Interna'onal  Research  Conference  on  Huanglongbing  

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Entomology  Presenta'ons  

Today’s  Summaries  

•  Psyllid  biology  /  ecology  /  HLB  pathogen  transmission  

•  Insec=cide-­‐based  management  of  ACP  /  HLB  

•  Molecular-­‐based  approaches  to  ACP  management  

Psyllid  Biology,  Ecology  and  HLB  Pathogen  Transmission  

Poster  5.11  The  stripped  mealybug,  Ferrisia  virgata,  a  new  vector  of  the  Huanglongbin  bacterium  “Candidatus  Liberibacter  asia=cus.”  Pi=no,  M.,  Hoffman,  M.T.,  Zhou,  L.,  Duan,  Y.P.  and  D.  Hall  

•  Found  to  readily  acquire  the  HLB  pathogen  •  Not  as  likely  to  inoculate  a  plant  with  the  pathogen  compared  to  psyllids  (reduced  inocula=on  success  rate)  

•  Pest  with  limited  dispersal…minimal  spread  capabili=es  •  Possible  headache  for  fresh  fruit  movement  

Poster  2.11    Occurrence  of  Diaphorina  citri  in  an  unexpected  ecosystem:  the  Lake  Kissimmee  State  Park  forest.    Mar=ni,  X.,  Addison,  T.,  Fleming,  B.,  Jackson,  I.,  Pelz-­‐Stelinski,  K.,  and  L.L.  Stelinski  

•  Yellow  s=cky  traps  used  to  survey  for  psyllids  in  Kissimmee  forest  (Jul  –  Oct  2012).  

•  Closest  citrus  grove  2.3  km    •  Low  levels  of  psyllids  trapped,  but  shows  they  are  present  •  No  psyllids  observed  on  wild  plants  in  the  swamp  •  Laboratory  no-­‐choice  tests  with  3  most  abundant  plant  species  found  in  

swamp  •  Gallberry,  Blueberry  and  Red  Bay  •  All  unsuitable  for  adult  psyllid  long-­‐term  survival  (dead  acer  3  days)    •  No  reproduc=on  found  on  these  hosts  

•  While  no  wild  hosts  iden=fied  in  this  study,  provides  indirect  evidence  that  psyllids  could  be  moving  from  swamps  into  adjacent  citrus  groves  

Talk 5.2. Induced release of a plant-defense volatile ‘deceptively’ attracts insect vector to plant infected with a bacterial pathogen. Stelinski, L.L., Mann, R.S., Ali, J.G., Hermann, S.L., Tiwari, S., Pelz-Stelinski, K.S., and H.T. Alborn.

•  Citrus  trees  produce  methyl  salicylate  in  response  to  HLB  infec=on  as  well  as  psyllid  feeding  

•  Methyl  salicylate  has  been  iden=fied  as  an  agractant  for  both  pests  and  beneficials  in  a  number  of  crops  

•  In  citrus,  it  serves  to  agract  psyllids  to  HLB  infected  trees  (to  pick  up  Las)  •  Psyllids  are  preferen=ally  agracted  to  newly  HLB-­‐infected  trees  •  As  trees  decline  (old  infec=on),  agrac=on  of  psyllids  to  HLB  infected  trees  decreases  

•  Increased  nutri'onal  sprays  to  ‘old  infec'on’  trees  increases  their  aErac'veness  to  psyllids  

Talk  7.4.  Vector  control  and  foliar  nutri=on  for  management  of  huanglongbing  in  Florida  citrus.  Stansly,  P.A.,  Arevalo,  H.A.,  Qureshi,  J.A.,  Jones,  M.M.,  Hendricks,  K.,  Roberts,  P.D.,  and  F.M.  Roka.  

•  Experiment  was  conducted  that  included  the  following  treatments:  1)  Control,  2)  Insec=cide  for  psyllids,  3)  nutri=onal  sprays,  and  4)  Insec=cide  for  psyllids  +  nutri=onal  sprays.  

•  No  treatments  reduced  HLB  infec=on  rate…increased  to  ~95%  •  Ct  values  were  higher  in  insec=cide  treated  plots  sugges=ng  reduced  disease  severity  

•  Yields  higher  in  vector  control  plots  in  yrs  2-­‐4  •  Increased  yields  in  nutri=on  plot  in  yr  4  only  •  Combined  insec=cide  +  nutri=on  treatment  was  best  in  all  4  yrs  

Talk  5.7    Low  acquisi=on  rates  of  ‘Ca.  Liberibacter  asia=cus’  by  Diaphorina  citri  Kuwayama  from  citrus  plants  exposed  to  high  temperatures  Lopes,  S.A.,  Luiz,  F.,  Mar=ns,  E.C.,  Fassini,  C.G.,  Sousa,  M.C.,  Barbosa,  J.C.,  Beape,  G.A.C.  

•  Observa=ons  in  Brazil  suggested  that  HLB  spread  was  more  slow  to  occur  in  the  warmer  areas  of  the  country  

•  Examined  the  effects  of  temperature  on  the  ability  of  psyllids  to  acquire  the  Las  bacterium  from  new  leaf  growth  

•  Found  when  temperatures  were  higher  (up  to  100F)  the  levels  of  Las  dropped  in  the  new  leaf  =ssues  

•  Acquisi=on  of  Las  by  psyllids  also  decreased  •  This  effect  declined  the  longer  a  tree  was  infected  with  the  HLB  pathogen  

•  The  longer  HLB  infected  trees  stay  in  the  field,  the  less  likely  bacterial  popula=ons  will  fluctuate  at  different  =mes  of  the  year,  thus  the  plants  will  be  more  likely  to  serve  as  a  year-­‐round  source  of  inoculum  for  psyllids  to  acquire  the  pathogen.  

Poster  5.9  Seasonal  shics  in  Candidatus  Liberibacter  asia=cus  prevalence  in  the  vector  Diaphorina  citri  in  Florida.  Ebert,  T.A.,  Brlansky,  R.H.  and  M.E.  Rogers  

•  (2008-­‐2012)  survey  of  the  incidence  of  psyllids  carrying  the  HLB  pathogen  (pcr  analysis  of  ~70,000  ACP)  

•  Are  there  =mes  of  the  year  psyllid  control  is  more  important  to  reduce  HLB  spread?  

•  Years  1-­‐2  :  more  pronounced  seasonal  effects,  par=cularly  in  the  cooler  fall  and  winter  months  (Sept-­‐Feb)  –  Also  =me  when  more  flush  was  present  for  nymphal  acquisi=on  

•  Years  2-­‐3:  seasonal  effects  on  psyllids  carrying  the  pathogen  were  less  pronounced.  –  In  loca=ons  with  a  high  incidence  of  HLB  infected  trees,  incidence  

of  psyllids  carrying  pathogen  was  high  (>60%)  year-­‐round  

Poster  5.9  Seasonal  shics  in  Candidatus  Liberibacter  asia=cus  prevalence  in  the  vector  Diaphorina  citri  in  Florida.  Ebert,  T.A.,  Brlansky,  R.H.  and  M.E.  Rogers  

•  In  loca=ons  with  lower  tree  infec=on  rates  (and  beEer  psyllid  control)  incidence  of  psyllids  carrying  the  HLB  pathogen  was  significantly  lower  (<5%  on  average)  

•  Overall,  the  results  show  that  as  the  rate  of  HLB  infected  trees  increases  over  'me,  so  does  the  incidence  of  psyllids  carrying  the  pathogen,  thus  the  need  for  year-­‐round  control  of  ACP.  

Insec=cide-­‐based  Management  of  ACP  /  HLB  

Poster  1.7  Incidence  of  Huanglongbing  in  commercial  orchards  in  northwest  Parana,  Brazil  Mula=,  F.,  Nocchi,  P.T.R.,  Zanugo,  C.A.,  Belasque,  J.,Nunes,  W.M.C.  

•  Examined  the  =ming  of  insec=cide  sprays  on  different  varie=es  and  the  effect  on  HLB  infec=on  rate  

•  (Once  infected  trees  were  detected  they  were  removed)  •  Applica=ons  =med  to  the  presence  of  psyllids  (scou=ng)  

•  0.1  –  0.4%  HLB  infected  trees  •  Applica=ons  made  only  when  new  flush  present  

•  2-­‐10%  HLB  •  Calendar-­‐based  applica=ons  (every  20  days)  

•  0.04-­‐0.12%  HLB  •  Results  showed  that  HLB  infec=on  rates  remained  lowest  using  the  preven=ve  calendar-­‐based  sprays  (every  20  days)  

Poster  6.16  Thresholds  for  HLB  vector  control  in  infected  commercial  citrus  and  compa=bility  with  biological  control  Monzo,  C.  Hendricks,  K.,  Roberts,  P.  and  P.A.  Stansly  

•  Calendar-­‐based  monthly  sprays  provided  best  control  of  psyllids  popula=ons  

•  No  difference  in  psyllid  popula=ons  where  no  insec=cide  used  compared  to  using  a  threshold  of  0.2  or  0.7  psyllids  

•  Conducted  a  ‘preliminary’  economic  analysis  which  suggested  that  while  yields  were  higher  in  the  calendar  spray  treatments,  the  costs  of  their  insec=cide  treatments  ($555.82/A)  did  not  jus=fy  the  yield  increase  

•  They  also  emphasized  the  fact  that  the  calendar  spray  program  had  hidden  costs  in  terms  of  killing  beneficials  

 •  1%    oil  sprays  were  evaluated  for  psyllid  control  •  There  was  an  ini=al  70%  reduc=on  in  psyllid  coloniza=on  of  oil  

 treated  plants.  •  The  authors  suggest  that  oil  has  a  repellent  effect  on  Asian  citrus  

 psyllid.  •  However,  psyllids  colonize  new  flush  acer  treatment  (dries),  which  

 reduces  effec=veness    of  mineral  oil  treatments.  •  Coverage  using  mineral  oil  is  paramount  for  it  to  be  effec=ve.  •  Immediately  following  treatment,  an  82%  reduc=on  of  egg  laying  

 was  observed  on  oil  treated  plants  •  Both  rain  and  development  of  new  flush  reduced  effec=veness.  •  1%  mineral  oil  treatment  acts  as  a  psyllid  repellent  and  causes  up  

 to  8%  mortality  of  adults.  

Talk  6.8    Effect  of  mineral  oil  on  host  selec=on  and  control  of  Diaphorina  citri  on  citrus.  Miranda,  M.P.,  Micelli,  M/L/,  Felippe,  M.R.,  Caldeira,  R.E.,  and  Yamamoto,  P.T.    

Poster  6.20  Frequent  low  volume  sprays  of  Hor=cultural  Mineral  oil  (HMO)  for  psyllid  and  leafminer  control  Jones,  M.M.,  and  P.A.  Stansly  

•  Field  trials  comparing  the  efficacy  of  low  volume  oil  sprays  versus  conven=onal  insec=cide  sprays  

•  Conven=onal  insec=cide  sprays  performed  beger  in  terms  of  reducing  overall  psyllid  popula=ons  throughout  most  of  the  year  

•  Most  pronounced  when  psyllid  popula=ons  were  at  their  highest  levels  

•  LV  oils  sprays  did  provide  some  suppression  of  psyllids,  par=cularly  the  nymphal  stage  

•  There  was  no  difference  between  any  of  the  treatments  in  terms  of  juice  quality  

Poster  6.24  Introducing  DuPont  Exirel  and  Verimark  new  insect  control  products  for  pest  management  and  op=mizing  yields  in  Florida  citrus  Por=llo,  H.E.,  Royal,  S.S.,  Taylor,  J.E.,  Temple,  J.H.,  Truszkowski,  A.T.,  Mares,  J.T.,  Cameron,  R.A.,  Anan,  I.B.  and  J.M.  Alvarez.  

•  Cyazypyr  is  a  new  mode  of  ac=on  that  provides  excellent  control  of  both  leafminer  and  psyllid  

•  Field  trial  results  show  dura=on  of  control  equal  to  if  not  beger  than  most  commercial  standards  

•  Presented  EPG-­‐based  feeding  data  showing  phloem  feeding  reduced  but  not  prevented,  psyllids  s=ll  spent  14%  of  =me  in  phloem  on  cyazypyr  treated  plants  

•  Pathogen  transmission  can  happen  at  any  =me  during  phloem  feeding,  there  is  no  =me  requirement  once  the  phloem  is  reached  

•  Two-­‐year  field  trial  using  primarily  soil  applica=ons  of  cyazypyr  on  young  trees  reduced  HLB  infec=on  rate  from  71%  (untreated)  to  25-­‐31%  in  cyazypyr  treatments.  

•  While  this  product  is  not  going  to  directly  prevent  pathogen  transmission  from  occurring,  this  will  be  a  valuable  new  tool  for  controlling  both  psyllid  and  leafminer  and  helping  to  prevent  insec=cide  resistance.  

Molecular  approaches  for  psyllid  control  

RNAi  approach  to  psyllid  control  

•  Citrus  psyllid  genome  project  provides  new  approaches  to  psyllid  control  

•  Once  you  know  what  a  certain  gene  does,  you  can  shut  off  that  gene  using  dsRNA  

Poster  6.27  Targe=ng  juvenile  hormone  metabolic  genes  in  the  Asian  citrus  psyllid  as  a  strategy  to  reduce  the  spread  of  citrus  greening  disease  Van  Ekert,  E.,  Borovsky,  D.,  Powell,  C.A.,  Cave,  R.D.,  Alessandro,  R.T.  and  R.G.  Shagers  

•  Iden=fica=on  of  gene  regula=ng  juvenile  hormone  •  Juvenile  hormone  regulates  nymphal  growth  and  development  •  Key  in  the  mol=ng  process  (one  nymphal  stage  to  the  next)  

•  Could  now  target  this  gene  to  prevent  nymphal  development  

•  Using  potato  psyllid  as  a  model  to  look  for  genes  to  target  in  ACP    

•  Important  found  genes  include  Hemolysin  A,  HLyE,  pore  forming  toxin,  biofilm  forma=on.  These  genes  may  implicated  in  Las  circula=on  and  propaga=on  within  ACP    

Talk  5.6  Transla=ng  Anatomical  Structures  and  Func=onal  Genomics  of  Candidatus  Liberibacter  asia=cus  and  solanacearum  Into  Circula=ve,  Propaga=ve  Vector-­‐Mediated  Transmission  Processes    Tonja  Fisher,  J.  Cicero,  M.  Vyas,  R.  He,  W.  Nelson,  M.  Willer,  C.  Soderlund,  D.  Gang,  and  J.K.  Brown  

Talk  4.2  Sequencing  and  annota=on  of  the  Wolbachia  endosymbiont  of  Diaphorina  citri  by  the  CGHLB  Genome  Resources  group  reveals  candidate  sources  of  interac=on  with  the  insect  host  Surya  Saha,  Wayne  Hunter,  and  Magdalen  Lindeberg  

•  Generated  and  annotated  drac  genome  of  the  Wolbachia  endosymbiont  of  ACP  

•  Annoted  genes  include:  – 54  predicted  ankyrin  proteins  (may  play  a  role  in  host  reproduc=ve  manipula=on)  

– Type  IV  secre=on  system  (linked  export  of  ankyrins,  which  regulate  host  gene  expression)  

– bacterioferri=n  (linked  to  host  iron  homeostasis)  

Poster  6.13  Genera=ng  Asian  citrus  psyllids  with  twis=ng  wings  to  prevent  the  spread  of  greening  disease  El-­‐Shesheny,  I.,  Harjeri,  S.  Gowda,  S.  and  N.  Killiny  

•  Topical  applica=on  of  dsRNA  to  psyllid  nymphs  targe=ng  wing  forming  gene  

•  Using  the  ctv  vector  approach,  created  plants  expressing  this  par=cular  dsRNA  

•  Wing  gene  may  not  be  suitable  for  use  in  transgenic  citrus  because  it  may  affect  other  insects  (including  beneficials)  

•  Have  iden=fied  11  unique  psyllid  genes  ready  for  screening  •  Currently  not  CRDF  funded,  so  unclear  how  quickly  this  will  move  forward  

Talk  5.3  Disrupt  the  bacterial  growth  in  the  insect  vector  to  block  the  transmission  of  Candidatus  Liberibacter  Asia=cus  to  citrus,  the  causal  agent  of  citrus  greening  disease  Killiny,  Nabil,  Hajeri,  S.,  Gowda,  S.,  and  Davis,  M.  J.  

•  Biofilm  forma=on  required  for  HLB  pathogen  transmission  

•  Acyl-­‐Homoserine  lactones  (AHLs)  produced  by  a  psyllid  endosymbiont  play  key  role  in  binding  of  Las  bacteria  (biofilm  forma=on)  

•  LuxR  protein  can  bind  to  AHL  preven=ng  Las  bacteria  from  agaching  and  forming  biofilm  

•  Can  introduc=on  of  extra  LuxR  protein  disrupt  biofilm  forma=on  causing  psyllids  to  be  unable  to  transmit  bacteria?  

•  Developed  plants  which  produce  LuxR  protein  •  Bind  to  available  AHLs  inside  insect  responsible  for  biofilm  forma=on  

•  Psyllids  which  feed  on  these    plants  lost  the  ability  to  transmit    

       the  HLB  pathogen    (biofilm  detachment      and  dispersal)  

Talk  5.3  Disrupt  the  bacterial  growth  in  the  insect  vector  to  block  the  transmission  of  Candidatus  Liberibacter  Asia=cus  to  citrus,  the  causal  agent  of  citrus  greening  disease  Killiny,  Nabil,  Hajeri,  S.,  Gowda,  S.,  and  Davis,  M.  J.  

•  Proof  of  Concept    -­‐  Targe=ng  AHLs  with  LuxR  proteins  can  prevent  pathogen  transmission  by  psyllids  

•  Posi=ve  Agributes:  safe  for  humans,  only  targets  endosymbiont  systems  within  the  psyllid  

•  Transgenic  plant  lines  will  be  produced,  that  will  also  target  other  systems  within  the  plant  preven=ng  pathogen  acquisi=on  from  plants  as  well.  

 

Talk  5.3  Disrupt  the  bacterial  growth  in  the  insect  vector  to  block  the  transmission  of  Candidatus  Liberibacter  Asia=cus  to  citrus,  the  causal  agent  of  citrus  greening  disease  Killiny,  Nabil,  Hajeri,  S.,  Gowda,  S.,  and  Davis,  M.  J.