their growth promotion and antagonistic effect in many agriculture systems and found success.

The rhizosphere is the region of soil that is immediately near to the root surface and that is affected by root exudates. There are different types of substances that diffuse from the roots and that stimulate the microbial activity, such as carbohydrates (sugars and oligosaccharides), organic acids, vitamins, nucleotides, flavonoids, enzymes, hormones, and volatile compounds. The result is a dense and active microbial population that interacts with the roots and within it. Rhizosphere bacteria can enhance the plant growth and crop yield by different ways.

In soil and rhizosphere region, many microorganisms live in close proximity and their interactions with each other may be associative or antagonistic. The dependence of one microorganism upon another for extra-cellular products (e.g. amino acids & growth promoting substances) can be regarded as an associative activity / effect in rhizosphere. There is an increase in the exudation of amino acids, organic acids and monosaccharide by plant roots in the presence of microorganisms. Gibberellins and gibberellin- like substances are known to be produced by bacterial genera viz Azotobacter, Arthrobacter, Pseudomonas, and Agrobacterium which are commonly found in the rhizosphere. Microorganisms also influence root hair development, mucilage secretion and lateral root development. Fungi inhabiting the root surface facilitate the absorption of nutrient by the roots. The biochemical qualities of root exudates and the presence of antagonistic micro organisms plays an important role in encouraging or inhibiting the soil borne plant pathogens in the rhizosphere region. Several mutualistic, communalistic, competitive and antagonistic interactions exist in the rhizosphere. Antagonistic microorganisms in the rhizosphere play an important role in controlling some of the soil borne plant pathogens. They produce variety of biologically active compounds such as plant growth substances, cyanides, antibiotics and iron chelating substances called "Siderophores"

The rhizosphere is rich in microbial diversity and harbours a variety of micro flora that influences plant growth development. Plant promoting rhizosphere microbes are free living microorganism that beneficially thrive the plant roots and even invade root tissue and capable of promoting plant growth .The major contributions of plant growth include increasing germination rate percentages, root growth, leaf surface area water and minerals uptake, tolerance or resistance to stresses. In the rhizosphere, important and intensive interactions occur among the plant, soil microorganisms and soil micro fauna. The major contributions of plant growth include increasing germination rate percentages, root growth, leaf surface area water minerals uptake, tolerance are resistance to stresses. Plant roots have strong effects on the physical environment in the rhizosphere. Shifts in the physical environment, which are influenced by root manipulation, have significant impacts on the availability and form of substrates used for microbial metabolism. In turn, this strongly affects the prevalence of different microbial functions, as well as their total biomass within the rhizosphere. This interplay goes both ways, as manipulation by microbes also plays a strong role in the ways that plant roots interact with, benefit from, and influence the soil environment around them.

Similarly, the leaf surface has long been considered a hostile environment for bacterial colonists. The leaf surface is exposed to rapidly fluctuating temperature and relative humidity, as well as repeated alternation between presence and absence of free moisture due to rain and dew. The leaf also provides limited nutrient resources to bacterial colonists. While other habitats probably offer more extreme conditions of desiccation or temperature, etc., they may not be subject to such rapid and extreme fluctuations in these several physical conditions. The phylloplane bacteria are utilised against plant pathogenic fungi as many of them possess antagonistic activities.

Similarly, plant constitutes diverse niches of endophytic microorganisms inhabiting in tissues without damaging the host. Endophytes have ability to enter the host system without stimulating pathogen indeed vulnerability responses but triggering host defense. They could provide barrier against invading pathogens directly or through production or bioactive compounds. The endophytes can be isolated from surface- disinfected plant tissues. It can be detected within the tissues of apparently healthy plants (Schulz and Boyle, 2006) and enter plant tissue primarily through the root zone; specifically, the bacteria enter tissues via germinating radicals. Endophytes inside a plant may either become localized at the point of entry or spread throughout the plant (Sharma et al., 2005). These microorganisms can reside within cells, in the intercellular spaces, or in the vascular system. Its indigenous population varies on plant source, plant age, tissue type, time of sampling, and environment. Generally, bacterial population found larger in roots and less in stems leaves and fruits. The levels of colonization by endophytes tend to be far less than the levels of colonization by pathogenic bacteria (Denise et al., 2002).

The main reason for the interest in endophytes is the realization that, these bacteria can develop more stable relationship between plant and endophytes than rhizospheric or epiphytic bacteria and plants. Therefore, endophytes with the plants beneficial traits are potentially excellent plant growth promoters and/or biological control agents for sustainable crop production (Di Fiore and Del Gallo, 1995). It is assumed that bacterial endophytes use the mechanisms as that of biological control agent and plant growth promoters (Berg and Hallmann, 2006). Widely recognized mechanisms of bio control mediated by plants growth-promoting microbes are antibiosis (Thomashow and Weller, 1995; Haas and Defago, 2005; Lugtenberg and Kamilova, 2009). For example, Bacillus cereus strain UW85 is known to produce both Zwittermycin (Silo-suh et al., 1994) and Kanosamine (Milner et al., 1996) helps to suppress diverse microbial domination; competition for niches and nutrients (Kamilo et al., 2006). Anderson et al. (1988) revealed that production of a particular plant glycoprotein called agglutinin is rhizosphere and a corresponding reduction in Fusarium wilt suppression in cucumber (Tari and Anderson 1988) Some can induce systemic resistance (Van Peer et al., 1991; Kloepper et al., 2004; Van Loon, 1998) and others show predation and parasitism (Ordentlich et al., 1998; Harman et al., 2004).

In the recent past, various methods are development mulberry disease, this includes mainly biological methods of the chemical control measures are given least preference due to adverse effect on soil, plants and also to human beings. The chemicals generally used for control of diseases accumulate in water bodies, pollute the air, and in some cases have other dramatic environmental effect on human beings. Various studies show that exploiting endophytes, phylloplane and rhizosphere have multifarious benefits since they provide protection as well as production increase in plant however, in mulberry the endophytes and exophytes are not exploited for control of diseases therefore, present study has been proposed to explore the possibility of exo and endophytic bacteria of mulberry for their antagonistic and growth promotion activities with following objectives.

Objectives

1. Isolation and identification of endophytic, phylloplane and rhizosphere bacteria of mulberry.
2. To study the antagonistic effects of endophytic, phyloplane and rhizosphere bacteria against certain soil borne pathogens of mulberry
3. To study the influence of endophytic, phylloplane, rhizosphere bacteria for seed germination and seedling growth of mulberry.

REVIEW OF LITERATURE

Inferior leaf quality due to diseases is a major concern in production of quality silk in sericulture. Since the production of mulberry leaves per unit area increased over the past few decades, producers became more and more dependent on agrochemical as a relatively reliable method for crop protection. However, use of chemicals end up in several harmful effects such as, development of pathogen strains resistant to plant protection chemical and environmental pollution. Further, most of the plant protection chemicals are not affordable for farmers due to higher cost. Several incidents of hazardous effect of pesticides in recent years created environmental awareness and hence there exists a negative public perception regarding the safety of chemicals used for plant protection measures. Search for alternate methods directed to quite a lot of environment friendly methods used in many crops. The use of biological control with bacterial bio control agents is one important method. However the introducing bio control agents should be related to the crop so that these bio control agents can establish easily. The rhizosphere, phylloplane and endophytic microbes are such organisms which can establish easily in that particular crop for its protection from invading pathogens. In the recent past, many studies were conducted to in this line.

Rhizobacteria

Rhizobacteria are ideal bio control agents since they inhabit the rhizosphere that provides the defence for root against pathogen. Pathogen encounters antagonism from rhizobacteria before and during infection on roots. Pseudomonas and Bacillus species are most diverse and versatile group of micro flora of almost all the horticulture and forestry crops and have potential to synthesize different metabolites with diverse biological activities. Several bacteria belonging to genera Bacillus and Pseudomonas have been intensively investigated as bio control agents due to their ability to produce antimicrobial metabolites and ecological fitness of soil (Plebon et al., 1997; Nielsen et al., 2000). It has been shown that the rhizobacteria exerts several mechanisms for suppression of root pathogens, and such mechanisms differed among the strains (Anjaiah, 2004). Two major mechanisms have been proposed to explain the suppressive and antagonistic effects of rhizobacteria (i) the pathogen is inhibited by competition for iron by excreting molecules called siderophores and (ii) Pseudomonas inhibit phytopathogens by producing secondary metabolites with antibiotic activity. Additional factors such as aggressive root colonization play an important role in rhizosphere competence and associated bio-control ability of a fluorescent pseudomonas.

Tripathi and Johri (2002) studied the biocontrol potential rhizobacteria of pea and wheat against maize sheath blight caused by Rhizoctonia solan i and found some isolates possess multiple disease control potential. Ramesh Kumar et al, (2004) reported fluorescent pseudomonas isolated from the rhizosphere of rice and sugarcane having strong antifungal activity against Fusarium oxysporum and R. bataticola, mainly through production of antifungal metabolites. Tiwari and Thrimurthy (2007) reported isolates of P. fluorescence from the rhizosphere of different source have ability to increase the shoot length and root length of plants. In vitro evaluation of the P. fluorescence isolates also confirmed their antagonistic ability against Pyricularia grisea and Rhizoctonia solani in dual culture tests.

Some rhizobacteria synthesize antifungal antibiotics, e.g. P. fluorescens produces 2,4-diacetyl phloroglucinol which inhibits growth of phytopathogenic fungi (Nowak Thompson et al.,1994). Certain PGPR degrade fusaric acid produced by Fusarium sp. causative agent of wilt and thus prevents the pathogenesis (Toyada and Utsumi, 1991). Some PGPR can also produce enzymes that can lyse fungal cells. For example, Pseudomonas stutzeri produces extracellular chitinase and laminarinase which lyses the mycelia of Fusarium solani (Mauch et al., 1988). Rhizobacteria has been suggested as potential biological control agent due to its ability to colonize rhizosphere and as black root-rot of pea (Papavizas and Ayers, 1974), root-rot of wheat (Garagulia et al., 1974), damping-off of sugar beet (Fenton et al., 1992; Shanahan et al., 1992; Kumar et al., 2002).Fluorescent pseudomonas exhibit strong antifungal activity against P.oryzae and R.solani mainly through the production of antifungal metabolites (Reddy and Rao, 2009), damping-off of cotton (Pal et al, 2000) etc.

On the other hand, Bacillus is the most abundant genus in the rhizosphere, and the PGPR activity of some of these strains has been known for many years, resulting in a broad knowledge of the mechanisms involved (Probanza et al., 2002 and Gutierrez et al., 2003). There are a number of metabolites that are released by these strains (Charest et al., 2005), which strongly affect the environment by increasing nutrient availability of the plants (Barriuso and Solano, 2008). Naturally present in the immediate vicinity of plant roots, B. subtilis is able to maintain stable contact with higher plants and promote their growth. In a micro propagated plant system, bacterial inoculation at the beginning of the acclimatization phase can be observed from the perspective of the establishment of the soil micro biota rhizosphere. Bacillus is also found to have potential to increase the yield, growth and nutrition of raspberry plant under organic growing conditions (Orhan et al., 2006). Bacillus megaterium is very consistent in improving different root parameters (rooting performance, root length and dry matter content of root) in mint (Kaymak et al., 2008). The PSB Bacillus megaterium var. phosphaticum and potassium solubilising bacteria, Bacillus mucilaginosus when inoculated in nutrient limited soil showed that rock materials (P and K rocks) and both bacterial strains consistently increased mineral availability, uptake and plant growth of pepper and cucumber, suggesting its potential use as fertilizer (Han et al., 2006 and Supanjani et al., 2006). The Bacillus pumilus 8N-4 can be used as a bio-inoculants for biofertilizer production to increase the crop yield of wheat variety Orkhon in Mongolia (Hafeez et al., 2006).

Phylloplane bacteria

Phyllosphere, or leaf surface as a microbial habitat, has gained considerable popularity as a subject of scientific enquiry. In the past decades, the number of studies has been done on phylloplane microbes. The phyllosphere refers to the habitat provided by the aboveground parts of plants and on a global scale supports a large and complex microbial community. Microbial interactions in the phyllosphere can affect the fitness in natural communities and the productivity of agricultural crops. The structure of phyllospheric communities reflects immigration, survival and growth of microbial colonists, which is influenced by numerous environmental factors in addition to leaf physico-chemical properties.

The microbial communities of leaves are diverse and include many different genera of bacteria, filamentous fungi, yeasts, algae, and less frequently, protozoa and nematodes which are important for plant health and growth (Whipps et al., 2008; Vorholt 2012). Leaves constitute a very large microbial habitat estimated about 6.4 x 108 km2 terrestrial leaf surface area that might be colonized by microbes (Rastogi et al., 2013; Bulgarelli et al., 2013). In most instances, the epiphyte population is dominated by bacteria which can be found in numbers ranging from 105 to 107 cells per gram of plant material (Yadav et al., 2010). Phyllosphere bacterial communities have potential to influence plant biogeography and ecosystem function through their influence on the fitness and function of their hosts.

Internal and external foliar micro biota has many other functions, including indirect protection against pathogens, through the interaction of commensally bacteria with the foliar plant pathogen (Arnold et al., 2003). These microbial communities may be produce antibiotic compounds and showed competition for resources (Berg 2009; Braun et al., 2010). Braun et al., (2010) showed that the toxin-producing isolate P. syringae pv. syringae 22d inhibited growth of the near isogonics foliar pathogen P.syringae pv. glycinea in vitro, but was not responsible for the antagonistic effects observed in plant. However, for other strains it was shown that antibiosis is involved in plant protection. For instance, a Tn 5 mutant of Pantoea agglomerans Eh 252 deficient in antibiotic production was not as effective as the wild type strain to control fire blight in the field (Stockwell et al., 2002). Antimicrobial compounds are produced by microorganisms to remain competitive in their environment by diminishing growth of other bacteria.

Research has been conducted on the plant growth-promoting abilities of various rhizobacteria. They differ from bio control strains in that they do not necessarily inhibit pathogens but increase plant growth through the improved cycling of nutrients and minerals such as nitrogen, phosphate and other nutrients. Endophytes also promote plant growth by a number of similar mechanisms. These include phosphate solubilisation activity (Verma et al., 2001; Wakelin et al., 2004), indole acetic acid production (Lee et al., 2004) and the production of a siderophore (Costa & Loper, 1994). Endophytic organisms can also supply essential vitamins to plants (Pirttila et al., 2004). Moreover, a number of other beneficial effects on plant growth have been attributed to endophytes and include osmotic adjustment, stomatal regulation, modification of root morphology, enhanced uptake of minerals and alteration of nitrogen accumulation and metabolism (Compant et al., 2005a, b). The recent areas where these plant growth-promoting bacterial endophytes are being used are in the developing areas of forest regeneration and phytoremediation of contaminated soils.

Endophytes

Endophytic bacteria are able to lessen or prevent the deleterious effects of certain pathogenic organisms. The beneficial effects of bacterial endophytes on their host plant appear to occur through similar mechanisms as described for rhizosphere-associated bacteria. These mechanisms have been reviewed in great detail by Kloepper et al. (1999) or, more recently, by Gray and Smith (2005) and Compant et al. (2005). Diseases of fungal, bacterial, viral origin and in some instances even damage caused by insects and nematodes can be reduced following prior inoculation with endophytes (Kerry, 2000; Sturz et al., 2000; Ping and Boland, 2004; Berg and Hallmann, 2006).

It is believed that certain endophyte bacteria trigger a phenomenon known as induced systemic resistance (ISR), which is phenotypically similar to systemic-acquired resistance (SAR). SAR develops when plants successfully activate their defence mechanism in response to primary infection by a pathogen, notably when the latter induces a hypersensitive reaction through which it becomes limited in a local necrotic lesion of brown desiccated tissue (van Loon et al., 1998). ISR is effective against different types of pathogens but differs from SAR in that the inducing bacterium does not cause visible symptoms on the host plant (van Loon et al., 1998). Bacterial endophytes and their role in ISR have been reviewed recently by Kloepper and Ryu (2006).

The review of literature shows that many studies were carried on the use of endo and exophytic microorganisms in many agricultural systems. The present study aims to explore potentiality of endo and exophytic bacteria for control of certain soil borne pathogens of mulberry.

MATERIALS AND METHODS

Collection of sample for isolation of rhizobacteria, phylloplane & endophytic bacteria

Samples were collected from the experimental mulberry garden of Central Sericultural Research &Training Institute, Mysore. Soil samples were collected by digging to a depth of 1-2 feet near the roots of mulberry plants grown in the experimental plots. The soil adhered to root was separated and transferred to separate clean polythene bags and carried to laboratory. The soil samples were then spread and air dried. The dried soil samples were powdered and sieved to get the fine powder of the soil and stored in separate polythene bags and labeled. Healthy mulberry leaves were collected in sterilized polythene bags from the mulberry gardens of Central Sericultural Research &Training Institute, Mysore. Mulberry plants were randomly selected from a healthy plantation and the plants were uprooted manually and washed under running tap water. Bits of 3cm length were excised using flame sterilized secateurs from 3cm above and below the soil line and from the main branch and all the samples were washed and then blotted using blotting paper. Leaves and fruits were collected in sterilised polythene bags.

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Fig. 1: Sources of isolation of bacteria

Preparation of media

The nutrient agar medium was used for isolation of rhizobacteria, phylloplane & endophytic bacteria. The medium and plates were sterilized by autoclaving at 121°C for 20min and cooled to 50°C. The sterile plates were exposed to UV light for 15 min. Thereafter 15ml media was poured in 9 cm diameter Petri plates, the plates were then exposed to UV light for 15 minutes and ensured the sterility.

Surface sterilization of the samples

For isolation of endophytic bacteria, the plant samples were washed with 0.2% HgCl, wiped the sample with sterile cotton, then washed the samples with 70% ethanol for 1 minute and then rinsed with sterile distilled water. The samples were then washed with 3% Sodium hypochlorite solution for 5 minutes and again rinsed with sterile distilled water. The samples were then washed in 70% ethanol for 1 minute and again rinsed with sterile water. This final water wash was collected for sterility test.

Isolation of rhizobacteria, phylloplane & endophyte bacteria

For the isolation of rhizobacteria, pour plate method were used. Soil samples were sprinkled on nutrient agar media and incubated for 24hours and the colonies obtained. For the isolation of phylloplane bacteria the leaf was imprinted on the nutrient agar media and incubated for 24hours and the colonies obtained. Similarly, for endophytic bacteria the outer layer of the cortex is peeled using sterilized sharp knife. Small pieces (0.2 cm) were taken from the inner cortical region of the plant cuttings and for leaf was cut in small pieces and for fruit small portion were cut and also juice of fruit were taken. All small pieces were aseptically placed on sterile nutrient agar media and juice was streaked on media. The plates were incubated in BOD incubator at 27°C for 72 hours in inverted position. To ensure the sterility of the samples, the final wash collected were inoculated on the nutrient agar medium separately by streak method using inoculation needle (Fig. 1).

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Fig. 2: Isolates of phylloplane, rhizospheric & endophytic bacteria from different samples

Preparation of pure culture

The individual colonies were sub cultured in nutrient media and again transferred to the nutrient agar slants. These cultures were considered as mother culture and used for further experiments (Fig. 2).

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Fig. 3: Pure culture of isolates of endophytic & rhizosperic bacteria of mulberry

Study of antagonistic effects of bacterial isolates against various root rot pathogens

The antagonistic effects of isolated rhizobacteria, phylloplane and endhophyte bacteria were tested against Rhizactonia batitaticola, Botryodiplodia theobramae, Fusarium oxysporum and Fusarium solani by streak method. The bacteria were purified and sub cultured. Three days old bacterial and seven days old fungi were used for antagonistic study. The bacteria were streaked on the PDA medium which was inoculated with 4mm diameter disk of fungal pathogens separately for each bacterium and pathogen. The plates without bacterial inoculation were served as control. Three replications were kept against each treatment and control. The plates were incubated at 29°C for 6-8 days.

The data on the antagonistic effect of the bacterial strains against R. batitaticola, B.theobramae, F. oxysporum and F.solani were taken. The plate without bacterial streak was kept as control. The radial growth of fungus towards the bacterial streak and radial growth away from the bacterial streak was recorded for evaluating the antagonistic effect of the rhizobacteria, the results were calculated considering the growth of fungi towards and away from the bacteria as follows:

Inhibition % = R-r/R x 100

Where,

R= growth away from bacterial streak

r= growth towards bacterial streak.

Gram staining

Rhizobacteria, phylloplane and endophytic bacteria which showed the antagonism against Fusarium solani, Botrydiplodia theobromae, Fusarium oxysporum and Rhizoctonia bataticola were Gram’s stained by using staining kit (Himedia) for Gram staining, loopfull of inoculums was taken from each test tube containing bacterium and placed on separate clean glass slide and made thin smear by using another clean glass slide. Heat fixed the smears on slides and then the smear was flooded with crystal violet and allowed it for 2-3 minutes and washed these slides with running tap water .The smear was then flooded with Gram’s iodine and allowed it for 1-2 minutes. Thereafter, the slides were washed with slow running tap water. Then all the slides were dipped in 90% alcohol and left for 2-3 minutes. Then the slides were washed with water. Flooded the sides with safranin and allowed it for 2-3minutes. All the slides were washed with water and allowed to dry and observed all the slides under microscope.

Extraction of mulberry seeds

Since mulberry is a perennial plant, seeds have to be collected in its fruiting season. Well ripened mulberry fruits were collected from mulberry plants maintained in the central sericulture research and training institute, Mysore. Fruits were washed and squeezed to come out of the seeds from pulp under running tap water in a beaker. The squeezed fruits were filtered using sieve to remove the pulp. The process was repeated until the seeds and remaining fruit material was left behind. The seeds and debris were rinsed and soaked in water. The seeds were rinsed thoroughly with water. They were allowed to air dry on a filter paper and preserved for priming study.

Bio priming of mulberry seeds with rhizosphere, phylloplane and endophytes

The rhizosphere, phylloplane and endophytes which was showed the antagonism against R.batitaticola, B. theobramae, F. oxysporum and F. solani were cultured on nutrient agar medium. Two days old bacterial culture was used for bio priming. The bacterial solutions were made in sterile distilled water adjusting the optical density at 600 nm (0.1 OD) under a spectrophotometer so as to get approximately 1X106 cells/ml. 5 ml of bacterial solution was taken in a test tube and mulberry seeds were soaked in the bacterial solution for 24 hours separately for each bacteria. Seeds soaked in sterilized distilled water served as control. After incubation, the soaked seeds were separated from the bacterial. Solution and inoculated on the pot containing sterile soil. Three replications were kept against each treatment and control. The seeds were observed for their germination and counted the germinated seeds till 12th day.

Seed germination test

Germination was considered to have occurred when the seeds developed at least 2mm long radical. In order to evaluate the germination rate, the germinated seeds were counted for 4th, 5th, 6th and 7th day after sawing. The final germination percentage was calculated based on the total number of seeds germinated on 9th day. The length of the root and shoot of germinated seeds was measured in millimetres. The weights of the germinated seeds were measured in grams. The germination (%) and vigour index were calculated following international rules for seed testing (ISTA, 1985) as follows (Fig. 3).

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Fig. 4: Bio priming of mulberry seeds with rhizosphere, phylloplane and endophyte

Statistical analysis of the data

The data were subjected for analysis of variance (ANOVA) and the means were compared for significance difference.

RESULTS

Effect of phylloplane bacteria on radial growth and suppression of F. solani

Treatment with phylloplane bacteria the reduction growth fungal pathogen F.solani showed PB-1 (10.44mm) followed by PB-4 (12.11 mm) and PB-5 (12.11 mm). However in control the growth was higher (19.67 mm). There was significant difference in growth of F.solani with days after inoculation. The highest growth (16.06 mm) was found 4th day after inoculation and least (11.27 mm) in 8th day after inoculation. Regarding suppression of pathogen the highest suppression was found (47.24%) in case of PB-1 followed by PB-5(38.87 %) and PB-4 (37.99 %). However the growth was least in the case of PB-8 (28.43 %) and PB-10 (29.54). There was no significant interaction between bacteria and days after inoculation for growth and suppression of the F.solani (Table 1).

Table 1: Effect of phylloplane bacteria of mulberry on radial growth and suppression of Fusarium solani

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