Impact and management strategies for Phellinus noxius in the urban environment

1. Impact and management strategies for Phellinus noxius in the urban environment Michelle Grose Agri-science Queensland

2. Presentation Outline • What is Phellinusnoxius? • Disease significance • Phellinus project • Symptom development • Aetiology of Phellinusin the urban environment • Management strategies • Disposal strategies • Industry recommendations • Further research

3. 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

4. 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

5. Phellinus noxius in the urban and peri urban environment • Developed to address increasing problem of Phellinusnoxiusin 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

6. 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.

7. 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

8. Progression of disease symptoms in Ficus macrophylla over time in artificially inoculated glasshouse trials

9. 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

10. 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)

11. 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

12. 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?

13. 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

14. Adapted from Ann et al.(2002) 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

15. 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

16. Aetiology of Phellinus noxius in the urban environment – sources of infection Somatic incompatibility

17. Aetiology of Phellinus noxius in the urban environment – sources of infection Isolates of P. noxius used in somatic incompatibility testing

18. Aetiology of Phellinus noxius in the urban environment – sources of infection Somatic incompatibility testing results after 48 days (experiment 1) • 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)

19. Aetiology of Phellinus noxius in the urban environment – sources of infection Somatic incompatibility testing results after 48 days (experiment 1) • 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)

20. Aetiology of Phellinus noxius in the urban environment – sources of infection Somatic incompatibility testing results after 48 days (experiment 1) • 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)

21. 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???

22. 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

23. Spore production from New Farm and Indooroopilly fruit bodies over time

24. 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 Heterobasidionannosum

25. Aetiology of Phellinus noxius in the urban environment – sources of infection Pathogen variability • DNA sequencing used to determine if single or multiple Phellinusspecies 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

26. 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 MacrogenInc 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

27. 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

28. Comparison of isolates of P. noxius using ITS sequence data

29. 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

30. Aetiology of Phellinus noxius in the urban environment – sources of infection Susceptibility of host species tested against two isolates of Phellinus noxius

31. 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 • Melaleucaalternifoliadid not become infected

32. Host range of Phellinus noxius based on field surveys and host range testing using controlled inoculation methods developed as part of this study

33. 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 Ficusseedlings • Assessed after 3 months

34. 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

35. 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

36. 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

37. 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

38. 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 microbialsmay limit survival by Phellinus • Mulching is useful for other diseases • Eg. Phythophoracinnamomiin avocados • Size of woodchip may be crucial in survival

39. 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

40. 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

41. Acknowledgements