ROLES AND BIOTECHNOLOGICAL APPLICATIONS OF BACTERIAL ENDOPHYTES
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There is a symbiotic relationship between the bacterial endophytes and plants as well as bacterial genes that are expressed with plant existence. These genes are necessary for various aims such as entering and colonizing plants, living in plant tissues and inducing plant growth, competing with pathogens and suppressing them or producing different substances that can be differently expressed by endophytes (Lodewyckx et al., 2002). Endophytic bacteria render important and rich models for studying the genetic expression of bacteria in a variety of natural niches or habitants that are much more different and richer from the culture medium under controlled laboratory conditions. However, there are few studies conducted on this issue (Lugtenberg et al., 2002).
The best example for the approach of biotechnological practice on plant production is the usage of Agrobacterium tumefaciens and Agrobacterium rhizogenes. These are soil bacteria and pathogenic for some plant varieties. They have genetic tools allowing colonization on plant. They have been used as vectors for transferring covetable genetic information to plant for about 40 years (Pacurar et al., 2011). Disarmed forms of bacteria can insert desired genetic information within a plant genetic construction. If genetic information is inserted on plant DNA, it can be transmitted to daughter cells. Therefore, genetically modified enriched plant cell provides us new genetic variations that can be used in plant breeding (Ziemienowicz, 2014).
Also, previous genomic studies are being made with various endophytic bacteria, including Azoarcus spp. (Battistoni et al., 2005), Klebsiella spp., Gluconacetobacter diazotrophicus, and Herbaspirillum sp., which could assist to reveal out the molecular interactions of endophytic bacteria with plants. Besides, in vivo expression technology that enables to search gene expression in various niches, including the rhizospheric area (Ramos-Gonzalez et al., 2005), can also be useful to study gene expression during life cycle of endophytic. Vermeiren et al. (1998), showed the expression of uifH gusA fusions of Azospirillum irakeuse and Pseudomonas stutzeri in rice roots. The expression of Azoarcus spp. nitrogenase genes inside the roots of field-grown Kallar grass was also demonstrated by in situ hybridization studies (Hurek et al., 1997). According to results, it seems that grass tissues assure a suitable environment for nitrogen-fixation gene expression; however, N fixation in plant appears to be carbon-limited (Christiansen-Weniger et al., 1992). In acetylene reduction assays, members of genera, Azospirillum, Herbaspirillum, Serratia, and Klebsiella that were inoculated with different grasses produced ethylene with only a carbon source presence (Egener et al., 1999; Gyaneshwar et al., 2002).
In recent years, production of PGPB or endophytes-based bio-pesti- cides, which are increasingly market share as plant protection products, is increasing in agricultural production. Because of being natural products, they are environmentally safe and have more advantage than chemical ones. According to targeted material, they have been improved and have used as fungicides, insecticide, herbicide together with various plant growth regulators (Yi et al., 2013). At this point genetic engineering has provided us a new approach to develop novel biological products by inserting the genes responsible for resistance to agricultural crops genome with using vector microorganism. Therefore, crops are modified by resistance protein-encoding genes, and resistance is acquired against biotic and abiotic stresses.
It seems like these bacteria which have integrative plasmids and colonize plant tissue epiphytically and endophytically are a good mediator for biological control. For example, Clavibacter xyli containing the Cry-toxins genes (cry 1 Ac) on integrative plasmid were used as biological control agent against com borers (Thomasino et al., 1995). Thus, Azospirillum spp., R. leguminosarum, P. capecia, and P.fluorescens, which are plant colonizing bacteria, were modified by cry-genes. Herbaspirillum seropedica, an endophytic bacterium that inhabits sugarcane tissue, was transformed by the gene crylAc7 stem from B. thuringiensis 234 to suppress growth of Eldaua saccharina larvae (Downing et al., 2000). Moreover, the Bt-toxin gene products can prevent and decrease both pest and insect vectors that are human disease-causing agents. Likewise, when a nitrogen-fixing cyanobacteria Anabaeno sp. PCC 7120 is transformed by multiple genes (cry44, cry’ll A, and p20), it becomes toxic against larvae of yellow fever mosquitoes (Wu et al., 1997).
On the other hand, one of the promising areas about endophytes studies includes their usage of in genetic studies for phytoremediation, such as degradation of organic pollutants from oil products and hazardous chemicals (Afzal et al., 2014). The endophytic bacteria associated with plant tissue may cleaned up soil environment contaminated with not only heavy metals but also complex chemical compounds. In a research regarding this issue, following genetical modification of endophytes with the genes encoding enzyme that can degrade organic pollutants, their ability of degradation was enhanced (Maksimov et al., 2015).
Another important example of the use of endophytic bacteria as a biotechnological tool is the degradation of mycotoxins phenomena. Therefore, it would be possible to gain more safe animal feed in terms of mycotoxins. It was found in a study that a bacterium named E3-39 strain had the ability of degrading the mycotoxin deoxynivalenol. The same bacterium can also reduce fusariotoxin. This characteristic was verified in vitro using mass spectrometry. The endophytic bacterium was identified to a group of Rhizobium-Agrobacterium (Maksimov et al., 2015).
Actually, acclimation of plantlets is a critical point of work where the beneficial bacteria have an important role. Adaptation of plantlets to acclimation conditions is difficult due to non-functional stoma, not fully efficient roots and photosynthesis organelles. If plantlets are inoculated with beneficial endosymbionts at the final stage of propagation, they can cope with this difficulty (Panigrahi et al., 2015). Azorhizobium, Azospirillum, Azoto- bacter. Bacillus, Burkholderia, Curtobacterium, Enterobacter, Halomonas, Methylobacterium, Microbacterium, Methilophylus, Paembacillus, Pseudomonas, Raistonia, Rhodococcus, Rhodopseudomonas, and Sphingopyxis belonging to 13 different genera trigger beneficial effect on micropropagation works. Some positive effects of these genera on plantlets were studied by shoot weight, leaf number, axillary shoot gr owth, rooting, rooted shoots, number, and length of roots, and acclimation of plantlets to ex vitro conditions (Orlikov’ska et al., 2017).
ENDOPHYTES AS BIOFERTILIZERS: FORESIGHT AND COMPLICATION
Recently, many studies on endophytic bacterial relationships with plants are carried out in restricted conditions. Actually, this approach does not reflect the real plant-endophyte interactions. For a realistic approach, beneficial effect of the bacteria should also be observed in the field (Riggs et al., 2001; Gyaneslrwar et al., 2002). Plant growth activity may be effected by conditions of plantation sites such as nitrogen content of soil (Muthukurnarasaury et al., 1999, 2002), soil type (De Oliveira et al., 2006), as well as age and variety of host plant (De Oliveira et al., 2006). Besides, many studies showed that use of nitrogen fixing bacteria and N-fertilizer combination decreased the requirement of extra fertilization on field (Saleh et al., 2001). At this point, actual challenge is the optimization of fertilizer amount and nitrogen fixing endophytic bacteria survival rate in rhizospheric soil. Sometimes high nitrogen fertilized soil cause reduction of bacterial colonization like sugarcane and their host bacteria G. diazothrophicus and H. seropedicae (Bueno dos Reis Junior et al., 2000; Muthukurnarasamy et al., 2002). In some events, like Azospirillum sp. adsorption on wheat root surface have negative effect if the concentration of Ca2+ and (РОд)3_ in the media is over 50 rnM (Pinlierio et al.,
2002). Therefore, indicative low soil fertility (Alfisol soil type) supports the endophytic inoculation and show's a better performance (De Oliviera et al., 2006). On the other hand, the survival rate of endophytic bacteria depends on concentration of nitrogen source especially ammonia in the media (25 mM NH4N03). High concentration of ammonia in the medium causes morphological changes in bacteria and may play a hazardous role in their survival. The use of a compost as a nitrogen source increases the number of bacterial colonization as it decreases the reducing effect of nitrogen fertilizer (Muthukumaraswamy et al., 2007). The genotype and age of the selected plant affect the performance of the bacteria improving the growth of the host plant (Munoz-Rojas and Caballero-Mellado, 2003; De Oliveira et al., 2006). It was reported that a significant reduction in the G. diazotrophicus population due to the age and genotype of the plant (Munoz-Rojas and Caballero-Mellado, 2003). In some sugarcane varieties, a large number of endophytes are known for a long time. In addition, it causes differences in the number of diazotrophic bacteria in environmental factors such as hydric stress and climatic changes in the soil (Bueno dos Reis Junior et al., 2000). However, additional field trials should be made for the optimization of parameters including time, method of application of the inoculant, and environmental factors.
CONCLUDING REMARKS AND FUTURE PROSPECTS
World’s population is expected to be close to 10 million in the next following 50 years. However, it is not an easy task to supply adequate food for the ever-increasing world population; therefore, various strategies and innovative approaches should be designed in order to achieve this purpose. The utilization of more agricultural land, increased use of herbicides, pesticides, and fertilizers, farm mechanization, transgenic crops and microorganisms to promote plant growth are among the strategies to be followed for more food production. However, many of these solutions are not sustainable, but seem to be beneficial in the short-term. Living with limited resources, forces humans to provide effective, longterm, sustainable, and eco-friendly solutions to provide enough supplements for the world. Hence, the versatile use of PGPBs in agriculture is a complementary approach to solve this problem. In the last 15-20 years, our knowledge about the mechanisms performed by the PGPBs has dramatically increased. Further understanding of the key mechanisms used by endophytic bacteria will enhance the effective employ of these organisms in agricultural applications. Bacterial endophytes with their inherent characteristics, genes, and metabolic pathways are increasingly attractive. Currently, synthetic biology has provided a genuine opening in the design of novel enzymes and microorganisms for biomass fermentation into fuels and different products. Hereafter, plants and their beneficial symbionts could also be altered to elevate the biomass for a rising population in respect of climate change. For this reason, efforts to increase the extensive use of PGPBs should focus primarily on clarification of how these bacteria elicit plant growth.