An   epidemiological   and   molecular   characteriza3on   study   was  carried   out   in   four   cassava   growing   provinces   of   Zambia   in  2013.  The  study  involved  cassava  mosaic  disease  (CMD)  survey,  virus  isolate  characteriza3on,  and  evalua3on  of     landraces  and  improved   cassava   varie3es   to   African   cassava   mosaic   virus  (ACMV)   and   East   Africa   cassava   mosaic   virus   (EACMV).   The  objec3ves  of  the  study  were  i)  to  determine  the  incidence  and  severity   of   Cassava   mosaic   disease   (CMD);   ii)   to   determine  gene3c   diversity   of   Cassava   mosaic   geminiviruses   (CMGs);   iii)    to  evaluate  the  effect  of  cassava  mosaic  disease  (CMD)  on  yield  of   cassava.   The   results   of   the   study   indicated   that   CMD  incidence   and   severity   was   61.5%   and   2.7,   respec3vely.   The  results   also   showed   that  most   of   the   diseased   cassava   plants  were   due   to   cuQng   infec3on   (57.7%)   compared   to   whitefly  infec3on   (3.8%).   Phylogene3c   analysis   categorised   variable  dis3nct   groups   of   EACMV   associated   with   CMD.   Among   the  germplasm  evaluated  for  CMD  response,  Manyopola  was  found  to   be   suscep3ble   (3.7)   whilst   Kampolombo   was   resistant   (1).  Mweru   had   the   highest   root   yield   (1.6   kg   plant-­‐1)   while  Kapumba  had  the  lowest  root  yield  of  (0.2  kg  plant-­‐1).  

Epidemiological  and  Molecular  Characterisa5on  of  Cassava  Mosaic  Geminiviruses  and  their  Effect  on  Yield  of  Cassava  in  Zambia

Mathias  Tembo1,3,  M.  Mataa1,  J.  Legg2,  P.C.  Chiko53  and  P.  Ntawuruhunga4  

1The  University  of  Zambia,  School  of  Agricultural  Sciences,  P.O.  Box  32379,  Lusaka,  Zambia,  2Interna5onal  Ins5tute  of  Tropical  Agriculture,  P.O.  Box  34441,  Dar-­‐es-­‐Salaam,  Tanzania,  3Zambia  Agriculture  Research  

Ins5tute,  P/Bag  7,  Lusaka,  Zambia,  4Interna5onal  Ins5tute  of  Tropical  Agriculture,  32  Poplar  Road,  Avondale,  Lusaka,  Zambia  

Abstract  

Fig   3:   Gel   electrophoresis   of   DNA   fragments   of  representa5ve   Zambian   isolates   of   (upper)   African  cassava  mosaic   virus   (774bp)   and   (lower)  East  African  cassava  mosaic  virus  (556bp)  using  the  specific  primers  JSP001/002  and  EAB555F/R,  respec5vely.    

There   has   been   a   marked   increase   in   cassava   produc3on   in  Zambia.  However  the  yields  are  s3ll  low  (5.8  t/ha)  (FAO,  2010),  compared  to  cassava  yields  of  25-­‐40t/ha  (Pluckne],  2000).  This  is   only   14.5-­‐23.2%   of   average   cassava   yields.   The   most  important   disease   affec3ng   cassava   is   cassava  mosaic   disease  (CMD)   caused   by   the   Cassava   mosaic   geminiviruses   (CMGs).  The   specific   objec3ves   of   the   study   were   to   determine   the  incidence  and  severity  of  CMD,  characterize  the  cassava  mosaic  geminiviruses   infec3ng   cassava   and   evaluate   the   response   of  improved  and   local  popular   cassava  varie3es  and   landraces   to  cassava  mosaic  geminiviruses  at  a  “hot  spot”.  

Introduction

Materials  and  Methods  A   total   of   80   fields   were   surveyed   in   2013   covering   Central,  Western   and   Eastern   and   Lusaka   provinces   using   the   method  described   by   Sseruwagi   et   al.   (2004).   Data   on   incidence,  severity,  type  of  infec3on  and  whitefly  popula3on  was  collected  from  cassava  fields  on  3  –  6  months  old  plants.  Geo-­‐coordinates  (longitude,   la3tude   and   al3tude)  were   recorded   for   each   field  loca3on   using   a   GARMIN   eTrex   20   Geographical   Posi3oning  System  (GPS)  handset.  Plant  genomic  DNA  was  extracted   from  186  fresh  leaf  samples  collected  from  CMD  symptoma3c  young  shoots  using  the  method  described  by  Dellaporta  et  al.   (1983).  Polymerase   Chain   Reac3on   (PCR)   was   carried   out   to   detect  presence  of  Cassava  mosaic  geminiviruses  (CMGs).  The  specific  primers   JSP   1/2     and   EAB555-­‐F/R   were   used   to   amplify   the  cassava  mosaic   geminivirus   (CMG)   isolates   for   African   cassava  mosaic  virus  (ACMV)  (Fondong  and  Pita,  1998)  and  East  African  cassava   mosaic   virus   (EACMV)   DNA-­‐B   par3al   fragment   with  expected  product  sizes  of  774bp  (Fig  7)  and  556bp,  respec3vely  (Ndunguru  et  al.,  2005).  89  amplified  products  were  sequenced  and  the  amplicons  were  analysed  using  CLC  Main  Workbench  7  (2014).   Aligned   nucleo3de   sequences   were   compared   with  corresponding   fragment   sequences   of   CMGs   generated   using  the   BLAST   programme.     A   field   experiment   (Fig   1)   was  established  in  order  to  assess  the  response  of  cassava  cul3vars  to   CMD   infec3on.   Nine   CMD   symptomless   cassava   varie3es  were   arranged   in   a   randomized   complete   block   design   (RCBD)    with   four   replica3ons.  Data  on  CMD   incidence,  CMD  symptom  severity,   whitefly   popula3on   and   weight   of   root   tubers   was  collected   and   analysed   using   GenStat,   16th   Edi3on   computer  Package  (VSN  Interna3onal,  2013).  

Fig  1:     Field  experiment  established  at  a   “Hot  Spot”  area   for  CMD  and  whiteflies  in  Rufunsa,  Zambia  

Results    

Fig  2:  CMD  Incidences  in  surveyed  provinces  Disease  incidences  were  significantly  different  (P>0.05)  between  the  provinces  (Figure  2).  Lusaka  province  had  the  highest  CMD  incidence  of  87.5%  while  Eastern  province  had  the  lowest  CMD  incidence  of  43%.  The  varia3ons  could  be  a]ributed  to  whitefly  popula3ons  and  the  effect  these  have  on  CMD  spread.  Average  CMD   incidence   recorded   over   the   areas   surveyed   was   61.5%  with   57.7%   being   due   to   cuQng   infec3on   and   3.8%   being  whitefly   infec3ons,   results   consistent   with   studies   done  elsewhere   in  Africa   (Legg  and  Raya,  1998).  The  primary  source  of  infec3on  of  CMD  in  farmers  fields  is  through  infected  cuQngs  as  a  result  of  farmers  recycling  and  exchanging  infected  cuQngs  among   themselves   .   CMD  severity  during   this  period  averaged  2.7.

774 bp

556 bp

ACMV-MAT-16-1

ACMV-MAT-21-1

ACMV-MAT-23-1

ACMV-MAT-12-1

ACMV-MAT-14-1

ACMV-MAT-13-1

ACMV-MAT-20-1

ACMV-MAT-24-1

ACMV-MAT-22-1

AF112352 ACMV-Cam

AY211884 ACMV

AF366902 ACMV Cameroon DO2

AY211885 ACMV Cameroon DO3

AY562429 ACMV Ugand mild 2

AF126800 ACMV Uganda mild

AF126802 ACMV Uganda severe

ACMV-MAT-1-1

ACMV-MAT-11-1

ACMV-MAT-17-1

ACMV-MAT-4-1

ACMV-MAT-8-1

ACMV-MAT-9-1

ACMV-MAT-7-1

ACMV-MAT-6-1

ACMV-MAT-18-1

ACMV-MAT-5-1

ACMV-MAT-3-1

ACMV-MAT-10-1

ACMV-MAT-15-1

ACMV-MAT-2-1

EACMV Tz AY795986

100

95

78

98

99

99

89

8470

0.05

Table 1 Incidence, symptom severity, whitefly population, number and weight of tubers

Genotypes

Average attributes per variety

Incidence (%)

Symptom severity (1-5

scale)

Whitefly population

Number of root tubers

Weight of root tubers

(kg) Bangweulu 85 3.3 16.6 5.5 0.9 Chila 25 1.4 30.7 8.6 1.2 Kampolombo 0 1 18.2 6.2 1.1 Kapumba 71.2 2 4.6 1.7 0.2 Katoba Mpunta 91.2 3.1 19.6 5.9 0.6 Manyopola 100 3.7 15.3 2.6 0.3 Mweru 40 1.5 28.5 9 1.6 Nalumino 15 1.2 17.5 6.8 0.7 Tanganyika 71.2 2 10.6 7.2 0.6

Mean 55.4

2.1

18

5.9

0.8

LSD(0.01) 26.96 0.3 7.21 1.4 0.27 CV % 7.3 8 11.5 4.8 11.5  

Fig  4:  Distribu5on  of  cassava  viruses  in    surveyed  areas,  2013  

Fig   5:   Phylogene5c   tree   (1000   boot   strap   replica5ons)  obtained   from   comparisons   of   the   par5al   A   (lee)   and   B  (right)   component   sequences   from   Zambia   and   available  NCBI   genebank   cassava   mosaic   geminivirus   DNA-­‐A   and  DNA-­‐B  component  sequences,  respec5vely.    

A  phylogene3c  analysis  of  the  CP  of  Zambian  CMGs  yielded  a  tree   (Fig   5,   len)   that   showed   lower   gene3c   divergence   but  with  substan3al  homology  with  sequences  of  ACMV-­‐UGMild  Uganda   (AF126800.1),   ACMV-­‐UGSvr   Uganda   (AF126802.1),  ACMV-­‐[MG:MG310A1]   Madagascar,   and   ACMV-­‐CM39  Cameroon  (AY211462.1)  with  sequence  iden33es  of  97  %,  97  %,   97   %   and   98   %,   respec3vely.   Comparisons   of   the  sequences  of  the  Zambian  EACMV  isolates  with  those  of  the  corresponding   DNA-­‐B   genomic   regions   of   other   CMGs   in  GeneBank  (Fig.  5,  right),  showed  greater  variability  within  the  EACMV   species   with   similarity   to   the   Kenyan   isolates  (EACMV-­‐KE),   Malawian   isolates   (EACMMV)   and   Tanzanian  isolates  (EACMV-­‐TZT)  with  sequence  iden33es  of  96  %,  90  %  and  96  %,   respec3vely.   There  was   sequence   iden3ty   to   the  isolates  of  EACMZV  at  84  %,  EACMCV  at  80  %  and  SACMV  at  89  %.  

Field   experimental   results   showed   CMD   incidences   differed  significantly  (P≤0.05)  among  the  cassava  genotypes  (Table  1).  CMD   severity   differed   significantly   between   the   genotypes  (P≤0.05)   6   months   aner   plan3ng   (MAP)   on   a   severity   1-­‐5  scale.   CMD-­‐affected   improved   cassava   varie3es   expressed  mild  symptoms.  Manyopola  was  found  to  be  suscep3ble  (3.7)  whilst  Kampolombo  was  resistant   (1).  Despite  a  significantly  high  number  of  whiteflies   (P≤0.05)  observed  on  Chila   (30.7)  and  Mweru  (28.5),  the  genotypes  had  the  highest  significant  root  yields  (P≤0.05)  of  1.6  and  1.2  -­‐kg  plant-­‐1  respec3vely,  12  MAP.   However,   results   indicated   a   lack   of   associa3on  between   whitefly   popula3ons   and   spread   of   CMD   in  improved  varie3es  as   found   in   studies  by   Legg  et  al.   (2003)  and  Sserubombwe  et  al.  (2001).  

SP02-­‐08  

Preliminary  iden3fica3on  of  the  CMG’s  with  differen3al  primers  dis3nguished   EACMV   and   ACMV.   The   amplifica3on   products  are  as  shown   in  figure  3.  94.6%  of  the  samples  tested  posi3ve  for  either  ACMV  or  EACMV.  PCR  results  showed  that  57.5%  of  the  isolates  analysed  had  dual   infec3on,  32.8%  of  the  sampled  plants  were  infected  with  ACMV,  and  4.3%  with  EACMV  (Figure  4.  The  results  show  a  significant  increase  in  dual  infec3ons  and  corresponding  significant  reduc3ons  in  both  single  infec3ons  of  ACMV  and  EACMV  when  compared  to  previous  studies  (Chiko3  et   al.,   2013;   Muimba-­‐Kankolongo   et   al.,   1997).   There   were  more  dual   infec3ons   in  Western  and  Central  provinces  than   in  Eastern   and   Lusaka   provinces.   This   may   be   due   to   whitefly  popula3on   (especially   in   Western   province)   and   recycling   of  infected  materials  by  the  farmers.  

Conclusions  It  was  established  that  cassava  mosaic  disease   is  s3ll  widely  prevalent   and   that   infected   cuQngs   remains   the   primary  source   of   CMD.   Phylogene3c   analysis   and   Restric3on  Fragment   Length   Polymorphism   (RFLP)   showed   less  variability  within  the  ACMV  isolates  from  the  surveyed  fields  compared  to  the  EACMV  isolates.  Among  the  nine  genotypes  evaluated,  Manyopola  was  suscep3ble  and  Kampolombo  was  resistant.   Other   cassava   varie3es,   which   are   recommended  and  released  for  cul3va3on,  are  also  moderately  suscep3ble  to  the  disease.    

Acknowledgements  This   work   was   supported   by   the   USAID   Feed   the   Future  programme   through   the   Interna3onal   Ins3tute   of   Tropical  Agriculture   (IITA)   who   provided   financial   and   logis3cal  support.