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Keywords:

  • Australia;
  • Corymbia;
  • Eucalyptus;
  • fungal disease;
  • plantation forestry;
  • spotted gum

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Quambalaria shoot blight, caused by the fungal pathogen Quambalaria pitereka, is a serious disease of eucalypt plantations in Australia. The aggressiveness of four Q. pitereka isolates was compared on a range of host genera, species, provenances and clones. Isolates differed substantially in their aggressiveness, with two consistently showing higher levels of aggressiveness based on incidence and severity of disease and lesion size. Isolates derived from Corymbia citriodora subsp. variegata (Ccv) and C. torelliana were shown to have a relatively restricted host range, with lesions but no sporulation found on Eucalyptus species, Angophora species other than A. costata and Corymbia species other than Ccv, the host of origin. The level of aggressiveness toward the different provenances of spotted gum and C. torelliana varied between isolates and there was evidence of some isolate × host interaction within provenances of Ccv. The two methods of inoculation used in this study, spray and spot inoculation, gave similar results. However, the fact that the spot inoculation method was labour-intensive was a disadvantage limiting the numbers of isolates and hosts that can be tested.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Quambalaria shoot blight, caused by the fungal pathogen Quambalaria pitereka, is a serious disease affecting the expanding eucalypt plantation estate in subtropical and tropical eastern Australia (Simpson, 2000; Self et al., 2002; Pegg et al., 2005; Carnegie, 2007). The pathogen is described as being endemic to the coastal forests of eastern Australia, where seedlings and young trees of Corymbia species can be severely damaged (Old, 1990). The implementation of hardwood plantation programmes in subtropical regions of eastern Australia saw the widespread use of spotted gum, primarily using provenances of C. citriodora subsp. variegata (Ccv) (Lee, 2007) and C. maculata, but also including Corymbia citriodora subsp. citriodora, (Ccc) and C. henryi for high-value solid-wood products. Quambalaria shoot blight was infrequently observed during the first few years of plantation development but was soon after found to be widespread and causing significant damage infecting young foliage and growing shoots (Self et al., 2002; Carnegie, 2007; Pegg et al., 2009a). Severe damage resulting in poor tree form, and in some cases tree death, resulted in the reduction in the use of spotted gum as a priority species in the subtropics (Carnegie, 2007; Pegg et al., 2009a). More recently Q. pitereka was discovered outside Australia on spotted gum plantations in China (Zhou et al., 2007).

Only an asexual state (anamorph) is known for Q. pitereka and this is also true for other Quambalaria spp. (Simpson, 2000; Pegg et al., 2009b). However, variability has been noted amongst isolates of Q. pitereka at the DNA level, with 10 haplotypes identified when comparing DNA sequences of isolates of the fungus collected from different locations and Corymbia species in northern New South Wales and Queensland (Pegg et al., 2008). Isolates representing the different haplotypes were found to occur within different geographic regions, and multiple haplotypes were identified from within a single plantation. The significance of this variability in relation to host range and pathogen aggressiveness has not been studied.

Variability in levels of susceptibility to Q. pitereka within species, provenances and families of spotted gum has been identified by a number of authors (Stone et al., 1998; Self et al., 2002; Dickinson et al., 2004; Johnson et al., 2009; Pegg et al., 2011). Spotted gum provenances have been selected as sources of seed for use in commercial plantations as well as development of seed orchards. These selections were based on field trial assessments focused on tree performance (growth rate, Q. pitereka tolerance) under conditions of natural infection by Q. pitereka. However, little attention has been afforded to the pathogen and the possibility that variability in its population might influence disease development or disease severity within spotted gum trials and commercial plantings.

Trials in subtropical and tropical Australia to select for spotted gum provenance material for use in plantation and seed orchard development are found in diverse locations where Q. pitereka disease levels are often inconsistent, primarily as a result of variable climatic conditions. The development and implementation of an artificial screening programme has been recommended (Pegg et al., 2011) to make selections of Q. pitereka-tolerant genotypes more consistent and reliable. This would enhance the future development of hardwood plantations using spotted gum by reducing damage by quambalaria shoot blight. In order to establish such an artificial screening procedure, investigations into the variability in host–pathogen interactions will be required. The aim of this study was, therefore, to determine whether there are differences in host range of Q. pitereka isolates and if there is variability in aggressiveness or specificity of these isolates to different host species, provenances and clones of spotted gum.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Quambalaria pitereka isolates

Isolates of Q. pitereka were selected from three different regions, including far-north Queensland, southern Queensland and northern New South Wales (Table 1). A series of preliminary studies was conducted using isolates from these regions, as well as those representing different haplotypes identified from DNA sequencing (Pegg et al., 2008) collected from a single property. Isolates showing higher levels of aggression were selected for use in this study. All isolates used in the study were inoculated onto spotted gum or Corymbia torelliana plants and re-isolated onto potato dextrose agar (PDA) (Difco – Bacto Laboratories Pty Ltd) and incubated at 25°C in the dark for 2–3 days before being subcultured onto PDA and incubated for a further 2 weeks to ensure culture age was uniform.

Table 1.   Isolates of Quambalaria pitereka used in host range and pathogenicity testing
Quambalaria isolate numberOriginIsolate’s original host taxon
Q107Mareeba, north QueenslandCorymbia torelliana × Corymbia citriodora ssp. citriodora
Q152Beaudesert, south east QueenslandC. citriodora ssp. variegata
Q298Beaudesert, south east QueenslandC. citriodora ssp. variegata
Q322Grafton, northern New South WalesC. citriodora ssp. variegata

Inoculation methods

Two methods of inoculation were used in this study. These included spraying the inoculum onto plants or inoculating leaves in a defined spot. Spores were removed from plates using a fine-haired paint brush and washed into 100 mL sterile distilled water (SDW). A concentration of 1 × 106 spores mL−1 was used for all inoculations with two drops of Tween 20 added to each suspension. The spore concentration used in this study was based on preliminary studies conducted to identify the optimum spore concentration for symptom development (G. S. Pegg, unpublished data).

For both inoculation methods, seedlings were germinated and grown in steam-sterilized soil mix and fertilized with slow release Osmocote® (Native Trees) (N 17·9: P 0·8: K 7·3) as required. Plants were irrigated twice a day for 10 min each time using overhead spray irrigation. Glasshouse temperatures were maintained at 28–30°C during the day and 22–24°C overnight. Following inoculation, seedlings were covered with plastic bags for 48 h to maintain high humidity levels and leaf wetness and placed on benches in a complete random design. Previous studies (Pegg et al., 2009b) identified that spore germination occurred optimally at humidity levels above 90% RH and penetration of the host was within 24 h. Uninoculated controls were treated in the same way as inoculated seedlings. Subsamples of the spore suspension applied to the trees were placed onto PDA and incubated at 25°C for 48 h to ensure that the spores were viable.

Spray inoculation

Seedlings were inoculated using a fine mist spray (2·9 kPa pressure) generated by a compressor driven spray gun (Iwata Studio series 1/6 hp; gravity spray gun RG3), to the upper and lower leaf surfaces of the seedlings until runoff was achieved. Uninoculated controls were sprayed with SDW with Tween 20 added as per spore suspensions. Disease incidence (I) was assessed as a percentage of leaves infected out of the total number of leaves present. Disease severity (S) was a subjective assessment of the percentage of the total area of infected foliage on diseased leaves only. Seedlings were assessed for disease incidence and severity 14 days after inoculation.

Spot inoculation

Preliminary studies on detached leaves were used to compare inoculation of leaves on adaxial and abaxial leaf surfaces. No lesion development occurred on leaves inoculated on the adaxial leaf surface and for this reason all further studies were conducted on abaxial leaf surfaces. A plastic tube (5 mm in diameter) with a cotton applicator placed inside was dipped into the spore suspension prior to application and then inoculated onto the abaxial leaf surfaces. On each leaf, isolates of Q. pitereka were inoculated onto one side of the midrib either at the apex, middle or base of the leaf (Fig. 1). The positioning of each isolate was rotated for each leaf with each isolate inoculated onto the leaf apex, middle or base, once per seedling. The youngest fully expanded foliage was selected for inoculation. Control seedlings were inoculated with SDW and Tween 20 solution. Disease levels were measured as a percentage of the area inoculated that was necrotic 14 days after inoculation.

image

Figure 1.  Lesion development 14 days after inoculation using the spot inoculation method with three isolates of Quambalaria pitereka onto Corymbia citriodora subsp. variegata provenance Presho (a) and C. henryi provenance Myrtle Creek (b–d).

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The aggressiveness of Q. pitereka isolates was compared on increasing ‘resolution’ of genotypes from species to provenances to clones. The aggressiveness of isolates was compared on Corymbia species known to be susceptible to Q. pitereka as well as on Angophora, Corymbia and Eucalyptus species on which Q. pitereka has not been identified. Variability of isolate aggression was also compared on provenances and clones of spotted gum. For the purpose of this study the term ‘aggression’ refers to the severity of disease caused by each isolate (Pariaud et al., 2009).

Comparisons of isolate host range

To determine the extent of variation in the host range within isolates of Q. pitereka, three isolates of Q. pitereka (Q107, Q298 and Q322) were inoculated onto Corymbia species not known to be susceptible to the pathogen (i.e. those other than spotted gum and C. torelliana) using the spot inoculation method. Species used were C. intermedia (seedlot 197), C. ptychocarpa (seedlot 2772) and C. tessellaris (seedlot 5915). A range of Angophora species were also tested: A. costata (seedlot unknown), A. leiocarpa (seedlot 3294) and A. melanoxylon (seedlot 2182). Two Eucalyptus species were also tested: E. drepanophylla (seedlot 95) and E. melanophloia (seedlot 2677). Ccv (Presho provenance seedlot 4928) was also included as a known host. Six replicates were used per isolate per host and placed in a complete random design in the glasshouse.

Comparisons of isolate aggressiveness on Corymbia species

The variability in aggressiveness of isolates of Q. pitereka towards Ccc, C. henryi, Ccv and C. torelliana (Table 2) was examined. Using the spray method, four isolates (Q107, Q152, Q298 and Q322) were inoculated onto Corymbia hybrids grown from seed (Table 2). Ten replicates were used per isolate per host and placed in a complete random design in the glasshouse. Ten control seedlings from each provenance were inoculated with SDW and Tween 20 solution.

Table 2.   Origin and species of spotted gum and Corymbia torelliana provenances and Corymbia hybrids
SpeciesaProvenanceOriginSeedlotLatitude (S)Longitude (E)MARb (mm)
  1. aCcc, Corymbia citriodora subsp. citriodora; Ccv, C. citriodora subsp. variegata. bMAR, mean annual rainfall.

CccBarronNorthern Queensland560917°15′145°30′1200
CccCheviot HillsNorthern Queensland350619°38′144°12′673
CccKirramaNorthern Queensland529818°12′145°46′2000
CccYeppoonCentral Queensland1124623°6′150°44′1325
Corymbia henryiMyrtle CreekNorthern New South Wales555429°9′152°59′1100
C. henryiNerangSouth east Queensland1025727°59′153°19′1200
CcvBrooyarSouth east Queensland1024826°10′152°30′1143
CcvCurraSouth east QueenslandK603526°4′152°39′1138
CcvGrangeNorthern New South WalesCVS9006229°31′152°32′1212
CcvHomeSouth east Queensland1023526°5′152°43′1143
CcvMt McEuanSouth east Queensland1280526°14′151°39′1200
CcvPreshoCentral Queensland492825°3′149°13′675
CcvRichmond RangeNorthern New South Wales19469a28°38′152°48′1267
CcvWondaiSouth east Queensland1025326°22′151°49′800
CcvWoondumSouth east Queensland11185-09626°15′152°49′1600
C. torellianaCairnsNorthern Queensland355216°57′145°45′1992
C. torellianaHelensvaleNorthern QueenslandK6340   
C. torelliana × Ccc  X213   
C. torelliana × C. henryi  X135   
C. torelliana× Ccv  X72   

Comparisons of isolate aggressiveness on Corymbia hybrids

The variability in aggressiveness of isolates of Q. pitereka towards Corymbia hybrids (Table 2) was examined. Using the spray method, four isolates (Q107, Q152, Q298 and Q322) were inoculated onto Corymbia species grown from seed (Table 2) as mentioned above. Ten control seedlings from each provenance were inoculated with SDW and Tween 20 solution.

Comparisons of isolate aggressiveness on provenances of Corymbia species

The variability in aggressiveness of isolates of Q. pitereka towards provenances of Corymbia species (Table 2) was examined. Using the spray method, four isolates (Q107, Q152, Q298 and Q322) were inoculated onto seedlings of 17 provenances within Corymbia species Ccc, C. henryi, Ccv and C. torelliana grown from seed (Table 2). Ten replicates were used per isolate per host and placed in a complete random design in the glasshouse. Ten control seedlings from each provenance were inoculated with SDW and Tween 20 solution.

As a comparison, and to reduce the influence of host variability, a spot inoculation technique was also used to investigate the interaction between isolates and provenances (Presho, Home, Yeppoon, Mt McEuan, Richmond Range and Myrtle Creek). Three isolates of Q. pitereka were compared (Q152, Q298 and Q322) on six spotted gum provenances. Ten trees were used per spotted gum provenance with controls treated with SDW and Tween 20 solution.

Comparisons of isolate aggressiveness on Ccv clones

Four Q. pitereka isolates (Q107, Q152, Q298 and Q322) were compared on seven Ccv clones (Forests NSW) using the spot inoculation technique. Plants for each clone were produced from micropropagation of archived cultures developed by Forests NSW (Lan et al., 2011). Ten replicates per clone per isolate were inoculated as described above and placed in a complete random design in the glasshouse.

Analysis of data

Normality of data was checked using an equality of variance F-test. All proportion data was arcsine square root transformed prior to analysis using anova and compared using Fisher's PLSD post hoc test (StatView®). Back-transformed data were used in all graphed and tabulated data.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Inoculation methods

Both methods of inoculation were effective in producing symptoms typical of Q. pitereka infection within 14 days after inoculation. Using the spray inoculation method allowed for the testing of a large number of host plants in comparison to the spot inoculation method, which was much more time consuming. Leaf size limited the number of isolates that could be tested on a single leaf using the spot inoculation method.

Comparisons of isolate host range

Small lesions developed on all species tested when inoculated with three isolates of Q. pitereka (Q107, Q298 and Q322), but A. costata and Ccv were most susceptible (Fig. 2). However, the presence of sporulation on lesions was recorded on Ccv and A. costata only. No disease symptoms were present on uninoculated controls. Significant differences were found between isolates (two-way anovaF2,297 = 3·5, = 0·03). Based on lesion size, isolate Q322 showed the highest level of aggressiveness on A. costata, A. leiocarpa, A. melanoxylon, Ccv, C. ptychocarpa, E. drepanophylla and E. melanophloia and showed a significantly higher level of aggressiveness than Q107 only. Isolate Q298 showed a higher level of aggressiveness on C. tessellaris and C. intermedia. There was a significant difference in host susceptibility (F8,297 = 19·6, < 0·0001) but no significant host × isolate interaction (F16,297 = 5·8, = 0·5).

image

Figure 2.  Comparison of host range of three Quambalaria pitereka isolates on species of Angophora, Corymbia and Eucalyptus showing mean lesion size (+1 standard error). Matching letters designate means that do not differ significantly (Fisher’s PLSD test < 0·0001).

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Comparisons of isolate aggressiveness on Corymbia species

Using spray inoculations significant differences in disease incidence and severity occurred when spotted gum species and C. torelliana (Table 3) were inoculated with Q.  pitereka isolates. Isolate Q322 showed the highest level of aggressiveness on Ccv, C. henryi and C. torelliana based on disease incidence and all spotted gum species and C. torelliana based on disease severity. Isolate Q322 was not significantly different from isolate Q298. Isolates Q107 and Q152 showed the lowest level of aggressiveness. No symptoms were present on control seedlings.

Table 3.   Comparison of mean incidence (a) and severity (b) of infection by isolates of Quambalaria pitereka 14 days after inoculation using the spray inoculation method onto species of spotted gum and Corymbia torelliana. Matching letters designate means that do not differ significantly
(a) Q. pitereka isolateCorymbia citriodora ssp. citriodora ACorymbia citriodora ssp. variegata BCorymbia henryi BCorymbia torelliana B
Q107 a51·7±5·447·4±3·228·3±7·233·8±6·6
Q152 b56·7±4·457·3±3·140·9±7·923·7±4·6
Q298 c80·6±4·470·2±2·852±7·448·6±5·7
Q322 c75·4±4·871·3±3·458·6±7·455±6·8
 d.f.FP    
Isolate 318·8<0·0001    
Host 318·1<0·0001    
Isolate*Host 9 0·7  0·7    
Residual635      
(b) Q. pitereka isolate C. citriodora ssp. citriodora AC. citriodora ssp. variegata BC. henryi BCC. torelliana C
Q107 a14·7 ± 2·418·3 ± 2·16 ± 1·65·5 ± 1·1
Q152 a13·2 ± 1·923 ± 2·211·7 ± 3·34·2 ± 0·7
Q298 b18 ± 221·9 ± 1·617·2 ± 411·2 ± 2·1
Q322 b17·6 ± 2·227·5 ± 2·421·3 ± 4·712·2 ± 1·8
 d.f.FP    
Isolate 3 9<0·0001    
Host 319·8<0·0001    
Isolate*Host 9 0·8  0·6    
Residual635      

Significant differences in susceptibility were identified between host species (Table 3). Disease incidence levels were lowest on C. torelliana for all isolates except isolate Q107. Disease severity levels were lowest on C. torelliana for all isolates. There was no significant host × isolate interaction (Table 3).

Comparisons of isolate aggressiveness on Corymbia hybrids

Using spray inoculations significant differences in disease incidence and severity occurred when Corymbia hybrids (Table 4) were inoculated with Q. pitereka isolates. Isolate Q298 showed the highest level of aggressiveness, but not significantly different from isolate Q322. Isolates Q152 and Q107 showed the lowest level of aggressiveness.

Table 4.   Comparison of mean incidence (a) and severity (b) of infection by isolates of Quambalaria pitereka 14 days after inoculation using the spray inoculation method onto Corymbia hybrids. Matching letters designate means that do not differ significantly
(a) Q. pitereka isolateCorymbia torelliana  × Corymbia citriodora ssp. citriodora AC. torelliana × Corymbia henryi BC. torelliana × C. citriodora ssp. variegata C
Q107 a52·2 ± 7·28·1 ± 3·839·4 ± 12·8
Q152 a28 ± 9·934·3 ± 11·663·9 ± 16·3
Q298 b66·5 ± 840·9 ± 9·385·8 ± 5·3
Q322 b64·5 ± 9·720·5 ± 10·175·8 ± 7·1
 d.f.FP   
Isolate 3 6  0·001   
Host 214·3<0·0001   
Isolate*Host 6 2·1  0·06   
Residual635     
(b) Q. pitereka isolateC. torelliana × C. citriodora ssp. citriodora AC. torelliana × C. henryi BC. torelliana × C. citriodora ssp. variegata A
Q107 a10 ± 1·72·1 ± 16·7 ± 2·5
Q152 a5 ± 1·76·4 ± 2·112·5 ± 5
Q298 b11 ± 1·27·9 ± 1·526·4 ± 5
Q322 b21·5 ± 97·1 ± 4·115 ± 1·5
 d.f.FP   
Isolate3430·007   
Host2 5·90·004   
Isolate*Host6 1·80·1   
Residual82     

Disease incidence and severity levels were significantly lower on the C. torelliana × C. henryi hybrids. There was no significant interaction between hybrids and isolates of Q. pitereka (Table 4). No disease was found on control seedlings.

Comparisons of isolate aggressiveness on provenances of Corymbia species

Corymbia citriodora subsp. citriodora (Ccc)

Using spray inoculations, significant differences in disease incidence were identified on Ccc provenances when inoculated with Q. pitereka isolates (Table 5). Disease severity levels were not significantly different. No single isolate showed a consistently higher level of aggressiveness towards all hosts, but Q298 and Q322 caused significantly higher levels of disease incidence on provenances of Ccc than isolates Q107 and Q152. Isolates Q298 and Q322 caused higher disease severity levels on all provenances, but differences from isolate Q107 and Q152 were not significant.

Table 5.   Comparison of mean incidence (a) and severity (b) of infection by isolates of Quambalaria pitereka 14 days after inoculation using the spray inoculation method onto Corymbia citriodora subsp. citriodora provenances. Matching letters designate means that do not differ significantly
(a)
Q. pitereka isolateBarron AKirrima ABYeppoon BCCheviot Hills C
Q107 a30·2 ± 10·945·2 ± 6·664·5 ± 11·565 ± 10·9
Q152 a46·8 ± 8·944·6 ± 8·975 ± 760·2 ± 8·3
Q298 b82·1 ± 8·781·4 ± 7·972·7 ± 12·486·3 ± 5·5
Q322 b54 ± 11·867·4 ± 8·982·3 ± 5·895 ± 7·4
 d.f.FP    
Isolate310·1<0·0001    
Host34·00·009    
Isolate*Host91·40·2    
Residual139      
(b)
Q. pitereka isolateBarron AKirrima AYeppoon BCheviot Hills B
Q107 a7·2 ± 2·58 ± 61·320·5 ± 622·5 ± 5·6
Q152 a9·5 ± 1·98 ± 1·921 ± 5·714·5 ± 3·1
Q298 a16 ± 416 ± 4·316 ± 4·124 ± 3·3
Q322 a7·2 ± 1·910 ± 1·832·5 ± 4·819 ± 2·2
 d.f.FP    
Isolate32·00·12    
Host310·1<0·0001    
Isolate*Host920·04    
Residual139      

Disease levels were significantly different on Ccc provenances with Yeppoon and Cheviot Hills most susceptible (Table 5). There was no significant host × isolate interaction for disease incidence, but for disease severity the host × isolate interaction was significant. No disease was found on control seedlings.

Corymbia henryi

Using spray inoculations, significant differences in disease incidence and disease severity were identified on C. henryi provenances when inoculated with Q. pitereka isolates (Table 6). Isolates Q298 and Q322 showed a significantly higher level of aggressiveness than isolate Q107. Isolates Q298 and Q322 were not significantly different from each other or Q152. Isolate Q152 was not significantly different from isolate Q107. Myrtle Creek provenance was significantly less susceptible than Nerang. There was no significant host × isolate interaction and no disease identified on control seedlings (Table 6).

Table 6.   Comparison of mean incidence (a) and severity (b) of infection by isolates of Quambalaria pitereka 14 days after inoculation using the spray inoculation method onto Corymbia henryi provenances. Matching letters designate means that do not differ significantly
(a)
Q. pitereka isolateMyrtle Creek ANerang B
Q107 a14·2 ± 6·942·5 ± 11·2
Q152 ab17·7 ± 864·2 ± 9
Q298 b33·7 ± 970·3 ± 7·7
Q322 b47·6 ± 11·270·9 ± 8·3
 d.f.FP  
Isolate33·8  0·005  
Host127·6<0·0001  
Isolate*Host30·7  0·5  
Residual67    
(b)
Q. pitereka isolateMyrtle Creek ANerang B
Q107 a3 ± 1·59 ± 2·6 
Q152 ab5 ± 2·218·5 ± 5·6 
Q298 bc6·9 ± 1·927·5 ± 5·8 
Q322 c19 ± 6·924 ± 6·7 
 d.f.FP  
Isolate3 5·2  0·003  
Host117·8<0·0001  
Isolate*Host3 0·98  0·4  
Residual67    
Corymbia citriodora subsp. variegata (Ccv)

Using spray inoculations, significant differences in disease incidence and disease severity were identified on Ccv provenances when inoculated with Q. pitereka isolates (Table 7). No single isolate showed a higher level of aggressiveness on all hosts with a significant isolate × host interaction (Table 7). Isolates Q298 and Q322 showed a significantly higher level of aggressiveness than Q107 and Q152, causing higher disease incidence levels on seven of the nine provenances tested. Isolates Q298 and Q322 also caused higher disease severity levels on six of the nine provenances, but there was no significant difference between these isolates and Q152 and Q107. Only isolate Q322 caused significantly higher disease severity levels than Q107.

Table 7.   Comparison of mean incidence (a) and severity (b) of infection by isolates of Quambalaria pitereka 14 days after inoculation using the spray inoculation method onto Corymbia citriodora subsp. variegata provenances. Matching letters designate means that do not differ significantly
(a) Q. pitereka isolateRichmond Range AGrange ACurra AWondai BMt McEuan BWoondum BHome BBrooyar BPresho B
Q107 a24·5 ± 10·524·4 ± 8·159·5 ± 727·8 ± 11·341·8 ± 8·665·2 ± 6·966·1 ± 6·552·5 ± 11·156·8 ± 7
Q152 b26·6 ± 10·950·8 ± 7·741·2 ± 8·172·1 ± 9·866·1 ± 7·849·2 ± 6·378·3 ± 10·371·6 ± 6·865·7 ± 7·1
Q298 c57·7 ± 7·447·9 ± 5·553·3 ± 11·374·8 ± 9·678·3 ± 5·471·6 ± 6·571·9 ± 6·975·9 ± 8·196·7 ± 3·3
Q322 c71·8 ± 9·757·7 ± 14·452·3 ± 10·281·7 ± 1068·1 ± 9·879·8 ± 5·560·4 ± 13·478·8 ± 5·392·1 ± 4·2
 d.f.FP         
Isolate316·0<0·0001         
Host87·6<0·0001         
Isolate*Host242·0  0·004         
Residual310           
(b) Q. pitereka isolateRichmond Range AWondai ABCurra ABCGrange ABCHome ABCMt McEuan BCDBrooyar CDWoondum CDPresho D
Q107 a17·5 ± 10·35 ± 1·826 ± 7·78 ± 2·820 ± 4·112·5 ± 3·717 ± 4·321·5 ± 5·232 ± 8·2
Q152 ab5 ± 2·126·4 ± 7·820·5 ± 715 ± 3·630·5 ± 7·430·5 ± 829 ± 6·520 ± 731·5 ± 5·4
Q298 ab12·5 ± 2·812·8 ± 2·216 ± 3·920 ± 4·319 ± 2·517·5 ± 3·826·5 ± 433 ± 6·637 ± 6·1
Q322 b21 ± 6·322·5 ± 5·318 ± 4·140 ± 13·113 ± 439·4 ± 5·234 ± 8·931·1 ± 6·930 ± 5
 d.f.FP         
Isolate34·3  0·005         
Host84·2<0·0001         
Isolate*Host241·8  0·01         
Residual310           

Significant differences in provenance susceptibility were also identified with disease incidence levels lowest on Richmond Range, Grange and Curra provenances (Table 7). Disease severity levels were lowest on Brooyar, Curra and Grange, with Presho and Woondum being most susceptible. No disease symptoms were detected on control seedlings.

Corymbia torelliana

Using spray inoculations, significant differences in disease incidence and disease severity were identified on C. torelliana provenances when inoculated with Q. pit-ereka isolates (Table 8). Disease incidence was highest when provenances were inoculated with isolates Q298 and Q322, significantly higher than with Q152. Disease incidence levels were also significantly higher on seedlings inoculated with Q322 than on those inoculated with Q107. Disease severity levels were significantly higher on seedlings inoculated with isolates Q298 and Q322 than isolates Q107 and Q152. Isolate Q152 showed the lowest level of aggressiveness, but not significantly different from Q107.

Table 8.   Comparison of mean incidence (a) and severity (b) of infection by isolates of Quambalaria pitereka 14 days after inoculation using the spray inoculation method onto Corymbia torelliana provenances. Matching letters designate means that do not differ significantly
(a)
Q.  pitereka isolateCairns AHelensvale A
Q107 ab23·5 ± 8·243 ± 9·6
Q152 a21·3 ± 6·226 ± 6·9
Q298 bc52·1 ± 9·345·1 ± 7·2
Q322 c48 ± 12·262 ± 5·9
 d.f.FP  
Isolate35·30·003  
Host11·70·2  
Isolate*Host30·70·6  
Residual67    
(b)
Q.  pitereka isolateCairns AHelensvale A
Q107 a6·1 ± 1·85 ± 1·3
Q152 a4 ± 14·5 ± 1·2
Q298 b11·9 ± 310·6 ± 3·2
Q322 b12·5 ± 3·112 ± 1·9
 d.f.FP  
Isolate36·20·0009  
Host10·030·8  
Isolate*Host30·20·9  
Residual67    

There was no significant difference in disease incidence and severity levels on the two provenances and there was no significant isolate × host interaction (Table 8). No disease symptoms were detected on control seedlings.

Using spot inoculations (Fig. 3), significant differences were identified between isolates of Q. pitereka on Presho (one-way anovaF2,87 = 90·6; < 0·0001), Home (F2,87 = 14·9; P <0·0001), Yeppoon (F2,87 = 19; P < 0·0001), Richmond Range (F2,61 = 15·6; < 0·0001) and Myrtle Creek (F2,66 = 20·4; < 0·0001). There was no significant difference when isolates were inoculated onto Mt McEuan seedlings (F2,87 = 1·7; = 0·2). Isolate Q298 showed a higher level of aggressiveness on Presho, Home and Yeppoon, whereas isolate Q322 showed a higher level of aggressiveness on Mt McEuan, Richmond Range and Myrtle Creek. Apart from the result for Yeppoon this matched the results from the spray inoculation study. Isolate Q152 showed the lowest level of aggressiveness of the three isolates. Isolate Q298 and Q322 did not show significantly different levels of aggressiveness on any of the host provenances. Both Q298 and Q322 caused larger lesions than Q152 on all spotted gum provenances. No lesion development occurred on controls inoculated with SDW and Tween 20.

image

Figure 3.  Comparison of mean lesion size (+1 standard error) on different provenances of spotted gum species 14 days after inoculation using the spot inoculation method with three isolates of Quambalaria pitereka.

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Comparisons of isolate aggressiveness on Ccv clones

Significant differences in isolate aggressiveness were identified when assessed on seven different spotted gum clones using spot inoculation with spores of Q. pitereka (two-way anovaF3,291 = 13·6; < 0·0001) (Fig. 4). No single isolate showed a different level of aggressiveness on all clones with a significant isolate × host interaction (F18,291 = 1·9; P = 0·02). Isolate Q322 showed a higher level of aggressiveness on four of the seven clones assessed and caused significantly higher disease levels than isolates Q107 and Q152. Isolate Q152 showed the lowest level of aggressiveness, significantly different from all other isolates. Isolate Q298 showed the highest level of aggressiveness on two of the seven clones and was significantly different from Q107.

image

Figure 4.  Comparison of Quambalaria pitereka isolate aggressiveness on seven spotted gum clones using the spot inoculation method showing mean lesion size (+1 standard error). Matching letters designate means that do not differ significantly (Fisher’s PLSD test < 0·0001).

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Significant differences in clone susceptibility were also identified (F6,291 = 42·8; < 0·0001) with disease levels greatest on clone 2. No lesion development was detected on control seedlings.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Results of this study are the first to consider possible variability in the important leaf and shoot pathogen Q. pitereka. The results show conclusively that isolates differ substantially in their aggressiveness to host species, provenances and clones of spotted gum, as well as Corymbia hybrids. While the number of isolates examined in this study was relatively limited, the differences in isolate aggressiveness shown will have significant implications for future breeding programmes. Interesting questions can also be raised regarding the source of the observed variability, particularly given that only the anamorph of Q. pitereka is known.

Results of this study indicate that Q. pitereka isolates derived from spotted gum and C. torelliana have a relatively restricted host range. While lesions developed on all host species, they were small and much less severe than those on Ccv. The lack of sporulation on these lesions could also be an indication of a noncompatible host–pathogen interaction, with the pathogen unable to complete its life cycle. Interestingly, significant lesion development was observed on A. costata. Why this species is so susceptible in comparison to other Angophora species and, more to the point, other closely related Corymbia species, is unknown. Previous studies (Walker & Bertus, 1971) suggest that Q. pitereka has a wider host range than that identified here, with infection following artificial inoculation occurring on C. ficifolia, C. exima and C. maculata. Simpson (2000) also determined that Q. pitereka infected species of Angophora and Corymbia and was responsible for shoot dieback of ghost gums, Blakella, in central Australia. Species of Eucalyptus in this study produced nonsporulating lesions, suggesting a noncompatible host–pathogen interaction.

Analysis of collections of Q. pitereka samples deposited in herbarium collections in Queensland (BRIP) and New South Wales (DAR) indicate that Q. pitereka has been identified on a very wide range of hosts, including numerous Corymbia species (C.  ficifolia, C. trachyphloia, C. tessellaris, C. calophylla, C. intermedia, C. polycarpa, C. watsoniana, C. zygophylla, C. bloxsomei, C. peltata, C. haematoxylon, C. gummifera, C. ferruginea, C. papuana and C. nesophila), several Angophora species (A. cordifolia, A. melanoxylon and A. floribunda) and two Eucalyptus species (Eucalyptus crebra and E. grandis) (G. S. Pegg, unpublished data). The holotype specimen used to describe the species was collected from C. exima (Simpson, 2000) and when examined along with isolates from spotted gum plantations using DNA studies, it resided within a common haplotype (Pegg et al., 2008). This would suggest that the host range for Q. pitereka is wide, although generally restricted to Corymbia spp. However, many of these specimens collected were identified prior to the use of molecular tools in fungal taxonomy, the identification of other Quambalaria species, and prior to the genus being reclassified (Simpson, 2000; de Beer et al., 2006). Therefore, records for Eucalyptus spp. could represent Q. eucalypti, recently found in Australia (Pegg et al., 2008). The fact that the present study showed Q. pitereka isolates from spotted gum and C. torelliana to have a relatively restricted host range suggests that more detailed population and host-range studies are required.

Of the four isolates in this study, two showed consistently higher levels of aggressiveness on spotted gum species, C. torelliana and Corymbia hybrids, but there was no evidence of host specificity. Isolates collected from spotted gum and C. torelliana were able to infect both host species in reciprocal transfer experiments. The fact that isolates selected from spotted gum in southern Queensland and northern New South Wales caused disease symptoms on C. torelliana in the glasshouse raises some interesting questions. Quambalaria pitereka has been found on C. torelliana in north Queensland (Pegg et al., 2008), but has not been detected on this host in south east Queensland or northern New South Wales, despite being planted widely as an amenity tree (Lee, 2007; Pegg et al., 2008). However, isolates collected from spotted gum in these regions showed a significantly higher level of aggressiveness on C. torelliana than those collected from C. torelliana in north Queensland. Conversely, the isolate originating from C. torelliana caused disease symptoms on spotted gum species. This is in contrast to a previous study (Pegg et al., 2008) where isolates collected from C. torelliana and Corymbia hybrids in north Queensland were limited to a haplotype specific to north Queensland and these taxa. Isolates from spotted gum within the same plantation did not share this haplotype nor did isolates taken from Corymbia hybrids in southern Queensland.

Isolates were different in their aggressiveness to Corymbia provenances, and the provenances displayed differences in susceptibility, tested both with spot and spray inoculation. While two isolates showed higher levels of aggressiveness in general, the level of aggressiveness toward the different provenances varied and there was evidence of some isolate × host interaction within provenances of Ccv. It was unclear from the results whether there was any definitive pattern of host specificity such as has been found for various other tree pathogens. For example, Powers & Matthews (1980) found that pine seedlings from different geographic sources were most susceptible to infection by their respective local fusiform rust fungal isolates, indicating host specificity. Likewise, Thompson & Burdon (1992) also suggested that locally derived pathogen isolates are more likely to be virulent in a given host population than those more distantly derived. Whether this phenomenon applies to Q. pitereka has not clearly emerged from the present study.

While variability in resistance to Q. pitereka has been identified at provenance and family level, no trials have been conducted using seeds derived from sources close to the trial sites and then replicated across various regions. However, spotted gum species and provenance trials have not as yet shown evidence of a region × pathogen interaction. In fact, the opposite was shown with recent trials in northern New South Wales (Pegg et al., 2011), where spotted gum provenances from northern Queensland were more resistant to infection by Q. pitereka at the trial site than provenances collected from regions closer to the trial site. This may reflect a host species difference rather than a level of host–pathogen interaction.

The two methods of inoculation used in this study gave similar results, particularly for the inoculation of provenances. However, the spot inoculation method reduced the influence of host variability and generally gave more consistent results. A disadvantage of this method is that it is labour-intensive and thus limits the numbers of isolates and hosts that can be tested. Leaf size can also limit the number of isolates that can be compared at any one time. The spray method in contrast provides an effective means to expose large numbers of individual hosts to a larger number of isolates. This method may be improved by limiting the assessment for disease levels to the first two newly developing leaves. In the present study, all leaves were assessed for disease and variable rates of leaf development may influence disease development and thus affect the accuracy of comparisons between seedlings. Sporulation and timing of spore production may also be a useful indicator to measure components of host–pathogen interaction in future assessments of both isolate variability and host susceptibility. Other quantitative traits that could be used in addition to lesion size and sporulation include infection efficiency, latent period and rate of lesion development.

While this study focused on a small number of isolates collected from only three regions, variability in aggressiveness was shown and this knowledge will be important when developing screening procedures. Identifying the level and extent of variation in relation to isolate aggression and host susceptibility is crucial in the development of a disease screening assay for plantation development using spotted gum. Detailed population studies are required to enhance the knowledge of Q. pitereka and aid in improving future evaluation of susceptibility of Corymbia germplasm for commercial development of spotted gum as a plantation species. Current breeding has identified variability in host susceptibility within spotted gum (Dickinson et al., 2004; Johnson et al., 2009) but has not considered the variability in pathogen aggression. Much of the selection is currently focused on quantitative traits, such as growth (Lee et al., 2009). The significance of the variability in isolate aggression, particularly in relation to disease development within plantation spotted gum, is unknown, as is the level of variability within plantations and native environments. The influence of the proximity and density of native spotted gum species to plantations in relation to disease development and the makeup of the pathogen population must also be considered. This is crucial for the development of the hardwood industry in Australia, where Corymbia species could play an important role in the future of timber production.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Queensland Department of Primary Industries Innovation and Biosecurity Program Investment, Forest Plantations Queensland, Elders Forestry (formerly Integrated Tree Cropping) and Forest Enterprises Australia for providing the necessary funding for this research. We also thank Forests NSW for providing spotted gum clones.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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