Department  of  Biotechnology Sri  Sathya  Sai  College  for  Women,  Bhopal,  India.

Corresponding author Email: orjitnandi@gmail.com

Article Publishing History

Introduction

Chemicals used to control  plant  diseases contaminate  the  soil  environment , degrade its fertility and  also defile underground  water,  causing  health  risk. Thus, biocontrol agents emerge as an alternate  to  those antifungal chemicals, these are inexpensive, eco-friendly  and  have  no  harmful  effects  on  human , animals and plants (Deshwal et  al.,  2003).  Legumes establish a symbiotic interaction with soil bacteria termed  as Rhizobia. These bacteria in association with legumes can fix atmospheric N and through this feature. Hence, they are introduced into agricultural systems to improve soil fertility, plant growth and limit the use of chemical fertilizers (Ouma et al., 2016 Srinivasan, 2017 Yassine, 2018).

Rhizobia not only fix nitrogen from atmosphere and supply it to plants but also promote  the  growth  of  plants. Rhizobia synthesize phyto-hormones  and  solubilization  of  minerals  act  as  a  biocontrol  agent  and can  inhibit  the  growth  of  pathogens.  Due  to  the  secretion  of  secondary  metabolites  such  as  antibiotics  and  HCN by rhizobia, they have the capacity to restrict the growth of fungal pathogen. In  iron  stress  conditions in rhizobia,  siderophore  production  provides  an  added  advantage,  resulting  in  the  exclusion  of  pathogen  due  to  iron  starvation. Rhizobacteria possessing all these features are also referred as plant growth promoting rhizobacteria (PGPR).Not only this but these bacteria also protect plants against flooding, drought, salt, flower wilting, metals, organic contaminants, and both bacterial and fungal pathogens, (Glick, 2014, Subramanium et al., 2015,  Srinivasan, 2017 and   Yassine, 2018).

Legumes fix atmospheric nitrogen only in association with a bacterial symbiont of the genus Rhizobium. Rhizobia have been arbitrarily divided into two classes: fast growing and slow growing. Normally, commercial cultivars of the soybean Glycine max are nodulated only by slow-growing Rhizobium japonicum (Keyser et al., 1982). The isolate appeared to be effectively nodulate only the wild soybean Glycine soja and an unbred soybean cultivar from China (G. max cv. Peking) Rhizobium japonicum 191 is a member of a salt-tolerant group of R. japonicum recently described by (Keyser et al., 1982).

Bradyrhizobium japonicum is a Gram negative bacterium belonging to rhizobia group associated with roots of soybean and have the capacity to fix N2 in the presence of nitrogenase enzyme (Baoling et al., 2007) .This bacterium and nitrogenase enzyme both are very sensitive for the environmental conditions. The commercially introduced strains must compete with highly adapted indigenous rhizobia for legume nodulation under specific physiological, biological and environmental soil conditions. Soil acidity limits symbiotic nitrogen fixation by limiting Rhizobium survival in soils, as well as reducing nodulation (Ibekwe  et al, 1995). Chakraborty  and  Purkayastha,  (1984)  reported  that  some  strains of Bradyrhizobium japonicum  produce rhizobitoxine   which can protect  soybean crops  from  the  infection  of  Macrophomina  phaseolina,  which is a  charcoal  rot  fungus  of  leguminous  crops.  Rhizobium  has  shown  to  reduce  root-rot  of  soybeans  caused  by  Phytophthora  megasperma. Rhizobial mechanism such as improvement in intake of plant nutrients by altering root morphology, production of siderophore (Antoun  et al.,  1998; Arora et al., 2001; Chabot et al., 1996; Srinivasan, 2017) to meet the iron requirement of the plant under iron stressed conditions and lowering of ethylene through ACC deaminase enzyme are some example with direct positive effects on non leguminious plant growth. B.  japonicum  strain inhibit the  growth  of  seven  pathogenic  microorganisms  causing disease in  soybean  was  studied  by (Balasundaram  and  Sarbhoy 1998)  The  fast  growing  rhizobial  strains  were  found  to  completely  inhibit  the  growth  of  white  sclerotia  of  S.  rolfsii. Different strains of  Rhizobium  and  Bradyrhizobium  have  been  reported  to  inhibit  the  growth  of  M.  phaseolina (Deshwal et al., 2003; Arora et al., 1998).  The  rhizobia  having  antagonistic property  showed  more  competency  in  root  hair  infection  in  host  plants  as  compared  to  non-  biocontrol  rhizobia. Rhizobia  also  appear  to  influence  the  plant  defense  mechanism  by  stimulating  the  production  of  phytoalexins  by  plants. Antibiotics  produced  by  rhizobia  have  been  found  to  play  an  important  role  in  disease  control.  HCN,  a  secondary  metabolite  produced  by  several  microorganisms,  has  deleterious  effect  on  the  growth  of  some  microbes (Knowles, 1996).

Studies conducted on  numerous  plant  microbe  interaction  have  shown  that  such  antagonistic  rhizobacteria  could  function  by  competition  and  antibiosis  i.e.  by  producing  antimicrobial  compounds  like  bacteriocin (Rodelas  et  al.,  1998;  Joseph  et  al.,  1983)  but  also  indirectly  induces systemic  resistance  against  plant  diseases. The  enzyme  system  of  bacteria  during nodulation in the roots supplies  constant  source  of  reduced  nitrogen  to  the  host  plant  and  the  plant  in  turn  provides  nutrients  and  energy  for  the  activities  of  the  bacteria  (Singh  et  al.,  2008).  It has also been evaluated that Rhizobium  increases  plant  growth  by  various  ways  such  as  production  of  plant  growth  hormones,  vitamins,  siderophores,  by  solubilisation  of  insoluble  phosphates,  induction  of  systemic  disease  resistance  and  enhancement  in  stress  resistance  (Hussain  et  al., 2009; Yassine, 2018).

Some Rhizobium spp.  have shown  antimicrobial  activities  towards  Pseudomonas  spp.  (Kacem et al., 2009)  Aspergillus  niger  (Yuttavanichakul  et  al.,  2012)  .In  the  present  study  Rhizobium  was  isolated  from  the  root  nodules  of  soybean  and  its  antagonistic  activity  was  studied  against  pathogenic  fungi  such  as  Aspergillus  niger  and  Fusarium  oxysporum.

Material  and  Methods

Isolation of nitrogen fixing bacteria  from  soybean  root  nodules

Soybean  plants  were  uprooted  carefully  from the soil so  that  intact  roots  can  be  obtained without destroying the nodules.  Healthy  soybean  nodules  were  detached  from  the  root  and  further  isolation  of  root  nodulating  rhizobia  was  carried  out  (Vincent,  1970).

The  separated  root  nodules  were  washed  in  tap  water  to  remove  the  adhering  soil  particles  from  nodule  surface.  Fresh  root  nodules  from  soybean  were  collected  and  surface  sterilized  with  70%  ethanol  and  0.1%  mercuric  chloride  and  washed  thrice  with  sterile  distilled  water.  Root  nodules  were  crushed  in  saline  solution. Test isolate was isolated  by  spreading  0.1ml  crushed  root  nodule  suspension  on  YEM  (Yeast  extract  mannitol,  pH.7.0)  agar  plate  and  incubated  at  36°C.Colonies of test isolate were observed  in  2-3  days  (Singh  et  al.,  2008)  were  further  used  for  morphological  and  biochemical  characterization.  To  check  the  antibacterial  activity,  rhizospheric  bacteria  were  isolated  by  serial  dilution  of  soil. All  the  experiments  were  carried  out  in  triplicates.Bradyrhizobium japonicum (RJ(s)TAL102) was collected from M.P. State Agro Industries, Bhopal.

Morphological characterization

Morphological  characters  such  as  shape,  colour, size elevation,  margin,  opacity    and  gram  staining  were  performed  for  identification  of  the  bacteria  (Gachande  and  Khansole,  2011) and for further biochemical test.

Biochemical characterization

All the tests were carried out with triplicates. For  characterization  of  bacteria, the  acid  production  test (Jordan, 1984),  oxidase  test (Kovaks, 1956), catalase  test (Graham  and  Parker, 1964), methylene  blue  test, starch  hydrolysis  test (Oliveria  et  al., 2007),  glucose  peptone  agar test (Kleczkowska  et  al., 1968), gelatin  hydrolysis  test (Sadowsky  et  al., 1983), growth  in  NaCl  (Sadowsky  et  al., 1983), citrate utilization test gram  staining  and  motility  were  performed.

Temperature tolerance

Effect  of  different  physical  parameters  on  the  growth  of test isolate  was  studied  by  keeping  plates  at  different    Differences  in  the  range  of  growth  temperatures  were  investigated  by  incubating  bacterial  cultures  in YEM  agar  at  32°,34°,36°,38°  and  40°C.

Antifungal  activity

The  antifungal  effect  of  test isolate were evaluated  against  pathogenic  fungi  (Aspergillus  niger, Alternaria alternate and  Fusarium  oxysporum)  by  filter  paper  method. Fluconazole  antifungal  was  used  as  a  positive  control  and  saline  as  negative  control. Bacterial suspension was made and filter paper was dipped into it and placed on the surface of assay plates labelled as Control.   The  plates  were  incubated  for  24 – 48 hour at  28°C  to observe antifungal  activity  and  the  zone  of  inhibition  were  recorded  (Arfaoui  et  al.,2006).

Results and Discussion

The  colonies  isolated from roots of soybean were  entire,  opaque  with  regular  margin, milky  white, translucent, circular  in  shape, shiny, raised (convex), sticky  consistency, musky  colour  of  the  colony  and  2-4mm  in  diameter. The  isolated  bacterium  was  aerobic,  non  spore  forming, pink  coloured  gram  negative  rods  and  motile. Rhizobium  showed positive results for acid  production  test, catalase  test, oxidase  test ,starch  hydrolysis  test, glucose  peptone  agar test and NaCl test. Also the negative result were seen for gelatin  hydrolysis  and  methylene blue test.The  optimum  temperature  was  28°C.There is a gradual decrease in a colony count was observed after 40°C  and growth was totally absent at temperature 44°C in broth.

Both the isolates of  Rhizobium  inhibited  the  growth  of  Aspergillus  niger  , Alternaria alternata and  Fusarium  oxysporum  which  is  pathogenic  fungi  and  affects  on  the  yield  of  crop  plants. The  zone  of  inhibition (in  mm) was recorded   and measured.

The  test isolates were identified as Rhizobium japonicum and  Bradyrhizobium  japonicum by morphological  and  biochemical  characterization. These  characteristics  were found to be  similar  with  the  standard  characteristics  of  the  Rhizobium  and thus this  indicates  that  the  isolated  microorganisms  are  Rhizobium japonicum and Bradyrhizobium japonicum.

Table 1:  Biochemical  characteristics  of  the  isolate

TEST	REMARK
	Rhizobium japonicum	Bradyrhizobium   japonicum
Acid  production  test	+ve	+ve
Catalase  test	+ve	+ve
Oxidase  test	+ve	+ve
Methylene  blue  test	-ve	+ve
Starch  hydrolysis  test	+ve	+ve
Glucose  peptone  agar test	+ve	+ve
Gelatin  hydrolysis  test	-ve	+ve
NaCl test	+ve	+ve
Gram  staining	Gram  negative  rod shaped	Gram  negative  rod shaped

Table 2: Antifungal activity of the isolate

S.no.	Treatments	Aspergillus niger	Fusarium oxysporum	Alternaria alternata
1.	Rhizobium japonicum	8.3	7.4	8.0
2.	Bradyrhizobium japonicum	9.2	7.6	8.5
3.	Fluconazole	10.1	10.0	10.3
4.	Control	0	0	0

*Note: Inhibition zone  measured in mm

Graph 1

The  zone  of  inhibition (in  mm)  recorded  was  8.3 for  Aspergillus niger  ,7.4  for Fusarium oxysporum  and 8 for Alternaria alternata from Rhizobium japonicum and for Bradyrhizobium japonicum the zone of inhibition was recorded 9.2 for  Aspergillus niger , 7.6  for Fusarium   oxysporum and for  8.5 Alternaria alternata.. Rhizobia have been reported to inhibit significantly the growth of Fusarium spp. and Aspergillus spp. (Srinivasan, 2107). Antifungal activity of Bradyrhizobium japonicum against Fusarium oxysporum has been reported by Mariastuti et al., (2018) that inhibition of Fusarium oxysporum by Bradyrhizobium  japonicum  ranged from 19% to 54.9%. Thus, indicating Rhizobium japonicum and Bradyrhizobium japonicum as a biocontrol agent.

Conclusion

The aim of the study was screening of Rhizobium spp. (Rhizobium japonicum and  Bradyrhizobium  japonicum) and determine its antifungal activity against Aspergillus niger , Alternaria alternata and Fusarium oxysporum causing various root rot diseases.In our investigation the antifungal activity of Rhizobium spp. were found to inhibit fungal growth showing inhibition zone, suggesting production of certain antifungal metabolites. Rhizobium spp. could be effectively used as a biocontrol agent in the form of bio-inoculant against fungal pathogen but enhancement in its antifungal properties would prove to be more efficacious. Therefore efforts are required to understand biocontrol mechanism of rhizobia against fungi. Genetic  engineering  approach  can  also  be  used  to  introduce  the  genes  coding  for  the  synthesis  of  antifungal and antimicrobial metabolites  into  rhizobial  strains  selected  for  use  in  biocontrol.

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