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Molecular re-evaluation of
Phytophthora species isolated during
   30 years of vegetation health
   surveys in Western Australia
Mike Stukely, Giles Hardy, Dianne White, Janet Webster,
          Juanita Ciampini and Treena Burgess
Jarrah Forest plain
       Northern sand



Swan Costal Plain

       Black gravel site




   Fitzgerald National Park
Impact of Phytophthora cinnamomi on plant species
              in Western Australia

Direct Impacts
• Out of 5710 described species in the South-West Botanical Province
• 2285 species susceptible (40%)
• 800 highly susceptible (14%)

Indirect Impacts
• Loss of biomass                Phytophthora is listed as a
                                 ‘KEY THREATENING PROCESS’
• Loss of litter
                                 to Australia’s Biodiversity by
• Loss of refugia for fauna      Commonwealth Government
• Loss of food resources
• Increased exposure
• Loss of pollinators
• Loss of nesting sites
Dieback mapping and vegetative health surveys

•For over 30 years aerial photography has been used to map the extent
 of disease in WA
•Many areas are uninterpretable aerially and ‘Dieback Interpreters’ are
 sent in to ground truth
•They are trained to recognise susceptible indicator species. First
 indication of the presence of P. cinnamomi is the death of susceptible
 indicator species from the families Proteaceae, Epacridaceae,
 Dilleniaceae, Xanthorrhoeaceae, Fabaceae.
•Usually ground truth areas of native vegetation due to be logged
•Samples are taken (usually soil from rhizosphere of dying plants) and
 sent to vegetation heath service (VHS)
•Soils are baited with cotyledons of Eucalyptus sieberi
•After 5(-10) days cotyledons are examined and plated onto selective
 media
•About 15% of phytophthora isolates are not P. cinnamomi
Phytophthora Management Map

                                                     Disease-free
Uninterpretable




                             Phytophthora infested

Green = Dieback free;   Purple = Uninterpretable; Red = Infested
Phytophthora species in natural ecosystems in WA

•Until recently indentified only by morphological features
P. citricola
P. megasperma
P. cryptogea
P. drechsleri
P. nicotianae
P. boehmeriae

•Other species are found in nurseries and in horticulture
•Over the past 2 years we have been conducting a molecular re-
 evaluation of Phytophthora spp. in natural ecosystems in WA
•Over 250 isolates so far examined
•Sequenced ITS region and constructed molecular phylogeny
97   P. inundata
                                                                                                     100
                                                                                    91                                100    P.sp.6 = P. sp. personii
                                                                                                    100     P.sp. 10
                                                                                         100         P.sp. 7
                                                                                                    100 P.sp. 3
                                                                                                        100                                                   Group 6
                                                                                              97              P.sp. 8
                                                                                      77             100
                                                                                                       P.sp. 11
                                                                                    66         100    P. megasperma
                                                               100
                                                                                         97    P. gonapodyides
                                        99
                                                                                       P. sp. hungarica
                                                                                    P. sp. sylvatica
                                                                100
                                                                                   P. sp. asparagi
                                                                                                                                    P. inflata
                                                                                                                            98
                                                                                                                       67         P. citricola
                                                                                                                            95     P.sp. 4
                                                                                  100
                                                                                                                                       96
                                                                                                                                             P. tropicalis
                                                                                                                            100
                                                                                                                                            100 P. capsici    Group 2
                                                         56
                            53                                                     100           P. multivesiculata
                                                                    98                                         100                      P. alticola
                                                                                            100
                                                                             100                        100 P. bisheria
                                                                                          100 P.sp. 2
                                                                                   100
                                             52                100                                P. arecae                                                   Group 4
                                                                                     100
                                                                               100         P. frigida
                                                                                    100
                  95
                                                                              100       P.sp. 1
                                 78
                                                         100                                                          P. nicotianae                           Group 1
                                                                            100          P. cactorum
                                                                            100
                                                                                                                                                              Group 3
                                                                                                      P. ilicis
                                              87
                                                                      100                    P. katsurae
                                                                                                                                                              Group 5
                                                                                           100            P. cambivora
                                                                    85
                                                                                         100         P. cinnamomi                                             Group 7
                                      100                                                             100
                                                                                                                 P. melonis
                                                                            100                           P. sp. niederhauseria
                                                                                               93     P. sojae
                                            100                  P. ramorum
                       71                                         100
100                                                                         P. drechsleri
                                            100
                                                                      P. cryptogea                                                                            Group 8
                                                               98
                                                               87     P.sp. 5 = P. cryptogea
                                                                                         100                            P. kernoviae
                                      100
                                                                                  100
                                                                                                          P. boehmeriae
                                                                                                                                             94 P. captiosa   Group 9
                                                                                                                  100
                                                   100                                                                                       93 P. fallax
                                                                                                           100
                                                                                                                                 P.sp. 9
      5 changes
96
                                                                                                  P. humicola
                                                                               93
                                                                                         P. inundata
                                                                     100

                                                                                        100         P. sp. personii
                                                 91

                                                                                   64        TCH 009 - P.sp. 10
                                                                              68
                                                                                             VHS 17779 - P.sp. 10
                                                                  100                         Phytophthora sp. P462 AF541911
                                                                                                Phytophthora sp. P532 DQ512952
                                                                   88
                                                                        Phytophthora sp. PgChlamydo
                                                             82
                                                                     P. gonapodyides
                                                                                    Phytophthora sp. P1049 AF541904
                                                                     100              Phytophthora sp. P896 AF541903
                                                                                    VHS 9854 - P.sp. 7
                                    100                                              DCE 68 - P.sp. 7
                                                                 DDS 3599
                                                                         91
                                                                               P.sp. 3
                                                       54
                                                                                         VHS 16108
                                                                                   67                     VHS 17350
                                                                   96                        97

                                                                                                   80   P.sp. 8
                                                                                    82            VHS 16115
                  100                             52                    51
                                                            65
                                                                                        77        P.sp. 11
                                                                                              VHS 5185
                                                                  100
                                                                              P. megasperma
                                            88
            100
                                                         P. hungarica

                                                      P. sylvatica
                                      100
      100
                                                             P. sp. asparagi
                        100
                                                                         P. citricola
                              100
                                                      P. nicotianae

                                                                                                         P. boehmeriae
5 changes
Often the morphological and molecular
          identifications do not match



Isolates     Morphology                  DNA

Many         P.   citricola              P.sp.4
Many         P.   citricola              P.sp.2
All tested   P.   drechsleri             P.sp.3/8
Two          P.   megasperma             P.sp.7
Many         P.   megasperma             P.sp.9
TCH009       P.   megasperma var sojae   P.sp.10
VHS17183     P.   megasperma             P.   megasperma
All tested   P.   nicotianae             P.   nicotianae
All tested   P.   cinnamomi              P.   cinnamomi
All Tested   P.   cryptogea              P.   cryptogea
Phytophthora species in natural ecosystems in WA

•New records for WA
P. inundata
P. gonapodyides
P. sp. asparagi
P. sp. niederhauseria
P. sp. personii
•At least seven new Phytophthora species which are genetically
 distinct to currently described species
Phytophthora species in natural ecosystems in WA
Species     No.   Host species
P. sp. 1    10    B. menziesii, B. littoralis,
                  B. attenuata, B media
P. sp. 2    21    Eucalyptus marginata, Corymbia calophylla, B. grandis,
                  Dryandra squarrosa, Patersonia xanthina
P. sp. 3    15    E. marginata, B. attenuata,
                  B. grandis; Pinus radiata (plantation)
P. sp. 4    64    E. marginata, B. attenuata, B. grandis, B. littoralis, B.
                  menziesii, B. prionotes, Conospermum sp., Leucopogon
                  verticillatus, X. gracilis, Podocarpus drouyniana,
                  Patersonia sp., Bossiaea sp., Gastrolobium spinosum;
                  Pinus radiata (plantation)
P. sp. 7    4     X. preissii; Pinus radiata (plantation)
P. sp. 8    3     Soil and water baits – native forest
P. sp. 9    23    B. attenuata, B. baxteri, D. cirsioides, D. falcata, A.
                  cuneata, Isopogon sp.; Pinus radiata (plantation)
P. sp. 10   2     B. prionotes, B. grandis
P. sp. 11   2     Banksia sp., X. preissii
O10 Treena
O10 Treena
O10 Treena
Significance of new phytophthora species

•Do they pose a threat to biodiversity?
•If yes, then management strategies will be developed
•Need to investigate
 host range/pathogenicity
 distribution
 factors affecting spread
•Are these species introduced?
•Are any of these species hybrids?
O10 Treena
O10 Treena

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O10 Treena

  • 1. Molecular re-evaluation of Phytophthora species isolated during 30 years of vegetation health surveys in Western Australia Mike Stukely, Giles Hardy, Dianne White, Janet Webster, Juanita Ciampini and Treena Burgess
  • 2. Jarrah Forest plain Northern sand Swan Costal Plain Black gravel site Fitzgerald National Park
  • 3. Impact of Phytophthora cinnamomi on plant species in Western Australia Direct Impacts • Out of 5710 described species in the South-West Botanical Province • 2285 species susceptible (40%) • 800 highly susceptible (14%) Indirect Impacts • Loss of biomass Phytophthora is listed as a ‘KEY THREATENING PROCESS’ • Loss of litter to Australia’s Biodiversity by • Loss of refugia for fauna Commonwealth Government • Loss of food resources • Increased exposure • Loss of pollinators • Loss of nesting sites
  • 4. Dieback mapping and vegetative health surveys •For over 30 years aerial photography has been used to map the extent of disease in WA •Many areas are uninterpretable aerially and ‘Dieback Interpreters’ are sent in to ground truth •They are trained to recognise susceptible indicator species. First indication of the presence of P. cinnamomi is the death of susceptible indicator species from the families Proteaceae, Epacridaceae, Dilleniaceae, Xanthorrhoeaceae, Fabaceae. •Usually ground truth areas of native vegetation due to be logged •Samples are taken (usually soil from rhizosphere of dying plants) and sent to vegetation heath service (VHS) •Soils are baited with cotyledons of Eucalyptus sieberi •After 5(-10) days cotyledons are examined and plated onto selective media •About 15% of phytophthora isolates are not P. cinnamomi
  • 5. Phytophthora Management Map Disease-free Uninterpretable Phytophthora infested Green = Dieback free; Purple = Uninterpretable; Red = Infested
  • 6. Phytophthora species in natural ecosystems in WA •Until recently indentified only by morphological features P. citricola P. megasperma P. cryptogea P. drechsleri P. nicotianae P. boehmeriae •Other species are found in nurseries and in horticulture •Over the past 2 years we have been conducting a molecular re- evaluation of Phytophthora spp. in natural ecosystems in WA •Over 250 isolates so far examined •Sequenced ITS region and constructed molecular phylogeny
  • 7. 97 P. inundata 100 91 100 P.sp.6 = P. sp. personii 100 P.sp. 10 100 P.sp. 7 100 P.sp. 3 100 Group 6 97 P.sp. 8 77 100 P.sp. 11 66 100 P. megasperma 100 97 P. gonapodyides 99 P. sp. hungarica P. sp. sylvatica 100 P. sp. asparagi P. inflata 98 67 P. citricola 95 P.sp. 4 100 96 P. tropicalis 100 100 P. capsici Group 2 56 53 100 P. multivesiculata 98 100 P. alticola 100 100 100 P. bisheria 100 P.sp. 2 100 52 100 P. arecae Group 4 100 100 P. frigida 100 95 100 P.sp. 1 78 100 P. nicotianae Group 1 100 P. cactorum 100 Group 3 P. ilicis 87 100 P. katsurae Group 5 100 P. cambivora 85 100 P. cinnamomi Group 7 100 100 P. melonis 100 P. sp. niederhauseria 93 P. sojae 100 P. ramorum 71 100 100 P. drechsleri 100 P. cryptogea Group 8 98 87 P.sp. 5 = P. cryptogea 100 P. kernoviae 100 100 P. boehmeriae 94 P. captiosa Group 9 100 100 93 P. fallax 100 P.sp. 9 5 changes
  • 8. 96 P. humicola 93 P. inundata 100 100 P. sp. personii 91 64 TCH 009 - P.sp. 10 68 VHS 17779 - P.sp. 10 100 Phytophthora sp. P462 AF541911 Phytophthora sp. P532 DQ512952 88 Phytophthora sp. PgChlamydo 82 P. gonapodyides Phytophthora sp. P1049 AF541904 100 Phytophthora sp. P896 AF541903 VHS 9854 - P.sp. 7 100 DCE 68 - P.sp. 7 DDS 3599 91 P.sp. 3 54 VHS 16108 67 VHS 17350 96 97 80 P.sp. 8 82 VHS 16115 100 52 51 65 77 P.sp. 11 VHS 5185 100 P. megasperma 88 100 P. hungarica P. sylvatica 100 100 P. sp. asparagi 100 P. citricola 100 P. nicotianae P. boehmeriae 5 changes
  • 9. Often the morphological and molecular identifications do not match Isolates Morphology DNA Many P. citricola P.sp.4 Many P. citricola P.sp.2 All tested P. drechsleri P.sp.3/8 Two P. megasperma P.sp.7 Many P. megasperma P.sp.9 TCH009 P. megasperma var sojae P.sp.10 VHS17183 P. megasperma P. megasperma All tested P. nicotianae P. nicotianae All tested P. cinnamomi P. cinnamomi All Tested P. cryptogea P. cryptogea
  • 10. Phytophthora species in natural ecosystems in WA •New records for WA P. inundata P. gonapodyides P. sp. asparagi P. sp. niederhauseria P. sp. personii •At least seven new Phytophthora species which are genetically distinct to currently described species
  • 11. Phytophthora species in natural ecosystems in WA Species No. Host species P. sp. 1 10 B. menziesii, B. littoralis, B. attenuata, B media P. sp. 2 21 Eucalyptus marginata, Corymbia calophylla, B. grandis, Dryandra squarrosa, Patersonia xanthina P. sp. 3 15 E. marginata, B. attenuata, B. grandis; Pinus radiata (plantation) P. sp. 4 64 E. marginata, B. attenuata, B. grandis, B. littoralis, B. menziesii, B. prionotes, Conospermum sp., Leucopogon verticillatus, X. gracilis, Podocarpus drouyniana, Patersonia sp., Bossiaea sp., Gastrolobium spinosum; Pinus radiata (plantation) P. sp. 7 4 X. preissii; Pinus radiata (plantation) P. sp. 8 3 Soil and water baits – native forest P. sp. 9 23 B. attenuata, B. baxteri, D. cirsioides, D. falcata, A. cuneata, Isopogon sp.; Pinus radiata (plantation) P. sp. 10 2 B. prionotes, B. grandis P. sp. 11 2 Banksia sp., X. preissii
  • 15. Significance of new phytophthora species •Do they pose a threat to biodiversity? •If yes, then management strategies will be developed •Need to investigate host range/pathogenicity distribution factors affecting spread •Are these species introduced? •Are any of these species hybrids?