Phytostimulating Potential of Endophytic Bacteria from Ethnomedicinal Plants of North-East Indian Himalayan Region

North-East Indian Himalayan Region has a humid subtropical climate having diverse ecosystems. The majority of the population of the region depends on agriculture for sustainable livelihood. However, it can produce only 1.5% of the country’s food grains, thereby importing from other parts of the country for consumption. To feed the increase in the population of the region, there is an urgent need to augment the agricultural and allied products to sustain the population and uplift the economic conditions. Plant beneficial endophytes isolated from ethnomedicinal plants of North-East India play an important role as a plant growth promoter by the production of phytohormones, solubilization and mobilization of mineral nutrients. It also indirectly promotes growth by protecting the plants from diseases through the production of antibiotics, enzymes and volatile compounds. The bacteria also have the potential to induce systemic resistance against various abiotic stresses. Since the region has various agro-climatic conditions, the plants are continuously affected by abiotic stress particularly, acidity, drought and waterlogging, there is a need to explore the indigenous endophytes that can mitigate the stress and enhance the sustainable development of agricultural products.


INTRODUCTION
North-East Indian Himalayan Region (NEIHR), with a landmass of 262,230 km 2 , contributing 8% of the total geographical area of the country and comprises of eight states is located between 87°32'E to 97°52' E longitude and 21°34'N to 29°50'N latitude. The region can be geographically divided into the Eastern Himalayan, the Patkai Naga Hills and, the Brahmaputra and the Barak valley plains. The region has a humid subtropical climate endowing with diverse ecosystems such as evergreen, tropical, temperate and alpine vegetation. The region is regarded as one of the last remaining rainforests of the Indian subcontinent's supporting different flora and fauna and several agricultural crop species.
About 70% of the population of the region depends on agriculture for sustainable livelihood. 1 However, the region contribute only 1.5% of the country's food grains production and is one of the major importers of food grain from other parts of the country for consumption. 2 The population of the region (excluding Assam) is projected to increase by 24% from 14.5 million in 2011 to 18.09 million in 2036. 3 There is an urgent need to augment the agricultural and allied products to sustain the population and uplift the economic conditions. The region, endowed with a range of diversity of climate, soil and vegetation, has the potential of achieving a much higher level of sustainable development if resources are properly and effectively managed.
Investigation on the bioactivity of medicinal plants is still ongoing, since, it represents a promising resource for production of unique secondary compounds for application in pharmaceutical, agricultural and other industries. Despite the importance of ethnomedicinal plants for human health for treating different diseases, a study on plant growth-promoting activity of their associated endophytes is scanty. The plant endosphere represents a multifaceted microenvironment hosting indigenous microbes that contribute to the growth of plants. 4 Plant beneficial endophytes reside inside the plant tissue without causing a negative effect to the host plants or the environment. They play an important role as plant growth regulators, induction of defence system, and protect the host from various biotic and abiotic stresses. 5 They augment the growth of plants by enhancing nutrient availability through nitrogen fixation, production of phytohormones, 1-aminocyclopropane-1carboxylate (ACC) deaminase and siderophore, and also solubilization and mobilization of mineral nutrients. [6][7][8][9][10] Endophytic bacteria can also indirectly promote the growth of host plants by the production of antimicrobial compounds, cell wall degrading enzymes, low molecular weight volatile compounds and induction of systemic resistance. [11][12][13][14][15] This review aims to elaborate on the plant growth-promoting (PGP) potential of the endophytic bacteria associated with the ethnomedicinal plants of NEIHR. Since the hilly region often faces water scarcity and soils are highly acidic and frequent occurrences of waterlogging in the plain valleys, the review also focuses on exploring important endophytes for application in diverse agro-climatic conditions of the NEIHR towards sustainable development of the agricultural sector.

Invasion, colonization and translocation of endophytic bacteria
Since plants and bacteria have co-evolved for millions of years, some bacteria possessed a necessary quality to invade, colonize and translocate to different tissue of plants. 16 Some bacteria from the plant rhizosphere invade and become endophytes as the latter may have an advantage over rhizospheric bacteria in accessing more nutrients without competing with other microorganisms and, better protected from various biotic and abiotic stresses. Endophytic bacteria may in turn exert direct or indirect beneficial effects to host plants. 7,17 Endophytic bacteria are generally believed to be originated from the rhizosphere and phylloplane bacterial communities, while some may be vertically transferred from endophyte-infected seeds or planting materials. 18 The entry of endophytic bacteria into the host plant particularly in roots occurs in several steps. The first step for the entry of bacteria into host tissue is the chemotaxis-induced motility that recruits bacterial to colonize plant roots. [19][20][21] The exudates rich in organic substrate released from roots serve as chemo-attractants that facilitate the communication between roots and microbes, ultimately helping in recruiting bacterial communities from the rhizosphere to start colonization of the root. 22,23 Bacteria defective in flagellar motility hinder the colonization of plant roots. For example, Pseudomonas mutant strains defective in flagella-driven chemotaxis (cheA or pilA) could not successfully colonize the root. 24, 25 Kost et al., 26 reported the role of root exudates such as oxalate in root colonization by Burkholderia strains. The plant beneficial strains were able to metabolize oxalate as a carbon source but pathogenic fails to degrade the oxalate. Similarly, the mutant strain Burkholderia phytofirmans defect in utilizing oxalate fails to colonize the maize root.
The next step is the entry of bacteria into the root through several mechanisms such as passive penetration at the root tip, primary and lateral root cracks, pathogen entry sites, or tissue wounds occurring as a result of plant growth. While others gain entry through active penetration by producing cell wall degrading enzymes, such as cellulase, glucanase, lipase, etc. 17,27-29 Hallmann et al., 17 reported that Enterobacter asburiae enter the host plant by hydrolyzing cellulose of plant cell wall. Phyllospheric bacteria also enter plant tissue through stomata, particularly on leaves and, also through wounds and cracks generated by wind, insect and pathogen attacks. 31,32 Successful colonization of endophytes into host tissue also occurs when bacteria can reduce or evade plant defence machinery since; plant defence responses may restrict endophytic colonization. For example, Bacillus subtilis colonized Arabidopsis thaliana by producing subtilomycin that binds to flagellin, thereby reducing the induction of plant defence mechanisms. Bacillus subtilis also colonize the host plant Amorphophallus konjac by producing subtilomycin. 33 After entering the root tissue, some endophytes can translocate to different tissue of the host plants such as stem, flowers, fruits and seeds. Translocation occurs through the vascular system especially xylem of the host plants facilitated by flagella movement. 29,[34][35][36] The population of endophytes in the root and stem starts to decline as the host plant continues to grow and reach a mature stage. The steady advance from roots towards stems and leaves, thereby reducing the population in roots and stems, was observed during colonization by Bacillus megaterium in maize and rice plant. 37 Mcinroy and Kloepper 38 also observed the decline in the bacterial population in the stem tissue of sweet corn after 10 weeks. The authors concluded that the decline in the population of bacteria, from the base of the plant to the top might be due to travelling of bacteria along with the water/nutrient in the vascular system during plant development. Devi et al. 40 studied the multiplication and distribution of endophytic bacteria with respect to the inoculation time of host plant grown in a greenhouse. They injected the bacteria into stem tissue and observed the increase in population and distribution at different plant tissue after 3 rd and 5 th days of incubation (DAI). The authors reported that the bacterial population significantly increase between 3 rd and 5 th DAI. Bacteria were not detected in leaves till 3 rd DAI; however, after 5 th DAI, it was recovered from the leaves of host plants.

Antifungal activities of endophytic bacteria isolated from ethnomedicinal plants of NEIHR
Numerous studies on the bioactivity of medicinal plants are still underway since they constitute a rich source for production of compounds having important ecological functions, protecting from pests, diseases and other environmental stresses. Recently endophytic bacteria isolated from medicinal plants received significant importance in the hunt for novel bioactive compounds having important ecological functions. Endophytic bacteria from ethnomedicinal plants have also demonstrated a broad range of bioactivity against many important pathogenic microorganisms. Antifungal Carnobacterium gallinarum and C. maltaromaticum isolated from root and leaves of Alstonia scholaris, a native of Indonesia, inhibit the growth of Fusarium sp. and Colletotrichum sp. The two strains promoted the growth of chilli plant and protected from Colletotrichum fruit rot and Fusarium wilt disease. 48 Investigation of endophytic bacteria Pseudomonas aeruginosa isolated from Curcuma longa L. rhizome collected from Karnataka, India displays good antagonistic effect against Pythium aphanidermatum and Rhizoctonia solani. The strain also produced cell wall degrading enzymes such as protease and cellulase. The strain reduced the leaf blight and rhizome rot disease of Curcuma longa plant. 49 Ramanuj and Shelat 50 also observed inhibition of Alternaria alternate and Aspergillus niger growth by chitinase producing Beijerinckia fluminensis, an endophyte associated with Curcuma longa.
Out Endophytic bacteria belonging to Streptomyces sp., Nocardia sp. and Streptosporangium sp. associated with Azadirachta indica collected from other parts of India have been reported to have antimicrobial effect against fungal pathogens. 58,59 Endophytic actinobacterial isolates from Emblica officinalis also exhibited antifungal, antibacterial and PGP activities. 60 Actinobacteria isolated from Rauvolfia serpentine have also been reported to exhibit antifungal against plant fungal pathogens and PGP activities. Bioactive microbial strains were also found to promote growth and reduce the mortality caused by pathogens in chickpea plants. 61

PGP activities of endophytic bacteria isolated from ethnomedicinal plants of NEIHR
To feed the increasing population in NEIHR in the future, there is a need to augment the production of agricultural and allied sectors. The production can be enhanced with the use of synthetic chemicals and fertilizers which in  Table 2.
Passari et al. 53 investigate the PGP potential of the 52 endophytic isolates associated with Clerodendrum colebrookianum Walp. that demonstrated antimicrobial activities. Of these, 44 isolates were found to be promising for solubilization of tricalcium P in the range of 8.2 to 92 mg/100 mL. Forty-eight isolates could produce IAA ranging from 14.2 to 80.6 µg/mL. Fifty-one isolates were found to be positive for the production of ammonia ranging from 12.8 to 68.3 mg/100 mL. Siderophore production was detected with 41 isolates. Of 10 isolates belonging to 3 Bacillus species, 2 Staphylococcus species, Paenibacillus sp., Bacillus thuringiensis, Lysinibacillus sphaericus, Pseudomonas sp., and Pseudomonas stutzeri showing multiple PGP traits were screened for their ability to colonize on the root surface. All the bioactive strains were able to excessively colonize on the root surface of a tomato plant. Three beneficial isolates Bacillus sp. BPSAC3, Bacillus sp. BPSAC6 and Paenibacillus sp. were chosen for studying plant growth promotion under greenhouse conditions. The treatment of seedlings with the bioinoculant significantly enhances the growth of tomato plants. Among the 3 bacteria, strain Bacillus sp. BPSAC6 which displays better PGP traits and antifungal properties also demonstrated better growth promotion in tomato plants.
Among the 22 bioactive endophytic actinobacteria isolated from 7 plants of Mizoram i.e., Eupatorium odoratum, Musa superba Roxb., Mirabilis jalapa, Centella asiatica, Curcuma longa, Clerodendrum colebrooklanum and Alstonia scholaris that showed antagonist against fungi, 14 were able to solubilize inorganic P varying from 3.2 to 32.6 mg/100 mL. Twenty strains could produce IAA in the range of 10 to 32 μg/mL. The hydroxamate and catechol-type siderophores were produced by 16 and 5 isolates ranging from 5.2 to 36.4 and 3.2 to 5.4 μg/mL. All the bioactive strains were positive for the production of ammonia ranging from 5.2 to 54 mg/mL. The majority of the PGP strains belong to Streptomyces species. Inoculation of chilli seeds with two promising strains Streptomyces sp. and Leifsonia xyli showing good antagonistic and PGP traits significantly enhanced the growth of seedlings under greenhouse conditions. Treatment of chilli plants with a mixture of two promising strains showed further enhancement in growth as compared to inoculation with the individual strain. 47 Patel et al. 63 documented that Bacillus sp., Enterobacter sp., Klebsiella sp. and Microbacterium sp. associated with Musa sp. produced IAA and siderophore, and solubilize P. The bioactive strains were able to promote the growth of banana plants grown in the greenhouse. Endophytic bacteria Pseudomonas aeruginosa associated with Curcuma longa rhizome significantly promote the growth and rhizome yield of host plant under greenhouse condition. 49 Carnobacterium gallinarum and C. maltaromaticum isolated from root and leaves of Alstonia scholaris exhibit antifungal and plant growth activities such as IAA production and solubilization of P. Growth promotion of chilli plants by bioactive strains was also reported by Ulfa et al. 48 Nongkhlaw and Joshi 41 compared the antimicrobial and PGP activities of endophytic and epiphytic bacteria collected from 10 medicinal plants in Meghalaya showed positive results for siderophore production ranging from 56 to 87.95%. P solubilization was detected in 11 isolates which ranges from 155.19 to 361.67 µg/mL and ACC deaminase was shown in 9 isolates. Treatment of rice seedlings with 12 promising actinobacterial strains stimulated the growth of plants under greenhouse conditions. The majority of the PGP isolates belong to Streptomyces species.
Endophytic strains Streptomyces thermocarboxydus, Streptomyces sp. BPSEAC8, Streptomyces sp. BPSEAC14, Tsukamurella tyrosinosolvens, Streptomyces sp. BPSEAC33 and Streptomyces violascens associated with endemic medicinal plants Costus speciosus, Mikania micranth, Ageratum conizoides L. and Scopariadulcis of Mizoram were able to solubilize P and, produce IAA and ammonia. 52 Similarly, 5 strains isolated from Garcinia lancifolia Roxb. could produce IAA and ammonia, and solubilize tricalcium P. 57 Devi et al. 40 isolated Serratia marcescens from Achyranthes aspera L. collected from the different habitats of Manipur for elucidating the potential of the strain for various plant growth parameters. It could produce IAA in the range of 0.8 to 133.2 µg/mL and about 75% units of siderophore. The beneficial strain was able to significantly solubilize P up to 259 µg/mL, able to produce ammonia and fix atmospheric N. Nitrogenase activity was found to be 2.523 nmol ethylene/µg/protein/hour. Priming of Serratia marcescens by injecting into the stem of Achyranthes aspera L. radically increases shoot length, fresh shoot weight, dry shoot weight, fresh root weight and dry root weight over control under pot trial experiment. 40 Similarly, non-pathogenic strain Pseudomonas aeruginosa associated with leaves and stems of the same medicinal plant produced 114.79 µg/mL of IAA and 72% unit of siderophore. It could also solubilize 383 µg/ mL of tricalcium P. Strain could fix N and the nitrogenase activity was found to be 1.8617 nmol ethylene/µg/protein/h. The authors confirmed the N fixing ability by Pseudomonas aeruginosa due to the presence of nifH gene. Pseudomonas aeruginosa could successfully colonize the internal tissue of Achyranthes aspera L seedlings. Priming of Pseudomonas aeruginosa by injecting into the stem of Achyranthes aspera L. also showed similar growth promotion as shown by Serratia marcescens. 55 Bacteria belonging to genus Acinetobacter, Citrobacter, Enterobacter, Pseudomonas and Serratia isolated from tissue, rhizoplane and rhizosphere of Achyranthes aspera from Rajasthan, India have been reported to solubilized P and produced IAA. The strains also improve the growth of the pearl millet plants under pot condition. 65 Endophytic bacteria Ochrobactrum intermedium isolated from rhizomes of Acorus calamus, a medicinal plant used by the ethnic group of Manipur that demonstrated good antagonist activity against phytopathogens was assayed for PGP potential in rice plants. The antagonist strain could produce 29.61 and 112.5 μg/mL of IAA and ammonia, respectively. The strain also produced a significant amount of siderophore (72%) and solubilized inorganic P up to 102 μg/mL. Priming of rice seeds with the bioactive strain significantly enhances seed germination and promotes the growth of rice seedlings under  54 Similarly, endophyte Enterobacter cloacae from the rhizome of Acorus calamus collected from Tamil Nadu, India was found to be positive for IAA production, P solubilization and siderophore production. Bioactive strain enhances germination and growth of the host plant. 66 IAA producing Streptomyces sp. isolated from the medicinal plant of Meghalaya has also been reported to enhance germination and growth of bean seedlings. 67

Plant growth promotion by endophytes from ethnomedicinal plants of NEIHR under abiotic stress
Plants are continuously affected by abiotic and biotic stress throughout their life cycle. Abiotic stresses affect microbial functional diversity and soil physicochemical properties contributing to yield losses up to 50% posing a serious challenge for global food production. The most common abiotic stresses affecting plant growth and development are drought, temperature, salinity and heavy metal. However, beneficial bacteria enhance plant growth under abiotic stresses by synthesizing plant hormones that promote plant growth through enhanced root development, increased water uptake and nutrient absorption. It also synthesizes 1-aminocyclopropane-1carboxylate (ACC) deaminase and rhizobitoxine that reduce the level of stress ethylene, induce production of stress-related enzymes and induce systemic resistance in plants. 68,69 Plant beneficial endophytic bacteria having the prospective to endure abiotic stresses can be used as a promising agent for climate-smart farming.
Heavy metal contamination in soil due to increased industrialization, intensive agricultural practices and other human activities severely affects the growth and yield of crops. Heavy metal contamination is non-degradable and toxic even at low concentration. 69,70 Heavy metals hamper the normal functioning of plants by forming a link with the sulfhydryl of the functional groups, causing structural disintegration of important molecules particularly protein. They compete with the essential metals (trace elements) and obstruct the normal function of enzymes or pigments upsetting the photosynthesis, respiration, and enzymatic activities. It also augments the generation of ROS that disturbs the redox homeostasis of cells resulting in the activation of apoptosis. [71][72][73] Metal tolerant bacteria mitigate stress by producing siderophore, exopolysaccharide and antioxidants that help in the sequestering, bioremediation of metals, and mitigation of the ROS generated by stress conditions. Phytohormones produced by the bacteria, in turn, promote the growth of plants. 73,74 Aluminium (Al +3 ) toxicity causes about 25-80% yield losses in several crops. 75 Endophytic bacterial isolates associated with the ethnomedicinal plants of NEIHR have the potential to promote growth of crops under heavy metal stress. Strains Pseudomonas sp. PAK2, Pseudomonas sp. PAK7, and Pseudomonas sp. PAK8 isolated from Mussaenda roxburghii of Arunachal Pradesh were able to tolerate 600 µg/ mL of Al +3 and all the isolates except Pseudomonas sp. PAK1 showed tolerance to 600 µg/mL of Mn +2 tested. Heavy metal tolerant endophytic bacteria demonstrated PGP traits such as IAA and siderophore production, P solubilization, and ability to grow on N-free medium. 6 Soil salinity is also considered as one of the major abiotic stresses globally that adversely affect the growth and yield of higher plants. The majority of the arable land to the tune of 3.6 out of 5.2 billion hectares is affected by salinity problems due to human and natural activities. 70 Salt stress has a detrimental consequence on plant growth due to induced ion toxicity, mineral deficiency, development of ROS, alteration of osmotic potential and elevation of ethylene level. It also reduces nutrient and water uptake, photosynthesis, respiration, nodulation, and nitrogen fixation activities. 69 and Pseudomonas sp. PAK8 showed good growth at the level of 2.0-10% NaCl 6 .

FUTURE PERSPECTIVES AND CONCLUSION
To increase food production in order to feed the escalating increase in the human population without affecting the environment, there is an urgent need to search for the bacteria that can contain the plant diseases and enhance crops production. Interestingly, there is a great potential for discovering unique endophytic bacteria from plants growing in tropical to temperate rainforests, unusual endemic regions, extreme environments and, oceans. 77,78 The NEIHR falls under two mega-biodiversity hotspots having diverse ecosystems. Moreover, there are several under and unexplored ecosystems and plants endemic to a particular region. The need of the hour is to search for beneficial endophytic bacteria from unexplored or under-explored ecosystems of NEIHR, which can be used as effective phytostimulating agents for sustainable production of important crops of the region especially rice, maize and horticultural crops.
Due to heavy rainfall, soils in the mountainous and hilly region of North East India are moderate to high acidic. Heavy rainfall and soil low pH causes a deficiency in the plant essential elements like Na, K, Ca and Mg, on the other hand, increasing the solubility of heavy metals such as Al, Fe and Si resulting in toxicity to plants. Al 3+ ions bind with phosphoric acid and form an insoluble compound limiting the availability of P to plants. 79 Soil acidity stunted the growth and development of root and plant biomass due to metal toxicity, poor fertility and low water-holding capacity. 80,81 It also contributes to a decline in the neutrophilic beneficial bacteria and on the other hand, increases the population of pathogenic fungi. 82 However, acid-tolerant PGP bacteria can support the growth of plants by the production of siderophore and phytohormones, solubilization of P and K, and production of antibiotics that inhibit the growth of pathogenic fungi. 81, 83 There are reports of exhibiting higher antifungal and phytostimulating effect by acid-tolerant than neutrophilic bacterial isolates. 84,85 In NEIHR of India, nearly 90% of the population lives in hilly rural areas and depends on agriculture for sustainable livelihood. Even though the region receives heavy rainfall annually of about 2000 mm, the hilly region often faces water scarcity since most of the rainwater is lost as runoff leading to moisture stress during lean periods. 86,87 Moreover, drought stress has increased in intensity over the past decades and is expected to adversely affect plant growth in >50% of the world's arable lands by 2050. 81,88,89 Drought stress is regarded as one of the most severe stress agents that negatively influence the germination, growth and yield of various crops. Drought largely affects the plants during the reproduction or flowering stage and severely reduces the yield. 90,91 Drought stress decreases nutrient diffusion, the mass flow of water-soluble nutrients, and the photosynthetic activity while enhancing the biosynthesis of 'stress ethylene' which is known for inhibiting plant growth. It also results in the generation of ROS negatively affecting membrane function, protein conformation, and induce lipid peroxidation resulting in cell apoptosis. 69,73,92 Drought tolerant bacteria have been reported to improve plant growth by phytohormone production that enhances plant growth through enhanced root development, increased water and nutrient absorption. They also synthesize ACC deaminase that reduces the level of ethylene in plant roots. 69 Frequent recurrence of floods in the valley areas of North East India, especially Assam and Manipur has caused tremendous loss of crops, livestock's and economy. During the year 1953 to 2004, the region suffered a loss of 2 billion rupees due to flood/waterlogging. 93 In the past few decades, waterlogging has caused major damage to the world's crop production to almost two to third; losing a billion dollars. 94 It is predicted to further aggravate in the coming decades posing a big threat to sustainable crop production. 95 Waterlogging affects the growth of plants by depleting the availability of oxygen and light, disturbing the photosynthesis activity and energy production mechanism. It also leads to the accumulation of ethylene thereby stunting the growth of roots and inhibiting plant growth due to a decrease in the absorption of the nutrients. 95,96 Beneficial bacteria mitigate the stress and promote the growth of plants under waterlogged conditions by the production of IAA and ACC deaminase that stimulate the growth and degrade the stress ethylene. The ammonia and α-ketobutyrate produced by the degradation of ACC precursor of ethylene are used by the plants as N and carbohydrate sources under waterlogged conditions.
As climate change and agriculture are inseparably linked, the crop loss due to a rapidly changing climate is expected to significantly reduce the food reserve and spike up the food price in near future. The global food deficit will force the nations to go for more agricultural intensification, which will, in turn increase the carbon and water footprint. Hence, with a rapid change in the climate and environment owing to uncontrolled anthropogenic activities, there is an urgent need for exploring alternative means for increasing the production of food crops to feed the rising human population. In this context, the researcher needs to explore the indigenous bacteria (endophytic) suited to the local environment that can promote the growth of agri-horticultural crops. Further surveys and bioprospecting for beneficial endophytic bacteria that can mitigate the common abiotic stress especially acidity, drought and waterlogging of the region are the need of the hour for climateresilient agriculture.

ACKNOWLEDGMENTS
The author gratefully acknowledges the financial support from the DBT-Research Associateship Programme in Biotechnology and Life Sciences of Department of Biotechnology, Government of India and physical support from ICAR Research Complex for NEH Region, Manipur Centre.