Quambalaria eucalypti found on Eucalyptus in Indonesia

The Eucalyptus plantation industry in Indonesia has expanded rapidly during the last few decades. During routine nursery disease surveys, symptoms of a leaf and shoot blight disease were detected on Eucalyptus mother plants. Isolates were obtained from symptomatic tissues and identified using DNA sequence analyses. Phylogenetic analyses showed that the isolates were those of Quambalaria eucalypti . Pathogenicity tests were conducted with isolates of Q. eucalypti on clones of E. pellita and E. grandis × E. pellita

Beginning in 2018, symptoms of a leaf and shoot blight disease resembling infection by a Quambalaria species were observed in nurseries in North Sumatra, Riau and in North Kalimantan (Indonesia).These infections were mostly on clonal mother plants used to vegetatively propagate Eucalyptus for plantation establishment.
The objectives of this study were to identify the causal agent of this disease and to test the pathogenicity of the putative pathogen on different Eucalyptus genotypes.

| Sample collection and fungal isolation
Symptoms of a leaf and shoot blight disease were observed on the leaves of Eucalyptus mother plants in nurseries in three regions, including North Sumatra, Riau and North Kalimantan.The disease was characterized by powdery white fungal spore masses on young leaves, shoots and stems (Figure 1).The symptoms were mainly observed on mother plants of E. pellita and its hybrids, including E. pellita × E. grandis, E. pellita × E. brassiana and E. pellita × E. urophylla.Symptomatic leaf and shoot samples (Figure 1) were collected from Eucalyptus clones in five nurseries in different regions including one in North Sumatra, three in Riau and one nursery in North Kalimantan (Figure 2).Each sample was placed in a separate brown paper bag and then transferred to the laboratory for isolation in culture.
White spore masses on the surface of the leaves and shoots were scraped from the samples with a sterile needle and transferred to the surface of potato dextrose agar (PDA Acumedia®: 40 g/L) in Petri dishes.The culture plates were then incubated at 27°C for 7 days, and single hyphal tips from primary isolations were subcultured on clean PDA to obtain pure isolates.The resulting isolates were stored in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.

| DNA extraction, PCR amplification and sequencing
DNA was extracted from mycelium of 7-day-old cultures using Prep-man® Ultra Sample Preparation Reagent (Thermo Fisher Scientific, Waltham, MA, USA).The internal transcribed spacer (ITS) regions 1 and 2, including the 5.8S rRNA region and the large subunit (LSU) of the rRNA, were amplified using primers ITS1F/ITS4 (Gardes & Bruns, 1993;White et al., 1990) and LR0R/LR5 (Rehner & Samuels, 1994;Vilgalys & Hester, 1990), respectively.Polymerase chain reaction (PCR) amplifications were performed in 13 μL reactions containing 1 μL of genomic DNA, 2.5 μL of 5× MyTaq buffer (Bioline, London, UK), 0.25 μL MyTaq DNA polymerases (Bioline), 0.5 μL of each primer (10 μM) and 8.25 μL sterile deionized water.Thermal cycling included an initial denaturation at 95°C for 5 min, followed by 10 primary amplification cycles of 30 s at 95°C, 30 s at 55°C and 60 s at 72°C, then 30 additional cycles of the same reaction sequence, with the annealing step increasing by 5 s per cycle.Reactions were completed with a final extension at 72°C for 10 min.Polymerase chain reaction products were purified using ExoSAP-IT PCR Product Cleanup Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced using BigDye terminator sequencing kit 3.1 (Applied Biosystems, Forster City, CA, USA) in both the forward and reverse primers.Sequencing was performed on an ABI PRISM 3100 DNA sequencer (Applied Biosystems, Forster City, CA, USA).Geneious Prime 2023.0.3 (https://www.geneious.com) was used to assemble and edit the raw sequences.All the sequences resulting from this study were deposited in GenBank (http://www.ncbi.nlm.nih.gov/)(Table 1).
F I G U R E 1 Symptoms of Q. eucalypti infection on leaves and stems.

| Phylogenetic analyses
Reference sequences for species closely related to those found in this study were downloaded from the GenBank database (Table 1).
All sequences were aligned using MAFFT v. 7 (http://mafft.cbrc.jp/align ment/serve r/) (Katoh & Standley, 2013), and then manually confirmed in MEGA v. 7 (Kumar et al., 2016) where necessary.Maximum likelihood (ML) analyses were performed on the individual regions as well as on the combined ITS and LSU data sets, using RaxML v. 8.2.4 on the CIPRES Science Gateway v. 3.3 (Stamatakis, 2014) with the default GTR substitution matrix and 1000 rapid bootstraps.Sequences for Microstroma juglandis (RB2042) were used as the outgroup.The resulting trees were viewed using MEGA v. 7 (Kumar et al., 2016).

| Relative aggressiveness of isolates
Initially, each isolate was subcultured on clean PDA and incubated at 27°C for 21 days.To induce the spore production, 10 mL of sterilized distilled water (SDW) was spread over the surface of the cultures.The water was then removed from the plates, and the cultures were further incubated at 27°C for 48 h, after which this technique was repeated three times.The spore suspension was then harvested and adjusted to 1 × 10 6 spore/mL using a haemocytometer.A drop of Tween 20 (Sigma-Aldrich) was added to the spore suspension prior to the inoculation to facilitate dispersion of the spores.
An initial inoculation trial was conducted using an E. pellita clone (clone ECL04) known to be susceptible to infection in the nursery, following the methods described by Mafia et al. (2009) and Bragança et al. (2016) with some modifications.Eight-week-old plants were used in this test that included nine different isolates chosen to represent the range of collection sites (Table 1).Five plants were inoculated with each isolate and where the leaves had either been wounded or not.Wounds were induced using a sterile hypodermic needle (disposable needle 21G, duraSurge) and four 10-mm-long scratches on four expanding leaves were made on each plant.The spore suspension was sprayed onto the surface of leaf until run-off.
For the controls, an equal number of plants were sprayed with sterilized distilled water.Each plant was then covered with a clear plastic bag to maintain a high relative humidity, and these were incubated at 30°C for 48 h.The plastic bags were then removed and the plants maintained at 30°C for 14 days.
A total of 20 leaves (5 plants × 4 leaves) were evaluated for each treatment.Disease severity of the leaves on each plant was assessed using the rating scale from 0 to 4 (Table 2).Isolations were made from inoculated tissue, and the resulting isolates were identified based on morphology.Data were analysed using Kruskal-Wallis tests to determine whether there were statistically significant differences between the treatments.Pairwise comparisons were then performed using the Wilcoxon rank sum test with continuity correction.All statistical analyses were performed in the R statistical software, version 3.2.0(R Core Team, 2020).sessment and re-isolation were carried out following the protocol described above.These data were analysed in the same way as the initial inoculation trial.

| Isolates
A total of 43 isolates were obtained from infected leaf and stem samples, morphologically resembling a Quamlabaria species.Of these, eight isolates were obtained from North Sumatera, 31 isolates were obtained from Riau, including Riau 1 (2 isolates), Riau 2 (18 isolates) and Riau 3 (11 isolates), while four isolates were obtained from North Kalimantan.

| Phylogenetic analyses
Nine isolates collected across the various sampling regions were selected for further studies.Amplicons of approximately 680 bp for the ITS and 870 bp for the LSU were generated.The ITS, LSU and combined sequence data sets used for phylogenetic analyses included 24 in-group taxa and contained 632, 561 and 1193 characters, respectively.Both the individual tree and combined trees were found to be congruent, having similar topologies.All isolates sequenced in this study were grouped in monophyletic clades with the ex-type and representative isolates of Q. eucalypti in all the analyses (Figure 3).These isolates were thus identified as Q. eucalypti.

| Relative aggressiveness of isolates
No symptoms of Quambalaria infection were found on plants inoculated without wounding, and there were also no symptoms on the control plants.In the case of the wounded leaves, white spore masses typical of Q. eucalypti were found on most of the inoculated plants 12 days after inoculation (Figure 4).The aggressiveness varied between isolates, with disease severity ranging from 0 to 4 (Figure 5).Isolate CMW 57605 was the most aggressive followed by CMW 57602 and CMW 57618 where the disease severity ratings were 4, 3.6 and 3.6, respectively.Some isolates produced less infection on the wounds with severity of 1, 0. Quambalaria eucalypti was easily re-isolated from the spore masses on the infected but never the control plants.
Based on the results of the Kruskal-Wallis test, disease tolerance in clone ECL02 was statistically different from the other five clones tested (H = 1659.7,df = 11, and p < 2.2e-16).Isolates morphologically typical of Q. eucalypti were easily recovered from all inoculated plants.No symptoms were observed on the control plants.

| DISCUSS ION
The results of this study showed that a new leaf and shoot disease emerging on Eucalyptus nursery plants in three regions of Indonesia was caused by Q. eucalypti.This was determined based on isolations made from symptomatic material, identification of the resulting isolates using DNA sequence analyses as well as pathogenicity tests.This is the first report of the pathogen in Indonesia as well as in Southeast Asia.
TA B L E 2 Disease severity scale used to score infections on leaves inoculated with Q. eucalypti, determined based on the presence of spore masses typical of Q. eucalypti.Several Eucalyptus species or genotypes have previously been reported to be affected by Q. eucalypti including E. grandis and E. nitens in South Africa (Roux et al., 2006;Wingfield et al., 1993), E. globulus and E. saligna × E. maidenii hybrids in Brazil (Alfenas et al., 2001), E. globulus and E. grandis in Uruguay (Bettucci et al., 1999), E. grandis, E. longirostrata, E. grandis × E. camaldulensis hybrids, E. microcorys and E. dunnii in Australia (Pegg et al., 2008), E. globulus in Portugal (Bragança et al., 2016), and E. urophylla × E. grandis hybrids in China (Chen et al., 2017).However, this is the first time that Q. eucalypti has been reported on E. pellita and its hybrids including hybrids with E. grandis, E. brassiana and E. urophylla.This is of substantial concern given the growing importance of E. pellita as a plantation species in the humid tropics (Bristow et al., 2006;Nambiar et al., 2018).
The pathway of entry of Q. eucalypti into Indonesia is unknown.
This pathogen is likely native to Australia (de Beer et al., 2006;Pegg, Carnegie, et al., 2009;Roux et al., 2006;Wingfield et al., 1993;Zhou et al., 2007) but has been accidentally introduced into countries in Africa, Asia, Europe and South America (Alfenas et al., 2001;Bragança et al., 2016;Chen et al., 2017;Pegg et al., 2008;Wingfield et al., 1993).Based on DNA sequence analysis, two ITS haplotypes were detected in the Q. eucalypti collection in this study.The majority of the isolates, collected in North Sumatra and Riau, shared the same haplotype as those known in China, while the isolates from Kalimantan shared the same haplotype as that in South Africa, Portugal and Uruguay (Chen et al., 2017).Future studies at the population genetic level are planned to understand the likely source of origin of the pathogen in Indonesia.
An inoculation trial showed that wounds were necessary for Q. eucalypti to infect leaves.This is an interesting result as the pathogen has previously been shown to easily infect young and unwounded leaf and shoot tissue (Pegg, Webb, et al., 2009;Wingfield et al., 1993).In contrast, our results are similar to those of  different Eucalyptus genotypes differ in their susceptibility to infection.It should thus be possible to select clones resistant to infection using a relatively simple screening procedure.This approach has been used previously (Bragança et al., 2016;Pegg et al., 2011;Roux et al., 2006)  Quambalaria eucalypti appears to be a pathogen of increasing prevalence in many regions of both the northern and southern hemispheres (Alfenas et al., 2001;Bettucci et al., 1999;Bragança et al., 2016;Chen et al., 2017;Pegg et al., 2008;Roux et al., 2006;Simpson, 2000;Wingfield et al., 1993).In Indonesia, its incidence is mainly in the nursery, and there are currently no reports of the pathogen causing problems on established trees.This situation could easily change, as it has in South Africa (Roux et al., 2006), making it important not to establish susceptible clones in plantations.
The pathogen could also undergo a host shift to infect commonly occurring native trees and shrubs in the Myrtaceae, as has been found in Uruguay (Pérez et al., 2008).

ACK N O WLE D G E M ENTS
We are grateful to Royal Golden Eagle (RGE) and the Forestry Agricultural Biotechnology Institute (FABI) at the University of Pretoria for financial support that made this study possible.This study was conducted as part of the RGE-FABI Tree Health Programme funded by both organizations and based both in South Africa and Indonesia.

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I G U R E 2 Geographic location of the sampling sites in Sumatra and Kalimantan, Indonesia.Number of samples collected from each site was indicated on the map.TA B L E 1 Collection details and GenBank accession numbers of isolates included in the phylogenetic analyses.Isolates obtained in this study are indicated in bold.CBS-The culture collection of Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands; CERC-China Eucalypt Research Centre (CERC), Chinese Academy of Forestry (CAF), ZhanJiang, GuangDong, China; CMW-culture collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; DAR-the plant pathology herbarium for the Department of Agriculture in NSW, Australia; WAC-Department of Agriculture Western Australia Plant Pathogen Collection, Perth, Australia.
Mafia et al. (2009) who showed that wounds favoured infection by Q. eucalypti.These results are intriguing given the fact this pathogen is clearly able to infect leaves via the stomata(Pegg, Webb, et al., 2009) and thus typical of a primary pathogen.Inoculation on Eucalyptus leaves of single susceptible genotype with numerous different Q. eucalypti isolates showed that these differed markedly in their ability to initiate infections.The most aggressive of the isolates used in an inoculation trial also showed that F I G U R E 4 Symptoms of Q. eucalypti infection on Eucalyptus leaves after inoculation showing typical white spore masses of the pathogen (a, b), while control produce no symptom (c, d).F I G U R E 5 Inoculation with unwounded and wounded methods on Eucalyptus clone ECL04 using nine Quambalaria eucalypti isolates.F I G U R E 6 Bar chart indicating the severity score resulting from inoculation trials of six Eucalyptus genotypes inoculated with Q. eucalypti (CMW 57605) and the controls.
providing an opportunity to manage the problem.Such screening is particularly relevant in the Indonesia situation where mother plant hedges are needed to mass propagate planting stock and leaf or shoot diseases, such as those caused by Q. eucalypti can significantly reduce productivity.The results of this study show the potential to manage Q. eucalypti in future by selecting materials tolerant to the disease.
Phylogenetic tree based on maximum likelihood (ML) analyses of ITS, LSU and combined sequences for Quambalaria spp.Isolates sequenced in this study are presented in boldface.Bootstrap values of ≥60% for ML analyses are indicated at the nodes.Bootstrap values <60% are marked with '*'.Microstroma juglandis (isolate RB2042) represents the outgroup.