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Department of Employment, Economic Development and Innovation
Impact and management strategies for Phellinus noxius in
the urban environment
Michelle Grose
Agri-science Queensland
Presentation Outline
• What is Phellinus noxius?
• Disease significance
• Phellinus project
– Symptom development
– Aetiology of Phellinus in the urban
environment
– Management strategies
– Disposal strategies
• Industry recommendations
• Further research
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
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What is Phellinus noxius?
• Fungus that causes brown root rot
– Roots are attacked, spreads up the root collar resulting in decay
and tree death
• Pantropical distribution
– Oceania
– Asia
– Caribbean,
– South America
• 200 known host species
– figs
– hoop pine
– avocado,
– poinciana
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
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Disease significance
•
•
•
•
Decline and tree death
Decay
Decrease in structural integrity in the roots and butt of a tree
Major concern in the urban environment due to tree failure and
resultant public liability issues
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
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Phellinus noxius in the urban and peri urban environment
• Developed to address increasing problem of Phellinus noxius in the
urban environment
• Funded by:
– Brisbane City Council
– Gold Coast City Council
– Redlands City Council
– In kind contribution by Sunshine Coast Regional Council
• Investigated:
– Symptom development
– Aetiology of P. noxius in the urban environment
– Management strategies
– Disposal strategies
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
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Symptom development
• Two studies were conducted to assess symptom development
processes
– Artificial inoculation under controlled glasshouse conditions
– Assessment of established plots in Mary Cairncross Scenic
Reserve, Maleny.
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
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Symptom Development – tree decline
Artificial inoculation
• Stages of tree decline
– Chlorosis on lower/older leaves, then on new
shoots
– Wilting then foliage death
– Tree death
• Symptoms first observed >15days after inoculation, at
which time tree growth ceased
• Tree death associated with fungal stocking
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
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Progression of disease
symptoms in Ficus
macrophylla over time
in artificially inoculated
glasshouse trials
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
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Symptom development – tree decline
Field infected trees
• Differences seen between tree size and host species
– Generally the smaller the tree, the faster the decline and death
• Large trees
– Thinning and chlorosis of tree crown
– Variable time from infection to tree death
• Small trees
– similar to glasshouse studies
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
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Symptom development – Stocking development
• Stocking is generally characteristic of the pathogen
• Height variation is seen between and within host
species, and at different locations
– Stocking <1m above soil level in Brisbane
– Stocking >2m in Mary Cairncross Scenic Reserve
• Seasonal differences
– White/cream margin more common in
warmer/wetter months
– Change to cinnamon brown/black occurs
in a short time
• Stocking not always present
– Acacia mangium (Indonesia) and jacaranda
(south east Queensland)
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
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Symptom development – internal staining
• Observed in naturally and artificially
infected wood
• Stocking is in advance of stain
• Stain concentrated on the side the
stocking is present
• Phellinus noxius only isolated from
stained timber
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
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Aetiology of Phellinus noxius
in the urban environment
– distribution
• Cape York to northern New
South Wales
• Brisbane and Gold Coast
mapped
• No specific distribution
patterns
- Close to river?
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
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Aetiology of Phellinus noxius in the urban
environment – site hazard rating system
• Development would aid in risk assessments
• Sites factors recorded –soil type, rainfall, previous vegetation type
etc.
• Not feasible – P. noxius found across a wide range of different
environments
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
13
Aetiology of Phellinus noxius in the urban
environment – sources of infection
• Two sources of infection
– Root to root contact
– Basidiospores
• Variability within a population has a direct correlation with
basidiospore spread
•
Determined by somatic incompatibility
Adapted from Ann et al.(2002)
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
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Aetiology of Phellinus noxius in the urban
environment – sources of infection
Somatic incompatibility
• Pairing of different isolates on agar plates and observing the
reaction between two colonies
• Reactions
– Compatible (intermingling and therefore clonal)
– Incompatible (isolates are different)
• Assessed according to mounding of mycelium between colonies
and pigmentation in agar
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
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Aetiology of Phellinus noxius in the urban
environment – sources of infection
Somatic incompatibility
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
16
Aetiology of Phellinus noxius in the urban
environment – sources of infection
Isolates of P. noxius used in somatic incompatibility testing
Isolate number
178
132
133
137
138
139
140
141
142
143
144
145
146
151
152
153
154
23a
26
43a
Host
Ficus benjamina
Ficus benjamina
Ficus benjamina
Persea americana
Persea americana
Persea americana
Persea americana
Persea americana
Persea americana
Persea americana
Persea americana
Persea americana
Persea americana
Persea americana
Persea americana
Persea americana
Persea americana
Araucaria cunninghamii
Araucaria cunninghamii
Araucaria cunninghamii
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
Location
New Farm Park
Ascot Park
Ascot Park
Mareeba
Mareeba
Mareeba
Mareeba
Mareeba
Mareeba
Mareeba
Walkamin
Walkamin
Walkamin
Tolga
Tolga
Tolga
Tolga
Ryan’s Area, Imbil
Ryan’s Area, Imbil
2nd Logging Area, Imbil
17
Aetiology of Phellinus noxius in the urban
environment – sources of infection
Somatic incompatibility testing results after 48 days (experiment 1)
Isolate
178 132 133 137 138
178 I
MB MB
132
I
I
133
I
137
I
OU
138
I
139
140
141
142
143
144
145
146
151
152
153
154
23a
26
43a
•
•
•
•
•
139 140
MB
OP
I
MB
MB
MB
A
141 142
MB
MB
MB
MB,MB,MB
I
MB
OP
OU
OP
OP
A
143 144 145
146 151
152 153 154 23a
26 43a
MB,MB
OU
OU
MB
OP
MB,MB,OP,MB,MB
I
A
OU
I
OP
OU
A
A
MB,MB MB
A
MB
A
MB
MB
MB
I
I
OP I
A
OP
I
Intermingling (I)
Barrage consisting of mounded mycelium along confrontation zone and dark pigmentation of agar
(MB)
Abrupt line between isolates, no barrage, sometimes sparse mycelium between isolates (A)
Isolates overlap about 5mm (OU)
Isolates overlap about 5mm with dark pigmentation in agar (OP)
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
18
Aetiology of Phellinus noxius in the urban
environment – sources of infection
Somatic incompatibility testing results after 48 days (experiment 1)
Isolate
178 132 133 137 138
178 I
MB MB
132
I
I
133
I
137
I
OU
138
I
139
140
141
142
143
144
145
146
151
152
153
154
23a
26
43a
•
•
•
•
•
139 140
MB
OP
I
MB
MB
MB
A
141 142
MB
MB
MB
MB,MB,MB
I
MB
OP
OU
OP
OP
A
143 144 145
146 151
152 153 154 23a
26 43a
MB,MB
OU
OU
MB
OP
MB,MB,OP,MB,MB
I
A
OU
I
OP
OU
A
A
MB,MB MB
A
MB
A
MB
MB
MB
I
I
OP I
A
OP
I
Intermingling (I)
Barrage consisting of mounded mycelium along confrontation zone and dark pigmentation of agar
(MB)
Abrupt line between isolates, no barrage, sometimes sparse mycelium between isolates (A)
Isolates overlap about 5mm (OU)
Isolates overlap about 5mm with dark pigmentation in agar (OP)
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
19
Aetiology of Phellinus noxius in the urban
environment – sources of infection
Somatic incompatibility testing results after 48 days (experiment 1)
Isolate
178 132 133 137 138
178 I
MB MB
132
I
I
133
I
137
I
OU
138
I
139
140
141
142
143
144
145
146
151
152
153
154
23a
26
43a
•
•
•
•
•
139 140
MB
OP
I
MB
MB
MB
A
141 142
MB
MB
MB
MB,MB,MB
I
MB
OP
OU
OP
OP
A
143 144 145
146 151
152 153 154 23a
26 43a
MB,MB
OU
OU
MB
OP
MB,MB,OP,MB,MB
I
A
OU
I
OP
OU
A
A
MB,MB MB
A
MB
A
MB
MB
MB
I
I
OP I
A
OP
I
Intermingling (I)
Barrage consisting of mounded mycelium along confrontation zone and dark pigmentation of agar
(MB)
Abrupt line between isolates, no barrage, sometimes sparse mycelium between isolates (A)
Isolates overlap about 5mm (OU)
Isolates overlap about 5mm with dark pigmentation in agar (OP)
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
20
Aetiology of Phellinus noxius in the urban
environment – sources of infection
Rate of infection within an infected site
• Mary Cairncross Scenic Reserve
– 2 x 10m plots centred on infected tree
– Each tree mapped
– Newly infected trees recorded quarterly
– Run for four years
• Results
– Disease progression was slow
– 3 trees in each plot became infected
– Centre tree died
• Conclusions
– Spreads slowly in natural environment
– Due to variability in tree species and resistance???
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
21
Aetiology of Phellinus noxius in the urban
environment – sources of infection
Spore production
• Traps set weekly under two fruit bodies (March-November 2009)
– Collected after 24hrs
– Distilled water and Tween 20 added to petri dishes
– Spores in solution counted using haemocytometer
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
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Spore production from New Farm and Indooroopilly fruit bodies over time
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
23
Aetiology of Phellinus noxius in the urban
environment – sources of infection
Spore production – conclusions
• Occurrence of basidiospores suggests that management strategies
may need to be modified
• Bolland (1984) showed ability of basidiospores to infect freshly cut
hoop pine stumps
– Can infect wounds
– Pruning needs to be done when spore production is low or
conditions are unfavourable for germination
• Investigate chemical treatments for wounds
– 35% urea for Heterobasidion annosum
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
24
Aetiology of Phellinus noxius in the urban
environment – sources of infection
Pathogen variability
• DNA sequencing used to determine if single or multiple Phellinus
species involved
– Determines order of nucleotide bases in DNA, to compare
similarities and differences
– ITS region (easy to amplify, high variability)
• Basidiospores introduce variation
– High variation suggests spread via basidiospores
– Low variability suggest clonal population
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
25
Aetiology of Phellinus noxius in the urban
environment – sources of infection
Pathogen variability
• DNA sequencing
– Isolates collected from different hosts and locations
– DNA extracted (modification of White and Kaper (1989)
– PCR reactions for ITS 1 and 4
– PCR products sent to Macrogen Inc for purification and
sequencing
– Sequences edited BioEdit Sequence Alignment Editor version
7.0.5.3 & Sequence Scanner version1
– MrBayes 3.1 used to create phylogenetic trees
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
26
Aetiology of Phellinus noxius in the urban
environment – sources of infection
Pathogen variability
• DNA Sequencing analysis
– All species P. noxius
– Not enough variability to divide them into haplotypes and form
phylogenetic trees.
– Were differences in base pairs between isolates
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
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Comparison of isolates of P. noxius using ITS sequence data
Host/
location
Isolate
183
184
279
280
290
293
294
295
296
297
298
299
300
363
365
392
477
554
582
636
639
647
686
687
706
822
823
830
71a
T
C
A
T
A
T
T
A
T
T
C
A
T
T
T
-
T
A
A
T
A
A
T
G
G
A
C
T
163
T
C
A
T
A
T
T
A
T
T
C
A
T
T
T
-
T
A
N
T
A
A
T
G
G
A
C
T
169
T
C
A
T
A
T
T
A
T
T
C
A
T
T
T
-
T
A
A
T
A
A
T
G
G
A
C
T
mango
Mutchilba
avocado,
Mutchilba
mango,
Mutchilba
Host/
location
Isolate
183
184
279
280
290
293
294
295
296
297
298
299
300
363
365
392
477
554
582
636
639
647
686
687
706
822
823
830
101
C
C
T
G
T
T
T
A
T
T
C
A
T
T
T
-
T
A
A
T
A
G
T
A
A
-
-
T
181
T
C
A
T
T
T
T
A
T
T
C
A
T
T
T
-
T
A
A
A
A
A
T
G
G
-
-
C
179
C
C
A
G
A
T
T
A
T
T
C
A
T
T
T
-
T
A
A
T
A
G
T
A
A
-
-
T
fig,
Toowong
fig, Gold
Coast
Fig/Byron
Bay
Host/
Isolat
location
e
183
184
279
280
290
293
294
295
296
297
298
299
300
363
365
392
477
554
582
636
639
647
686
687
706
822
823
830
hoop, Derrier
12a
T
C
A
T
T
T
T
A
T
T
C
A
T
T
T
-
T
A
A
T
A
G
T
A
G
A
C
T
17b
C
C
T
G
T
T
T
A
T
T
C
A
T
T
T
-
T
A
A
T
A
G
T
A
A
-
-
T
23a
T
C
A
T
T
T
T
A
T
T
C
A
T
T
T
-
T
A
A
A
A
A
T
G
G
-
-
C
26
T
C
A
T
T
T
T
A
T
T
C
A
T
T
T
-
T
A
A
A
A
A
T
G
G
-
-
C
hoop, 2nd
Logging
Compartment
hoop, Ryan's
Area
hoop, Ryan's
Area
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
28
Aetiology of Phellinus noxius in the urban
environment – sources of infection
Isolate Pathogenicity
• Important to know if there’s host specificity or differences in
aggression towards host genera
– Pathogenicity tests
• Artificially inoculated 5 host species using 2 different isolates
•
•
Plants assessed after 3 months
Infection, tree death or state or tree decline recorded
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
29
Aetiology of Phellinus noxius in the urban
environment – sources of infection
Susceptibility of host species tested against two isolates of Phellinus noxius
Species
151 (avocado, Tolga)
102 (Ficus
Toowong)
Acmena ingens
Infected 6/6
Infected 5/6
Callistemon salignus
Infected 5/6
Infected 3/6
Harpullia penduala
No infection 0/6
Infected 2/6
Pittosporum undulatum
Infected 1/6
No infection 0/6
Ficus macrophylla
Infected 6/6
Infected 6/6
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
benjamina,
30
Strategies for the management of Phellinus in the
urban environment
Susceptible/resistant species for the urban environment
• Knowledge of susceptible/resistant species is important for
rehabilitation
– Strong chance of reinfection due to root contact
• Field inoculated
– New hosts recorded
• Glasshouse inoculation procedure
– Assess 14 ‘urban’ species for susceptibility/resistance
– Differences in severity of infection occurred
– Melaleuca alternifolia did not become infected
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
31
Host range of Phellinus noxius based on field surveys and host range testing
using controlled inoculation methods developed as part of this study
Field susceptible species
Glasshouse susceptible species
Musa acuminata x Musa balbisiana (banana)
Acmena ingens (red apple)
Hibiscus sp.
Callistemon salignus (willow bottlebrush)
Caesalpinia ferrea (leopard tree)
Pittosporum undulatum (native daphne)
Mangifera indica (mango-honey gold)
Lomandra hysterix (slender mat rush)
Cupaniopsis anacardiodes (tuckeroo)
Buckinghamia celsissima (ivory curl flower)
Brachycyiton acerifolius (flame tree)
Mellicope elleryana (pine euodia)
Podocarpus elatus (brown pine)
Livistona australis (cabbage palm)
Tristaniopsis laurina (water gum)
Corymbia maculata (spotted gum)
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
32
Strategies for the management of Phellinus in the
urban environment
Chemical control
• Currently no chemical control options
• Would be useful for treatment and protection
• Previous studies
– In vitro
– Limited hoop pine field trials
• Preliminary glasshouse studies
– Folicur® (a tebuconazole)
– Alamo® (a propiconazole)
– Adding chemical as soil drench at set application times
– Inoculating Ficus seedlings
– Assessed after 3 months
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
33
Strategies for the management of Phellinus in the
urban environment
Chemical control
• Experiment one treatments
– 4 weeks prior
– 2 weeks prior
– 1 week prior to inoculation
• All folicur and alamo treatments showed symptoms of infection
• One week prior treatment had lower infection rates
– Alamo – 1/3 infected
– Folicur – ½ infected
– Poor uptake through soil application??
• Inoculated controls had poor infection rates
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
34
Strategies for the management of Phellinus in the
urban environment
Chemical control
• Experiment two treatments
– Multiple chemical applications
– 4 weeks, 2 weeks, 1 week prior
– 2 weeks, 1 week prior
– Double chemical concentration 1 week prior
• No infection occurred, even in inoculated controls
– Needs to be repeated with fresh isolates?
• Need more research into chemical control
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
35
Methods for disposal of infected tree stumps and
roots
Survival in woodblocks
• Survival within dead roots/debris for >10 years
• Very difficult to remove all infected material
– Treatment for this infected material would be useful
• Study to assess effect of:
– Urea (placed in soil/woodchips with 3000p.p.m, or 300p.p.m)
– Desiccation (woodblocks placed on tray at room temperature)
– Microbial activity (placed in autoclaved/non-autoclaved
woodchips)
• Assessed by isolating after 1 month, 3 months and 6 months after
treatments imposed
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
36
Methods for disposal of infected tree stumps and
roots
Survival in woodblocks
• Few hitches in first 2 samplings
• Six month sampling
– P. noxius isolated from all treatments except for desiccation
• Further work needs to be done on other woodblock sizes and
possibly other treatments
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
37
Methods for disposal of infected tree stumps and
roots
Survival in woodchips/mulch
• Disposal of infected trees is a major problem
• Current method involves deep burial – expensive & time consuming
• Attempted to impose treatments on woodchips from infected tree
– Pathogen could not be isolated
– Attempted to inoculate woodchips in large bins
– Trichoderma sp. became an issue
• Suggests presence of other microbials may limit survival by
Phellinus
• Mulching is useful for other diseases
– Eg. Phythophora cinnamomi in avocados
– Size of woodchip may be crucial in survival
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
38
Industry Recommendations
•
•
•
•
Remove all infected material from a site
Don’t replant with susceptible species
Install root barriers
Infected trees should not be chipped and used as mulch
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
39
Further research
• Continue host testing & develop susceptibility rating system
• Chemical controls
– Prevent spread
– Slow down decline of infected trees
• Investigate decay processes/rates of decay
• Develop methods to determine internal decay
• Structural changes caused by decay agents
– Methods of detection
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
40
Acknowledgements
© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010
41