Microsoft Word - Ngumah_Proofreaded.docx                                                                                                                    Issue 2, Vol 6, 2015, 24-29 ANTIFUNGAL  POTENCIES  OF  ETHANOL  LEAF  EXTRACTS  OF  FOUR  PLANTS  FOUND  AROUND   TRADITIONAL  YAM  BARNS  IN  SOUTH  EASTERN  NIGERIA  ON  FIVE  YAM  PATHOGENS  IMPLICATED  IN   POSTHARVEST  YAM  ROT Chima    Ngumah1*,  Sarah  Umeh1,  Etienne  Chinakwe1,  Jane  Ngumah2   1Department  of  Microbiology,  Federal  University  of  Technology  Owerri,  P.M.B  1526  Owerri,  Nigeria.   2Department  of  Biology,  Federal  University  of  Technology  Owerri,  P.M.B  1526  Owerri,  Nigeria.   *e-­‐mail:    ccngumah@yahoo.com   ABSTRACT The  susceptibilities  of  five  fungi   implicated  in  postharvest  rot  of  yams,  namely,  Aspergillus  niger,   Penicillium   oxalicum,   Mucor   circinelloides,   Fusarium   oxysporum,   and   Rhizopus   nigricans   were   evaluated  using  ethanol  leaf  extracts  from  four  plants  commonly  found  around  traditional  yam  barns   in   South   Eastern   Nigeria.     The   plants   screened   were   Fagara   rubescens,   Neubouldia   laevis,   Pterocarpus   soyauxii,   and   Vernonia   amygdalina.   Antimicrobial   susceptibility   was   measured   as   an   index  of  turbidity  of  broth  cultures  after  an  incubation  period  of  72  hours  at  ambient  temperature   (29  –  310C).  The  optical  densities  (OD650)  were  measured  by  spectrophotometry.  None  of  the  tests   showed   susceptibility   to   any   of   the   plant   extracts   screened   irrespective   of   the   concentration   employed.     This   study   also   revealed   that   although   all   the   plants   contained   bioactive   secondary   metabolites,  no  antifungal  potency  was  detected  for  any  of  the  plant  extracts  on  the  test  organisms.   Keywords: yam  pathogens,  plant  extracts,  yam  rot,  susceptibility,  phytochemicals INTRODUCTION                    Yams  are  monocotyledonous  plants  belonging  to   the   genus   Dioscorea   in   the   family   Dioscoreaceae.     Yams  serve  as  a  staple  food  for  millions  of  inhabitants   of  the  tropics  and  sub-­‐tropics.    Dioscorea  species  are   important   food   crops   in   West   Africa,   the   Caribbean,   and   some   parts   of   Asia   (including   China,   Japan,   Malaysia  and  Oceania).1                      Shrines   and   traditional   yam   barns   in   South   Eastern   Nigeria   serve   as   home   for   various   plants,   some  of  which  have  been  proven  to  be  very  valuable.     Some   of   these   plants   are   used   for   therapeutic   purposes.2   This   study   investigates   the   antifungal   properties  of  the  leaves  of  some  of  these  plants  found   around   traditional   yam   barns   on   common   fungi   implicated  in  the  postharvest  rot  of  yam.  The  plants   screened   in   this   study   were:   Fagara   rubescens   (Planch),   Neubouldia   laevis   (Seem),   Pterocarpus   24 All Res. J.Biol, 2015, 6, 24-29     soyauxii   (Taub),   and   Vernonia   amygdalina   (Linn).     These   plants   were   chosen   because   they   are   commonly  used  in  constructing  traditional  yam  barns   in  South  Eastern  Nigeria,  and  are  unofficially  believed   to  play  a  role  in  the  preservation  of  stored  yams.   MATERIAL  AND  METHODS   Sample  collection                      Rotten   yam   tubes   were   collected   from   yams   stored   on   horizontal   bamboo   platforms   (improvised   yam   barns)   at   Okwuaba   village   in   Okpofe,   Ezinihitte   Mbaise  Local  Government  Area  of  Imo  State  (Nigeria).     Fresh  leaf  samples  were  plucked  at  about  midday  at   Okwuaba  Okpofe.    The  leaves  were  authenticated  by   the   Department   of   Forestry,   Imo   State   Ministry   of   Agriculture,   Owerri.   The   leaves   were   air   dried   at   ambient   room   temperature   (280C   –   310C)   until   constant  weight  was  achieved.    The  dried  leaves  were   then  ground  to  powder  using  a  mechanical  grinder.     Extraction  of  plant  materials                        70%  Ethanol  was  used  for  the  extraction  of  the   plant  materials.    For  the  cold  ethanol  extraction,  100g   of   each   powdered   plant   material   was   steeped   in   500ml   of   70%   ethanol   for   48   hours,   while   the   hot   ethanol  extraction  was  carried  out  by  seeping  100g  of   each   powdered   plant   material   in   500ml   of   70%   hot   ethanol  which  was  maintained  at  600C  for  1  hour  in  a   water  bath.2  The  slurries  were  filtered  through  folds   of  sterile  cheese  cloth.    The  filtrates  were  evaporated   to   dryness   by   forced   air   pressure   using   a   rotary   evaporator   to   a   yield   of   about   12.5%   w/w   (with   respect  to  the  powdered  plant  material).3     Preparation  of  plant  extract  diluent                      1000mg  quantity  of  each  ethanolic  extract  (hot   and   cold)   was   reconstituted   with   5   ml   of   10%   dimethyl  sulfoxide  (DMSO)  to  obtain  a  concentration   of  200  mg/ml.4      A  two  fold  serial  dilution  was  used  to   obtain  the  following  concentrations  in  sterile  distilled   water:   100   mg/ml,   50   mg/ml,   25   mg/ml,   and   12.5   mg/ml.    These  were  stored  by  refrigeration  at  40C  in   sterile  amber  coloured  bottles  until  required.     Isolation  of  fungal  pathogens  from  rotten  yam                      The   rotten   yam   tubers   were   rinsed   in   sterile   distilled   water   and   surface   sterilized   with   95%   ethanol.    The  rotten  yam  tubers  were  then  cut  open   with  a  sterile  knife  (flamed).    About  3  pieces  (3  mm   diameter)   of   each   infected   tissue   were   picked   with   flamed  sterilized  forceps  and  inoculated  on  separate   sterile  solid  Sabouraud’s  Dextrose  agar  (SDA)  plates.5     The   plates   were   incubated   at   ambient   room   temperature   (29   –   310C)   for   up   to   5   –   7   days   and   examined   daily   for   growth   of   moulds.     The   isolates   were  then  sub-­‐cultured  to  obtain  pure  cultures  of  the   organisms,  which  were  eventually  transferred  to  SDA   slants  and  stored  at  40C  until  required.         Characterization  of  fungal  isolates                      The   fungal   isolates   were   identified   using   their   growth  (colonial)  morphology  on  SDA  and  microscopic   morphology.     The   colonial   morphology   on   SDA   was   determined   by   observing   the   surface   and   reverse   views  of  the  fungi  growing  on  each  agar  plate.    The   25 All Res. J.Biol, 2015, 6, 24-29     colour,  shape,  elevation,  and  spore  head  colouration   were   noted.     To   view   the   microscopic   morphology,   two  drops  of  cotton  blue  lactophenol  were  placed  on   a  clean  grease-­‐free  glass  slide.    Then,  sterile  (flamed)   inoculating   needles   were   used   to   transfer   a   small   portion   of   mycelial   growth   to   the   cotton   blue   lactophenol.    The  fungal  growth  was  then  teased  out   in   the   cotton   blue   lactophenol   and   covered   with   a   clean   grease-­‐free   coverslip   and   then   examined   microscopically  using  x10  and  x40  objectives.6     Preparation  of  inoculum                        All   fungal   isolates   were   aseptically   inoculated   onto  sterile  SDA  slants  prepared  in  McCartney  bottles   and   incubated   at   ambient   room   temperature   (29   -­‐   310C)   for   4   days   to   obtain   young   actively   growing   cultures   consisting   of   mycelia   and   conidia   /   arthrospores   /   blastospores.   The   fungal   growth   on   each  agar  slant  was  aseptically  scraped  off  and  placed   in   10   ml   sterile   saline   (0.9%   w/v)   and   shaken   vigorously   using   a   vortex   mixer   until   the   fungal   filaments  were  broken  into  small  colony  forming  units   (cfu).   Each   suspension   was   standardized   using   a   haemocytometer  to  obtain  104  -­‐  106  cfu/ml  and  this   was  used  as  the  inoculum.       Testing  for  anti-­‐fungal  potency  of  the  plant  extracts                      The   susceptibility   of   the   isolated   fungal   pathogens   to   different   concentrations   of     the   plant   extracts   were   examined   using   a   spectrophotometer     by   measuring   their   optical   density   (absorbance)   at   620nm.3    0.5  ml  of  the  standardized  fungal  suspension   was  inoculated  into    a  separate  test  tube  containing   9.0   ml   of   sterile   Sabouraud’s   Dextrose   broth   (SDB).     Then,   0.5   ml   of   the   reconstituted   plant   extract   was   finally  inoculated  into  each  test  tube.    This  was  carried   out   for   the   following   concentrations   of   each   plant   extract:   100   mg/ml,   50   mg/ml,   25   mg/ml,   and   12.5   mg/ml.   The   test   tubes   were   incubated   at   ambient   room  temperature  (29  –  310C)  for  72  hours  and  then   transferred   to   a   spectrophotometer,   where   their   optical   densities   were   measured.   For   each   set   of   experiments  there  was  a  control  test  tube  containing   SDB   and   plant   extract   without   fungal   isolate.     The   control  was  used  to  blank  the  spectrophotometer.               Phytochemical  analysis                      Powdered  leaf  plant  samples  were  used  to  carry   out  phytochemical  tests  using  standard  procedures.7                   26 All Res. J.Biol, 2015, 6, 24-29       RESULTS  AND  DISCUSSION          Table  1.    Optical  densities  of  72  hours  Sabouraud’s  Dextrose  broth  cultures  of  five  yam  (postharvest)  pathogens   treated  with  four  leaf  extracts.     Each   leaf   extract   recorded   the   same   optical   density   for   all   the   concentrations   screened   per   test   isolate.     The   presence   of   fungal   growth   was   confirmed   using   microscopy   by   viewing   samples   from   the   broth   cultures   using   x10   and   x40   objectives,   and   then   plating  0.1mL  on  solid  SDA.    None  of  the  leaf  extract   concentrations   screened   in   this   work   showed   antifungal   potency   on   any   test   organism,   this   is   at   variance   with   the   reports   of   researchers   who   demonstrated   that   ethanol   leaf   extracts   of   Carica   papaya,  Glyphaea  brevis,  and  Spondias  mombin  were   potent   against   the   same   test   organisms   used   in   this   work  (Ngumah,  2012;  and  Ngumah  et  al.,  2013).8,9     Comparison   using   analysis   of   variance   (ANOVA)   showed  no  significant  difference  in  the  OD650  (P>0.05):     within   different   concentrations   of   the   same   leaf   extract  for  each  test  isolate,  and  among  the  different   plant  extracts  screened  on  each  test  isolate.     Although   the   plant   leaves   screened   in   this   work   contained   bioactive   secondary   metabolites   (namely:   alkaloids,   tannins,   and   flavonoids)   reported   by   workers   to   confer   antimicrobial   activity   to   plant   extracts,   no   antifungal   activity   was   recorded   in   this   work   by   these   leaf   extracts.10,11,12     This   lack   of   antifungal  activity  may  be  attributed  to:  probable  low   concentration  levels  of  phytochemicals,13,14  resistance   levels  among  strains,15  age  of  leaves,16  state  of  leaf  at   point   of   extraction-­‐dry   or   fresh,17,18   and   method/extraction  solvent.19         Plant  Extracts                                                                                                                                      Optical  Density  at  620nm  (OD620)  ±  SD                                                                                        A.  Niger                        P.  oxalicum                      M.  circinelloides                  F.  oxysporum                        R.  nigricans   Fagara  rubescens                      0.78±0.023              0.8±0.03                                  0.8±0.042                                        0.8  ±0.042                                  0.77±0.035     Neuboldia  laevis                        0.82  ±0.025              0.8±0.042                              0.79±0.041                                  0.77±0.035                                0.8±0.042   Pterocarpus  soyauxii        0.77±0.031                0.78±0.046                          0.8±0.042                                        0.82±0.012                              0.8±0.042   Vernonia  amygdalina      0.81±0.023                0.81±0.017                        0.77±0.038                                    0.8±0.02                                        0.82±0.006   Control                                                                              0.56                                          0.56                                              0.56                                                          0.56                                                    0.56   27 All Res. J.Biol, 2015, 6, 24-29     CONCLUSION   Although   data   obtained   in   this   work   revealed   the   presence  of  bioactive  substances,  none  of  the  plants   screened  showed  antifungal  activity  on  any  of  the  test   isolates.     This   suggests   that   the   mere   presence   of   bioactive  secondary  metabolites  does  not  necessarily   guarantee  the  antimicrobial  potency  of  plant  extracts.     Secondly,  though  there  was  no  antimicrobial  activity   recorded   for   the   test   organisms,   the   same   plant   extracts  may  exhibit  antimicrobial  properties  on  other   organisms.     In   addition,   quantitative   analysis   should   be   done   to   ascertain   the   phytochemical   levels   in   these  extracts  and  compare  them  with  levels  found  in   other   plant   extracts   that   have   been   documented   to   exhibit  antifungal  activity  on  the  same  test  organisms   used  in  this  work.     REFERENCES   1. 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