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Front. Agr. Sci. Eng. 2017, 4(4): 433–447 https://doi.org/10.15302/J-FASE-2017173 Available online at http://engineering.cae.cn RESEARCH ARTICLE Quambalaria species associated with eucalypt diseases in southern China Shuaifei CHEN1,2, Qianli LIU1, Guoqing LI1, Michael J. WINGFIELD (✉)2 1 China Eucalypt Research Centre, Chinese Academy of Forestry, Zhanjiang 524022, China 2 Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria 0028, South Africa Abstract The genus Quambalaria includes several mainly cloned hybrids of Eucalyptus urophylla and important pathogens of species of Eucalyptus and E. grandis, other Eucalyptus species include E. camaldu- Corymbia, mainly causing leaf and shoot blight. Recently, lensis, E. dunnii, E. globulus, E. pellita, E. smithii, extensive shoot and leaf dieback and stem cankers E. urophylla, as well as their hybrids and clones[2–4]. suspected to be Quambalaria diseases have been found Corymbia citriodora (Myrtaceae), previously classified as on young Eucalyptus urophylla  E. grandis trees in a species of Eucalyptus, has also been widely planted in Guangdong and Hainan Provinces. The occurrence of southern China[2,3] and the two genera are collectively Quambalaria species and their association with eucalypt referred to in this paper as eucalypts. hosts within China needs to be investigated for tree The extensive development of eucalypt plantations in diseases management. The isolates from the diseased China and the relatively limited numbers of clones planted samples were identified based on their morphological in the past two decades has resulted in the appearance of structures and phylogenetic analyses with DNA sequence numerous pests and pathogens that have caused increasing data for the internal transcribed spacer region and large levels of damage[5]. Consequently, extensive surveys of ribosome subunit RNA of the nuclear rDNA. This eucalypt plantations have been undertaken in southern work revealed that three species of Quambalaria were China, resulting in several important diseases being present: Quambalaria pitereka from Corymbia citriodora, recorded. These include stem diseases caused by Terato- Q. eucalypti from E. urophylla  E. grandis, both isolated sphaeria zuluensis[6,7], species of Botryosphaeriaceae[8], from young eucalypt leaves and shoots in Guangdong species of Cryphonectriaceae[9,10] and Ceratocystis[11]. Province, and Quambalaria simpsonii, which was isolated Leaf diseases caused by Calonectria spp.[12–14], species of from stem cankers of E. urophylla  E. grandis at four Mycosphaerellaceae and Teratosphaeriaceae[15,16] have different sites across Guangdong and Hainan Provinces. also emerged as serious problems. The leaf and shoot These results confirmed that Quambalaria agents were pathogen, Quambalaria pitereka has been found on associated with the diseases occurring on eucalypt hosts in Corymbia citriodora in the Guangdong Province of South China. This is the first report of Q. eucalypti in Asia southern China[17]. and the first report of Q. simpsonii in China on Eucalyptus Six species of Quambalaria occur on eucalypts. They trees. include Q. coyrecup, Q. cyanescens, Q. eucalypti, Q. pitereka, Q. pusilla and Q. simpsonii and all appear to Keywords Corymbia, Eucalyptus, forest pathogens, be native to Australia where their host trees also occur plantations, Myrtaceae naturally[18–27]. Quambalaria eucalypti has also been found on native Myrceugenia glaucescens (Myrtaceae) trees in Uruguay, although it seems likely to have been 1 Introduction introduced into that country[28]. Of the six Quambalaria spp., Q. eucalypti and Q. pitereka cause leaf and shoot In China, Eucalyptus spp. (Myrtaceae) have been widely blight on eucalypts[20,21,24], Q. coyrecup causes cankers established in commercial plantations which cover about and shoot blight on Corymbia spp.[23], and Q. cyanescens 4.5 million hectares in southern China[1]. They include is generally regarded as a saprophyte[22]. It remains unknown as to whether Q. simpsonii is pathogenic to Received May 2, 2017; accepted September 28, 2017 eucalypts[26], and the taxonomic status of Q. pusilla [22,29] Correspondence: mike.wingfi[email protected] remains unresolved . © The Author(s) 2017. Published by Higher Education Press. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0) 434 Front. Agr. Sci. Eng. 2017, 4(4): 433–447 Recently, leaf and shoot blight with symptoms typical of 2 Materials and methods those caused by species of Quambalaria was observed on young C. citriodora and E. urophylla  E. grandis trees in 2.1 Collections of fungal isolates southern China. In addition, a fungus with morphological characteristics typical of Quambalaria was isolated from Leaf and shoot blight was observed on C. citriodora trees cankers on the stems of E. urophylla  E. grandis trees. of different provenances in two experimental plantations The aims of this study were to identify these Quambalaria and an E. urophylla  E. grandis plantation in Guangdong spp. based on comparisons of DNA sequence data and Province in southern China (Figs. 1a, 1c, 1d and 1e). White morphological characteristics. masses of conidia and conidiophores characteristic of the Fig. 1 Symptoms of infection by Quambalaria spp. on eucalypt trees. Shoot (a) and juvenile leave (b) of Corymbia citriodora infected by Quambalaria pitereka covered in white masses of conidia and conidiophores. New shoot (c) produced from the infected C. citriodora, and reinfected by Q. pitereka. Death of apical shoot (d) of Eucalyptus urophylla  E. grandis clone infected by Quambalaria simpsonii. Mature leaf (e) and young apical shoot (f) of E. urophylla  E. grandis clone infected by Q. simpsonii. Arrows indicate infected sites. Shuaifei CHEN et al. Quambalaria species in China 435 Quambalaria[21,22] were common on the surface of the To characterize the haplotypes from ITS sequences, all infected leaves and shoots (Figs. 1b and 1f). Isolations haplotypes designated by Pegg et al.[24] were determined were made by scraping conidial masses from the leaf and for all isolates of Quambalaria spp. from this and previous shoot surfaces and transferring these to 2% malt extract studies (Table 1). For phylogenetic analyses, two isolates agar (MEA) medium (20 g malt extract and 20 g agar per representing each haplotype were used. Where only one liter water) and incubated at 25°C. During the process of isolate was available for a particular haplotype, this isolate isolating the stem canker pathogen Teratosphaeria zuluen- was duplicated in the phylogenetic analyses to determine sis (unpublished data) from cankered E. urophylla  whether it would reside in an independent clade. Micro- E. grandis hybrid trees, a fungus with the morphological stroma juglandis was used as the outgroup taxon (Table 1). characteristics of Quambalaria species was isolated and For haplotype determination using LSU sequences, these cultures were included in the present study. All representative Chinese isolates which included all the Quambalaria isolates were collected during August 2015 haplotypes determined based on the ITS sequences, and all and June 2016. isolates for which the LSU had been sequenced in previous After the fungi had been cultured for 10 d on 2% MEA, studies were included (Table 1). All isolates used for single germ tubes emerging from colonies were subculture haplotype determination by LSU sequences were used in on 2% MEA media to obtain pure cultures. Cultures were the phylogenetic analyses. Where only one isolate was deposited in the culture collection of the China Eucalypt available for a particular haplotype, the isolates were Research Centre, Chinese Academy of Forestry, Zhan- duplicated in the phylogenetic analyses. M. juglandis was jiang, China. Representative isolates were also deposited at also used as the outgroup taxon for the LSU analyses the China Forestry Culture Collection Centre, Beijing, (Table 1). China (Table 1). Sequences in ITS and LSU data sets were aligned using the iterative refinement method (FFT-NS-i settings) of the 2.2 DNA extraction, PCR and sequence reactions online platform of MAFFT v. 5.667[34]. The alignments were further edited manually in MEGA version 4 soft- Isolates collected from eucalypt trees in this study were ware[33]. All alignments were deposited in TreeBASE. identified based on DNA sequence comparisons (Table 1). The phylogenetic analyses were conducted using the For DNA extraction, isolates were grown on 2% MEA at maximum likelihood (ML) method, the ML tests were 25°C for 10 d after which actively growing mycelium for conducted with PHYML v. 3.0[35] and the best models of each isolate was scraped from the surface of the medium nucleotide substitution were established with MODELT- using sterile scalpel blades and transferred to 1.5-mL EST v. 3.7[36]. The analyses were conducted using Eppendorf tubes. DNA was extracted using “method 5” PHYML v. 3.0[35]. Additional ML parameters in described by Van Burik et al.[31]. The concentration of PHYML included retention of the maximum number of resulting DNA was checked using a Nano-Drop 2000 1000 trees and the determination of nodal support by Spectrometer (Thermo Fisher Scientific Inc., Waltham, nonparametric bootstrapping with 1000 replicates. The MA, USA). phylogenetic trees were viewed using MEGA version 4 Two gene regions, the internal transcribed spacer (ITS) software[33]. regions including the 5.8S gene of the rDNA operon and the conserved nuclear large subunit

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