Efficacy of micronutrients in influencing growth behavior of Rhizobium of Pigeonpea (Cajanus cajan  L. [Millsp.])

INTRODUCTION

Trace  elements  are  essential  for  key  physiological  activity  of  photosynthesis and  respiration¹.  Metals  such  as copper,  zinc  comprise  as  trace elements  for  redox  process  to  balance  molecules  through  electrostatic interaction, as  a  part  of  many  enzymes  and  for  standardization  of  osmotic  pressure².  The  combination  of microelements  in  soil  suspension  confides  on  the physical  and  chemical  characteristic  specifically  pH  and  PE ³. Zinc  accomplishes  as  trace  mineral  as  well  as  it  also  plays  roll  as  circumstantial poisonous  constituent, influences  nodulation  and  nitrogen  fixation^4&5.  Boron is a  necessary  micronutrient  required  for  routine  plant growth⁶.  Insufficiency  of  boron leads  to  aberrant  nodule  formation  with  convincing  minimization  of  infection⁷.

Rhizobium  is  one  of  the  valuable  bacterium  species,  which  fixes  50%  of 175  million  tons  of  total  biological  N2  annually  worldwide  with  legume  species⁸.  Chick pea  and  Rhizobium  leguminous  symbiotic  association per  year  harvest  up  to  176  kg  N/ha-1  which  depends  on  bacterial  strain  and circumstantial  determinants^9&10.  The bacteria  of  the  genus  Rhizobium  is  an  inherently  varying  and  physiologically heterogenus  faction  of  microbes  which  has  the  potentiality  to  nodulate leguminaceae  family  plants¹¹.  Rhizobium  has  the capability  to  convert  atmospheric  N2  into  ammonia  by  symbiotic  association  which  is  again  transferred  and  taken  up  by  the  plants³.  In crop  species,  induction  of  root  nodules  is  affected  by  various  atmospheric determinants¹².

The  common  media  used  for  culturing  rhizobia  is  yeast  extract  mannitol  media,  which  is  made  up  of  yeast  extract  powder  or  pastes¹³.  Yeast  extract  mannitol  media  is  found  to  be  best  suiting  nutrient  media  for Rhizobium  growth.  The  ideal  medium  contains  mannitol  and  sucrose  as  the  source of nitrogen,  growth  factors  and  mineral  salts¹⁴.

Other  than  these  elements  for  the  appropriate  growth  of  rhizobia,  search  of  other  required  elements  are  also  essential.  Thus,  the  object  of  this  work is  to  find  out the  effect  of  other  trace  elements  on  rhizobia,  by  which  they  can  be incorporated  in  media  for  Rhizobium.

MATERIALS AND METHOD

Isolation  of  Rhizobium  from  root  nodules  of  pigeonpea

Pigeonpea  plants  were collected  from  pigeonpea  research  experimental  area  of  College  of  Agriculture Sehore  and  root  nodules  were  separated.  The  nodules  were  washed  under  running  tap  water,  surface  sterilized  using  liquid  detergent  2%  (V/V)  Savlon,  then treated  in  6  drops  of  tween  2o  for  15  min,  rinsed  with  70%  ethanol  for  30  sec and  disinfected  by  0.05%  (W/V)  HgCl₂¹⁵.  After  sterilization, nodules  were  crushed  with  a  sterile  glass-rod  and  suspension  was  serially  diluted to  10^-9  dilution.  0.1ml  of  each  dilution  was  spread  on  Petri-plate  containing  yeast extract  mannitol  agar  media  (YEMA).  The  inoculated  plates  were  incubated  in  BOD  incubator  at  37^. C  for  2  to  3  days.

Preparation  of  Yeast  Extract  Mannitol  Agar  with  different  concentrations  of micronutrients

The  normal  culture  media  for  isolation  and  proper  growth  of Rhizobium  is  Yeast  Extract  Mannitol  Agar  with  Congo  red.  YEMA  (/L)  (control) contains  K₂HPo₄  (0.5g),  Nacl  (0.1g) , MgSO4 x 7H2O  (0.2g),  CaCO₃  (3g),  Mannitol (10g),  yeast extract  (0.2g),  Congo red  (2.5ml)  (1% W/V),  Agar  (12 g). Congo  red  is an  indicator  for  isolation  and  has  capacity  to  inhibit  gram   positive organism.

As  a  source  of  micronutrients  (Zn,  Mn,  B,  Cu,  Mo,  Fe),  salts  of  these elements  were  used  i.e.  ZnSO₄,  MnO,  H₃BO₃,  CuSO₄,  AmMo,  FeSO₄.  To  prepare  the concentrations  (0.1,  0.3,  0.5,  0.7,  0.9,  1.0,  1.1,  1.3  and  1.5)  of  micronutrients, composition  of  basic  Yeast  Extract  Mannitol  media  was  added  with  100ml  distilled water,  and  in the  above  prepared  composition  a  concentration  was  mixed  from  the  above  concentration  of  micronutrients  and  thus  each  concentration  of  all  the micronutrients  was  prepared  with  YEMA.  Prepared  control  and  micronutrient medium  were  autoclaved  at  121  ⁰C  for  20  minutes.

Each  media  plate  was  inoculated  by  spread  plate  technique  from  Rhizobium  culture  and  plates  were  incubated  for  2-3  days.

Preparation  of  multi-micronutrient  Yeast Extract  Mannitol  media  (YEM)

Concentrations  (g/100ml)  of  micronutrients  those  exhibited  increased  number  of CFU  against  the  control  were  dissolved  in  100ml  distilled  water  with  YEM composition.  This  multi-micronutrient  yeast  extract  mannitol  broth  media  was inoculated  with  Rhizobium  culture  and was  allowed  to  incubate  on  rotatory  shaker at  37^oC  for  4-7  days.   For  viability  test,  0.1ml  of  the  multi-micronutrient  broth culture  was  spread  on  YEMA  plates  and  incubated  for  2-3  days.

All  the  experiments  were  repeated  at  three  times  and  following  formula was  used  to  calculate  the  number  of colonies  formed:

(Colony Forming Unit) = number of colonies * inverse dilution * 10

RESULT AND DISCUSSION

In present investigation, colony counts on effect of micronutrient (Table – 1) revealed that 0.1 % Zinc (5711.66 ± 173.00), 0.3% Manganese  (5771.33 ± 6.79), 0.1% Boron (6127 ± 5.25) (Fig 1), 1.5% Copper (4522.33 ± 7.68), 0.1% Molybdenum (6485.66 ± 11.10), 0.3% Iron (4456 ± 7.28) were the most efficacious concentrations with yeast extract mannitol media to Rhizobium growth, and exhibited maximum number of population against rest of the concentrations in terms of CFU. However the  highest CFU (6485.66 ± 11.10) was recorded in 0.1% Molybdenum  concentration (Fig 2).

The maximum growth of 5711.66 CFU was recorded in 0.1% concentration of zinc (Fig 1). With increase in concentration thereafter, there was a significant drastic decrease in Rhizobium count.

 

Fig 1: Growth behavior of Rhizobium influenced by zinc, manganese and boron concentration (gm/100 ml).  Click here to View Figure

 

Increase in the concentration of manganese from 0.1% to 0.3% significantly increased the CFU count (5771.33) by 27.62 percent over the control (4522.33) (Fig 1). Increase the manganese concentration to the level of 0.5% also had significant CFU count (4582.00) as compared to the mean concentrations of manganese (1925.62). Although this increase in CFU is significantly greater than control, the percent increase is meager (1.32 percent) (Table 1).

Table 1: Colony forming units (CFU) of pigeon pea Rhizobia as influenced by different concentrations of  Zn, Mn, B, Cu, Mo and  Fe. Click here to View table

 

CD 5% for interaction of micro nutrient x concentration = 26.10

Through the application of boron the higher CFU count (6126.00), was observed in 0.1% application which was 35.46 percent higher than the control (Fig 1). The next dose of 0.3%, although had significant growth rate of 31.07 percent over the control, but this growth was neither at par or superior over 0.1% application of boron.

As regard the effect of copper, there was a linear increase in CFU of rhizobia from 76.00 to 4522.33, with the increase in concentration from 0.1% to 1.5% respectively. However the highest CFU (4522.33) was equal to the control (Fig 2) (Yeast extract mannitol media). It seems that copper did not have influential role to increase rhizobia population.

 

Fig 2: Growth behavior of Rhizobium influenced by copper, molybdenum and iron   concentration (gm/100 ml). Click here to View Figure

 

Molybdenum plays a vital role in nodulation and nodule growth in leguminous crops, because, it is constituent of the nitrogenase enzyme for every bacterium which fixes nitrogen. Also, molybdenum enhances plant growth as well as seed yield and mineral composition¹⁶ The concentration of 0.1% molybdenum has superiority of enhancing rhizobium CFU to the maximum of 6485.67. This increase is not only significantly superior amongst the concentrations of molybdenum; it also has the superiority over all other elements used in this study (Fig 2). It was closely followed by 0.3% concentration of molybdenum (6214.00 CFU count). There was only a 6 percent decrease of CFU count as compare to 0.1% concentration of molybdenum. It is also evidence that application of molybdenum at the rate of 1kg/ha as soil application and 1 gm/kg of as seed coating enhance the nodules numbers and nodule weight in chickpea¹⁷

A reduction in specific rates of nitrogenase activity has been observed in iron limited peanut nodules indicating a possible direct limitation by iron deficiency on nodule function¹⁸. In the present study, as per the activity of manganese, the iron also showed enhanced growth from 0.1% concentration to 0.3% concentration and thereafter decreased (Fig 2). However it did not show any incremental effect on enhancement of rhizobium count by increasing the iron concentration further.

Multi-micronutrient broth prepared from the best individual concentrations of zinc, manganese, boron and molybdenum, when used for rhizobium growth, it enhance the CFU count to highest of 6970.00 (Table 2). The percent increase in CFU was highest of 22.03% over 0.1 percent zinc. It was 7.48 percent higher than the best individual micronutrient i.e. molybdenum (6484.67). This increase in CFU count, even over the best individual component (molybdenum) was significantly higher (Table 2).

Table 2. Percent increase in CFU of rhizobia by multi-micronutrient media over individual best micronutrient

	Concentration of individual best micronutrient and multi-micronutrient	CFU count	Percent increase in CFU by multi-micronutrient media
1	0.1% Zn	5711.67	22.03
2	0.3% Mn	5771.33	20.76
3	0.1% Bo	6126.00	13.77
4	0.1% Mo	6484.67	07.48
5	Mean	6023.41	15.71
6	Multi-micronutrient	6970.00*	–
	SD +	475.54

 

The quantum of quality Rhizobium is a very basic requirement to enhance the production of food legumes in Asian continent, especially in India, because large population solely depends upon food legumes for their protein requirement the small quantity of micronutrients can trigger the enhancement in Rhizobium production, which would be the cost effective and economically viable it may also increase the plant vigor at seedling stage enabling the plants to survive better. This study can further be rectified by using various cross combinations of micronutrients, so that further effective cross combinations can be worked out.

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