The Interaction between Endophytic Actinomycetes and Rhizobium in Leguminous Plants

Biological N xation represents the major source of 2 N input in many agricultural soils including those in arid regions where little arti cial fertilizer is applied. The major N xing systems in agriculture are the 2 symbiotic systems, where bacteria such as rhizobia interact with legumes to x atmospheric nitrogen which plays a signi cant role in improving the fertility and productivity of low-N soils. The symbiotic association of legume-rhizobium is initiated by the colonization of the rhizosphere by the rhizobia and subsequent attachment to the root hairs of the host plant. Furthermore, the host will produce avonoids, such as luteolin in alfalfa and diazedin in soybean, which interact with nod protein in the rhizobia. Moreover, this process then elicits the expression of a cluster of nodulation genes such as and nod, nol, noe in the rhizobia The interaction is potentially of . great importance to the health and growth in nature of this nodulating legume. The interaction between endophytic Actinomycetes and rhizobia in leguminous plants is one way to improve the capability of leguminous plant to s x atmospheric n in plant roots and contribute to itrogen the plants nutrition. From other studies, we know that certain types of Actinomycetes, for example Streptomyces, interact with peas to form healthy roots as an effective site to form nodules and improve biological nitrogen xation. Knowledge about this activity against fungal pathogens might lead to nding biocontrol agents for use in sustainable agricultural practices. Root-colonizing soil borne Actinomycetes might in uence root nodulation in leguminous plants by increasing root nodulation frequency, possibly at the sites of infection by spp. Actinomycetes Rhizobium also colonize and sporulate within the surface cell layers of the nodules. This colonization leads to an increase in the average size of the nodules that form and improves the vigor of the bacteroids which generate the red color within the nodules by enhancing nodular assimilation of iron and possibly other soil nutrients.

N input in many agricultural soils including those in arid regions where little arti cial fertilizer is applied. The major N -xing systems in agriculture are the 2 symbiotic systems, where bacteria such as rhizobia interact with legumes to x atmospheric nitrogen which plays a signi cant role in improving the fertility and productivity of low-N soils. The symbiotic association of legume-rhizobium is initiated by the colonization of the rhizosphere by the rhizobia and subsequent attachment to the root hairs of the host plant. Furthermore, the host will produce avonoids, such as luteolin in alfalfa and diazedin in soybean, which interact with nod protein in the rhizobia. Moreover, this process then elicits the expression of a cluster of nodulation genes such as and nod, nol, noe in the rhizobia The interaction is potentially of . great importance to the health and growth in nature of this nodulating legume.
The interaction between endophytic Actinomycetes and rhizobia in leguminous plants is one way to improve the capability of leguminous plant to s x atmospheric n in plant roots and contribute to itrogen the plants nutrition. From other studies, we know that certain types of Actinomycetes, for example Streptomyces, interact with peas to form healthy roots as an effective site to form nodules and improve biological nitrogen xation. Knowledge about this activity against fungal pathogens might lead to nding biocontrol agents for use in sustainable agricultural practices.
Root-colonizing soil borne Actinomycetes might in uence root nodulation in leguminous plants by increasing root nodulation frequency, possibly at the sites of infection by spp. Actinomycetes Rhizobium also colonize and sporulate within the surface cell layers of the nodules. This colonization leads to an increase in the average size of the nodules that form and improves the vigor of the bacteroids which Introduction A symbiotic, associative, or symbiotic biological nitrogen xation (BNF) is a free and renewable process (Jensen and Nielsen, 2003) which should constitute an integral part of sustainable agroecosystems. Yet there has been a rapid increase in use of fertilizer N and a parallel decline in the cultivation of leguminous plants and BNF. BNF in various agro ecosystems has been extensively reviewed (Boddey et al., 1998;Giller and Wilson, 1991;Ladha et al., 1996;Ledgard, 2001) and since atmospheric N is an almost inexhaustible 2 resource, BNF is a sustainable source of N in agricultural cropping systems As BNF is largely restricted to occurring in legumes, replacing fertilizer N in agricultural systems with symbiotically xed N BNF may require a legume 2.
crop to be grown as a green manure crop before, for example, maize, intercropped with maize, or grown alone. N xed by heterotrophic diazotrophs in 2 sugarcane (Boddey et al., 1995) or Anabaena azollae applied in ooded rice may complement soil and fertilizer N as sources of nutrient for these crops. 2 3 October 5 www.j-tropical-crops.com 30 Asmiaty Sahur Biological Nitrogen Fixation nd ts se n a U I i Agriculture

Nitrogen Cycles
The Nitrogen (N) and carbon (C) cycles are regarded as the driving forces in acidi cation of farming soils (Bolan et al., 1991;Helyar, 1976 . Transformations ) of N, xation of N by legumes, leaching of nitrate (NO ), and the effects of ionic forms of N taken up by 3 plants are particularly important processes in the gain and loss of organic matter.

Nitrogenize Reaction
The Society for General Microbiology called nitrogenize an enzyme which catalysis the conversion of nitrogen gas to ammonia in nitrogenxing organisms as one of the critical enzymes in nature. In legumes, nitrogenize only occurs within the bacteroids and the reaction requires hydrogen as well as energy from ATP. This nitrogenize complex is sensitive to oxygen, becoming inactivated when exposed to it. However, this is not a problem with free living anaerobic nitrogen-xing bacteria such as Clostridium because they have a variety of different mechanisms to protect nitrogenize complex, including high rates of metabolism and physical barriers . for (Sprent and Sprent, 1990) Azotobacter, example, overcomes this problem by having the highest rate of respiration of any organism, thus maintaining a low level of oxygen in its cells.
The nitrogenize reaction is supplied with energy in the form of ATP and reducing power from electron (e-) carriers, usually ferredoxin:

Rhizobium
Bradyrhizobium and Rhizobia (species of , , Rhizobium Mesorhizobium Bradyrhizobium Azorhizobium Allorhizobium , , , and Sinorhizobium) form intimate symbiotic relationships with legumes by responding chemically to avonoid molecules released as signals by the legume host. These plant compounds induce 'expression of nodulation' ( ) genes in the rhizobia, which in turn nod produce lipo-chito-oligosaccharide (LCO) signals that trigger mitotic cell division in roots, leading to nodule formation (Dakora, 1995;Lhuissier et al., 2001).
Bradyrhizobium japonica was rst isolated from a soybean nodule in Florida in 1957.
sp. Rhizobium NGR234 has a host range of more than 112 genera of legumes (Viprey 2000). Rhizobia can also et al., be found in the roots, or rhizosphere, where they cause the formation of nodules.
T h e b e n e c i a l e f f e c t o f a n d R h i z o b i u m Bradyrhizobium in legumes, in terms of biological nitrogen xation, has been a main focus of interest in the past. Rhizobia are known to cause nodulation and increase nodule weight in legumes along with an increase in growth and development of the host plant (Kraus et al., 1987), and this nodulation requires temporal and spatial regulation of genes and gene networks (Hayashi et al., 2012). In addition, they protect the roots from pathogen attack due to production of diverse microbial metabolites like siderophore (K et al., ), rhizobitoxin, loepper 1980 plant growth enhancement through IAA production, and uptake of phosphorus and other minerals ( et al., 09). Gholami 20

Actinorhizal Plants
Actinorhizal root nodules result from the interaction between nitrogen-xing Actinomycetes called Frankia and the roots of dicotyledonous plants belonging to 8 plant families and 25 genera (Benson and Silvester, 1993). These plants offer striking differences with the -legume symbiosis Rhizobium (Franche et al., 1998;Wall, 2000). While is Frankia l a m e n t o u s , b r a n c h i n g , g r a m -p o s i t i v e Actinomycetes, Rhizobia are gram-negative, unicellular bacteria.
can interact with a Frankia diverse group of dicotyledonous plants (Diagne et al., 2013) whereas Rhizobia only form symbiotic relation with plants from the legume family and with one non-legume.

The functional relationship between
and Frankie plants is far from simple. The microbe and the plant may show complete compatibility as far as establishment of infection is concerned, but the resulting association may not provide optimal bene t to either partner.

Symbiotic Nitrogen
The association between the legume host plant including peas, lentils, and alfalfa and the nodule bacteria is mutually bene cial (symbiotic), due to xation of atmospheric nitrogen and its availability to the plant. The progress in using the rhizosphere bacteria, including their mechanism of action related to plant growth-promoting traits has been reported by Bhattacharyya and Jha (2012) and Shahzad et al. . Nod factors are speci c lipo-chitooligosaccharides (Lerouge et al., 1990) that affect the host plant in a number of ways. The rst visible effect of Nod factors on the plant root is the curling of root hairs. Nod factors also trigger cell division in cortical cells, which leads to the formation of the nodule meristem (Cohn et al., 1998;Denarie et al., 1996).
The symbiotic legume-rhizobium relationship is initiated by the colonization of the rhizosphere by the rhizobium and subsequent attachment to the root hair of the host plant. Furthermore, the host will produce avonoids, such as luteolin in alfalfa and diazedin in soybean which are products that will interact with Nod in Rhizobia. Moreover, this process then elicits the expression of a cluster of nodulation genes such as and in the Rhizobium nod, nol noe (Denarie et al., 1996)

Molecular Interaction of Leguminous Plants and Rhizobia
Rhizobia elicit the formation of new organs, called nodules, on their leguminous host plants, in which they x nitrogen. The early steps in the symbiotic reactions between the rhizobia and leguminous plants are mediated by the bacterial secretion of substituted lipochito oligomers, the factors nod (Prome et al., 1998). This lipo-chito-oligosaccharide signal is produced by the bacteria in response to the phenolic acids produced by the host plant. Flavonoids, the inducers of genes in rhizobia are nod chosen in evolution because they are unique selected markers for the hormonal balance of the root (Bladergroen and Spaink, 1998). Flavonoids are produced and sent out by legume plant roots and attract both mutually bene cial and pathogenic to the roots (Phillipot et al., 2013), but only some of their constituents act as signals that induce a response in the symbiotic rhizobia. Flavonoids, which include is avones, chalcones, avonols, avones, and anthocyanin amongst other related compounds, induce genes for nodulation in rhizobia to the host plants.

Plant
-promoting rhizobacteria (PGPR) were growth rst de ned by Kloepper and (1978) include Schroth soil bacteria that colonize the roots of plants following inoculation of the seed and which enhance plant growth. These plant-growth promoting rhizobacteria (PGPR) have been identi ed as a biological control agent that could be an alternative to pesticide use for disease suppression without negative effects on the user, consumer, or the environment (Johnsson et al., 1998).
K e y f e a t u r e s o f p l a n t -g r o w t h -p r o m o t i n g rhizobacteria are their ability to colonize the root systems of plants and to modulate plant growth by enhancing the availability of nutrients. The rhizobacteria can also induce metabolic activities shift the phyto hormonal balance, induce defence mechanisms such as systemic acquired resistance (SAR) and induced systemic resistance (ISR), or by reducing phytotoxic microbial communities (Mc Cully, 2001).The by which PGPR mechanisms promote plant growth s ha been studied by several research Mordukhova et al. 1991) who ers including ( reported rhizobacteria have the ability to that produce or change the concentration of the plant hormones indole acetic acid (IAA gibberellic acid, ), cytokinin, and ethylene (Arshad and Frankenberger, 1991;Glick et al 1995) ., . The PGPR mechanism also performed a symbiotic N xation (Boddey and 2 Do¨bereiner,1995;Kennedy et al., 1997), a n t a g o n i s m a g a i n s t p h y t o p a t h o g e n i c microorganisms e.g., spp by production of Fusarium siderophores (Scher and Baker, 1982), -1,3glucanase chitinases, antibiotics (Shanahan et al., , 1992), cyanide and solubilisation of producing a mineral phosphates and other nutrients (De Freitas et al.,1997).
Biological control, i.e. the use of speci c microorganisms that interfere with plant pathogens and pests, is a nature-friendly, ecological approach to overcome the problems caused by standard chemical methods of crop protection. Bio control involves harnessing disease-suppressive microorganisms to improve plant health. Disease suppression by bio control agents involves sustained interactions among the plant, the pathogen, the bio control agent, the microbial community on and around the plant, and the physical environment (Handelsman and Stabb, 1996). Therefore, despite its potential in agricultural applications, bio control is one of the most poorly understood areas of plant-microbe interactions.
A bio control agent should grow and persist, or "colonize," the surface of the plant it protects, and colonization is widely believed to be essential for bio control (Weller, 1983;de Wager et al., 1987;Parke, 1991). However, colonization, or even the initial size of the population of the bio-control agent, has been shown to be signi cantly correlated with disease suppression in only a few instances (Parke, 1991;Bull et al., 1991).
The use of plant-growth promoting rhizobacteria (PGPR) as inoculants to achieve various objectives The Interaction between Endophytic Actinomycetes and .......... such as rapid early growth is a recent area of interest. Inoculants that promote rapid early seedling growth have potential to play an important role in agriculture, for example by promoting early growth of g r a s s a n d l e g u m e p a s t u r e s p e c i e s . T h e establishment of pastures in acidic soils can be slow, resulting in greater weed competition and reduced opportunities to exploit soil resources. Opportunities for the use of PGPRs on wheat and other cereal crops are being investigated for similar reasons (Wakelin and Rider, 2004).