Citrus Viroid Diseases

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Citrus Viroid Diseases
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What are Viroids?

• Small infectious, circular (under non denaturing
  conditions), single-stranded RNA molecules with
  the nucleotides paired to form stable structures
• Cause several important citrus diseases
• Implicated in several other diseases of
  undetermined etiology
• Five distinct viroids have been discovered in
  citrus

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Citrus Viroids

• Exocortis
• Cachexia (caused by variants of CVd-II)
• CVd-I
• CVd-III
• CVd-IV
• Variants are common within groups and mixed
  infections of several groups occur

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• Knowledge of the viroids first came from field
  observations to and eventually ones based on the
  inoculation of Etrog citron (Citrus medica L.) as
  an rapid indicator host
• Knowledge further came from the use of
  polyacrylamide gel electrophoresis (PAGE)
  procedures to separate and identify the
  individual viroids

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• Variations of the symptoms of viroids encountered
  in field trees on trifoliate orange (Poncirus
  trifoliata) rootstocks and in citron indicators
  was attributed to strain variation of the
  exocortis viroid
• Now known to be the presence of distinct viroids
• In addition to the exocortis bark scaling and the
  cachexia symptoms CVd-I, CVd-II, CVd-III and
  CVd-IV all reduce growth of trees on susceptible
  rootstocks

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Use of Viroids for Dwarfing

• Dwarfing produced by these viroids can be
  considered a disease however these dwarf trees
  are healthy in appearance and produce yields
  comparative to or better than healthy trees (when
  based on fruit per unit of canopy volume)
• In high density plantings viroid inoculated trees
  may give high yields while restricting tree size

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Differentiation of Viroids on Symptom Expression
in Citron
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Viroid Classification -Proposed
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Cachexia

• Affects mandarins and some mandarin hybrids
  (tangors and tangelos), alemow, Rangpur lime and
  kumquat
• Described in 1948 as a discoloration, gumming and
  browning of phloem tissues wood pitting and
  bark cracking on Orlando tangelo
• Related to the condition called xyloporosis in
  Palestine sweet lime

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• Problems when budwood is grafted on sensitive
  rootstocks or when infected trees are topworked
  with the aforementioned sensitive cultivars
• This viroid is widespread in old-line cultivars
  in the Mediterranean region
• Degree of injury varies with slight to severe
  stunting and can cause chlorosis, tree decline
  and tree death

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Host Range of Cachexia

• Most citrus species and cultivars can be infected
  but many are symptomless
• Highly susceptible include Orlando tangelo,
  Ellendale tangor and alemow
• Mild symptoms in sweet lime, rough lemon and
  Rangpur lime
• Experimental transmission to cucumber and other
  non citrus hosts

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Symptoms

• Discoloration and gumming of the bark which is
  seen when the bark is cut or scraped from the
  outer bark
• Inner phloem surface is bumpy with numerous
  rounded bumps or pegs that fit into depressions
  in the wood (xylem)
• Bark projections are not sharp like with tristeza
  stem pitting or cristicortis
• Warm conditions favor symptoms severely infected
  trees are stunted or chlorotic and may decline or
  die

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• Similar symptoms are found with gummy bark in
  sweet orange, gum pocket or gummy pitting in
  trifoliate orange and Kassala disease of
  grapefruit

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Causal Agent and Transmission

• Sequence variants of citrus viroid II (CVd-II)
• These are citrus variants of the hop stunt viroid
  (HSVd) group
• Isolates of CVd-IIa do not cause cachexia and
  differ from the pathogenic isolates of CVd-Iib
  and CVd-Iic by a few nucleotides in the variable
  domain
• Transmission is by grafting and as a contaminant
  of cutting tools apparently no vector of seed
  transmission

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Detection

• Identified in susceptible cultivars and
  rootstocks by symptoms
• Indexing of symptomless hosts by grafting of
  indicator plants such as Parsons Special
  mandarin grafted on a vigorous rootstock or
  Ellendale tangor on trifoliate orange
• Gum deposits develop within 6-12 mo near the base
  of the scion but must be under warm conditions
• sPAGE analysis, hybridization of extracts or
  tissue imprints with labeled n.a. probes or
  retrotranscription and PCR of extracts with
  specific primers

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Control

• Use viroid-free budwood
• Cachexia viroid is resistant to thermotherapy but
  can be eliminated using shoot-tip grafting
• Decontaminate cutting tools with sodium
  hypochlorite (1 chlorine)
• Avoid topworking trees with susceptible cultivars

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Exocortis

• Known in Australia as scaly butt
• A bark scaling and stunting disease of trees on
  trifoliate orange and trifoliate hybrids and
  Rangpur lime rootstocks
• Rarely lethal and fruit quality is not affected
  but productivity is severely affected

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Causal Agent

• CEVd viroid contains 370-375 nucleotides,
  largest of the citrus viroids
• Field isolates may contain a mixed complex of
  closely related sequence variants
• CEVd is the only viroid to cause the severe bark
  scaling symptoms but the others can cause
  significant stunting
• CEVd is highly resistant to heat inactivation and
  to chemicals that inactivate viruses

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• Can remain infectious for long periods
  inactivated by hydrolysis, ribonucleases and
  exposure to hypochlorite solutions
• Transmission by grafting and dissemination
  primarily by symptomless budwood
• Mechanically transmitted by on cutting/prunig
  tools
• Seed and vector transmission not demonstrated
• Wide range of experimental non citrus hosts (e.g.
  grape, tomato, eggplant, etc.

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Host Range and Symptoms

• Most citrus species and cultivars and some citrus
  relatives and some noncitrus hosts
• Sensitive trifoliate orange (Poncirus
  trifoliata), Rangpur lime, and some citrons and
  lemons all of which show stem blotching and bark
  splitting
• Some citrons show leaf epinasty and vein necrosis
• Sweet oranges, grapefruit, and mandarin are
  symptomless

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• However if grafted on sensitive rootstocks the
  rootstock will show bark scaling and the tree
  will be stunted
• Bark shows longitudinal splits and patches of
  dead scales of the outer bark will appear
• Inner bark remains alive and a new layer of
  scales can form as the outer ones slough off
• General decline of the canopy of the tolerant
  cultivar may occur even without rootstock scaling

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Detection

• Bark scaling on rootstock and stunting of the
  tree but usually not until trees are 4-8 yrs old
• Stunting may not be obvious unless comparisons
  are made
• Indexing on Etrog citron Arizona 861-S1on a
  vigorous rootstock severe leaf epinasty and bark
  splitting 1-3 mo.
• PAGE or sPAGE assays, hybridization of extracts
  or tissue imprints with labeled nucleic acid
  probes, retrotranscription and PCR using specific
  primers

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Control

• Use CEVd-free budwood on sensitive rootstocks
• Shoot-tip grafting to free materials of the
  viroid
• Decontamination of cutting and pruning tools
  including hedging and harvesting equipment with
  sodium hypochlorite (1 chlorine)

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Gummy Bark

• Graft transmissible disorder of sweet orange in
  eastern Mediterranean, North Africa, and the Near
  East
• Discoloration and gum impregnation of the bark
  and appears similar to that seen with cachexia
  viroid
• In mild cases few gum pockets near the budunion
  but with severe cases the gummy bark are
  widespread on the trunk and may extend to
  scaffold limbs

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• Occurs on trees 8-10 years old which has hampered
  identification
• Most trees with this also have other viroids and
  virus-like pathogens
• Most consistent associated with CVd-II

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Gum Pocket and Gummy Pitting

• Reported in Argentina and South Africa on trees
  grafted on trifoliate rootstock
• Similar to a disease described in Australia and
  Italy
• Symptoms include pits with a pocket of gum in the
  trifoliate orange rootstock usually more severe
  on the trunk exposed to the sun
• Trees are stunted
• Causal agent related to CVd-II variants

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Shell Bark

• Occurs in old-line lemon trees over 10 yrs old
• Symptoms outer bark dies and cracks into
  vertical strips which may enlargen on the trunk
  and the major limbs
• Inner and cambium remain intact
• Trees may show some decline
• On lemons with rootstocks other than lemon will
  have symptoms only on scion
• Other scions include smooth flat Seville oranges,
  bitter sweet orange, shaddock and other specific
  cultivars

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• Trees have a number of citrus viroids including
  exocortis
• Shoot-tip grafting eliminates problem
• Budwood from know disease-free trees
• Pruning after the bark shells off and before new
  bark is produced

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Control of Viroid Diseases

• Use clean certified disease free budwood
• Resistant rootstocks
• Avoid propagation from scions on resistant
  rootstocks unless certified disease free
• Clean pruning and cutting tools with sodium
  hypochlorite (1 chlorine content)

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• The distribution of citrus viroids Citrus
  exocortis viroid (CEVd), Citrus bent leaf viroid
  (CBLVd), Citrus viroid (CVd)-I-LSS, Hop stunt
  viroid (HSVd), Citrus viroid III (CVd-III),
  Citrus viroid IV (CVd-IV), and Citrus viroid OS
  (CVd-OS) and Apple stem grooving virus (ASGV
  synonym Citrus tatter leaf virus) were
  investigated using the multiplex reverse
  transcription polymerase chain reaction on
  samples from 217 citrus trees in Japan. HSVd and
  CVd-III were the first and second most frequently
  detected, respectively. CVd-I-LSS and CVd-OS were
  also frequently detected from some species or
  varieties, whereas CEVd, CBLVd, CVd-IV, and ASGV
  were detected only occasionally