Quambalaria species associated with eucalypt diseases in southern China

Abstract

The genus Quambalaria includes several important pathogens of species of Eucalyptus and Corymbia, mainly causing leaf and shoot blight. Recently, extensive shoot and leaf dieback and stem cankers suspected to be Quambalaria diseases have been found on young Eucalyptus urophylla × E. grandis trees in Guangdong and Hainan Provinces. The occurrence of Quambalaria species and their association with eucalypt hosts within China needs to be investigated for tree diseases management. The isolates from the diseased samples were identified based on their morphological structures and phylogenetic analyses with DNA sequence data for the internal transcribed spacer region and large ribosome subunit RNA of the nuclear rDNA. This work revealed that three species of Quambalaria were present: Quambalaria pitereka from Corymbia citriodora, Q. eucalypti from E. urophylla × E. grandis, both isolated from young eucalypt leaves and shoots in Guangdong Province, and Quambalaria simpsonii, which was isolated from stem cankers of E. urophylla × E. grandis at four different sites across Guangdong and Hainan Provinces. These results confirmed that Quambalaria agents were associated with the diseases occurring on eucalypt hosts in South China. This is the first report of Q. eucalypti in Asia and the first report of Q. simpsonii in China on Eucalyptus trees.

Keywords

Corymbia / Eucalyptus / forest pathogens / plantations / Myrtaceae

1 Introduction

In China, Eucalyptus spp. (Myrtaceae) have been widely established in commercial plantations which cover about 4.5 million hectares in southern China^[1^]. They include mainly cloned hybrids of Eucalyptus urophylla and E. grandis, other Eucalyptus species include E. camaldulensis, E. dunnii, E. globulus, E. pellita, E. smithii, E. urophylla, as well as their hybrids and clones^[2–4^]. Corymbia citriodora (Myrtaceae), previously classified as a species of Eucalyptus, has also been widely planted in southern China^[2,3^] and the two genera are collectively referred to in this paper as eucalypts.

The extensive development of eucalypt plantations in China and the relatively limited numbers of clones planted in the past two decades has resulted in the appearance of numerous pests and pathogens that have caused increasing levels of damage^[5^]. Consequently, extensive surveys of eucalypt plantations have been undertaken in southern China, resulting in several important diseases being recorded. These include stem diseases caused by Teratosphaeria zuluensis^[6,7^], species of Botryosphaeriaceae^[8^], species of Cryphonectriaceae^[9,10^] and Ceratocystis^[11^]. Leaf diseases caused by Calonectria spp.^[12–14^], species of Mycosphaerellaceae and Teratosphaeriaceae^[15,16^] have also emerged as serious problems. The leaf and shoot pathogen, Quambalaria pitereka has been found on Corymbia citriodora in the Guangdong Province of southern China^[17^].

Six species of Quambalaria occur on eucalypts. They include Q. coyrecup, Q. cyanescens, Q. eucalypti, Q. pitereka, Q. pusilla and Q. simpsonii and all appear to be native to Australia where their host trees also occur naturally^[18^–27^]. Quambalaria eucalypti has also been found on native Myrceugenia glaucescens (Myrtaceae) trees in Uruguay, although it seems likely to have been introduced into that country^[28^]. Of the six Quambalaria spp., Q. eucalypti and Q. pitereka cause leaf and shoot blight on eucalypts^[20^,21^,24^], Q. coyrecup causes cankers and shoot blight on Corymbia spp.^[23^], and Q. cyanescens is generally regarded as a saprophyte^[22^]. It remains unknown as to whether Q. simpsonii is pathogenic to eucalypts^[26^], and the taxonomic status of Q. pusilla remains unresolved^[22^,29^].

Recently, leaf and shoot blight with symptoms typical of those caused by species of Quambalaria was observed on young C. citriodora and E. urophylla× E. grandis trees in southern China. In addition, a fungus with morphological characteristics typical of Quambalaria was isolated from cankers on the stems of E. urophylla× E. grandis trees. The aims of this study were to identify these Quambalaria spp. based on comparisons of DNA sequence data and morphological characteristics.

2 Materials and methods

2.1 Collections of fungal isolates

Leaf and shoot blight was observed on C. citriodora trees of different provenances in two experimental plantations and an E. urophylla× E. grandis plantation in Guangdong Province in southern China (Figs. 1a, 1c, 1d and 1e). White masses of conidia and conidiophores characteristic of the Quambalaria^[21,22^] were common on the surface of the infected leaves and shoots (Figs. 1b and 1f). Isolations were made by scraping conidial masses from the leaf and shoot surfaces and transferring these to 2% malt extract agar (MEA) medium (20 g malt extract and 20 g agar per liter water) and incubated at 25°C. During the process of isolating the stem canker pathogen Teratosphaeria zuluensis (unpublished data) from cankered E. urophylla× E. grandis hybrid trees, a fungus with the morphological characteristics of Quambalaria species was isolated and these cultures were included in the present study. All Quambalaria isolates were collected during August 2015 and June 2016.

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.

Full size|PPT slide

After the fungi had been cultured for 10 d on 2% MEA, single germ tubes emerging from colonies were subculture on 2% MEA media to obtain pure cultures. Cultures were deposited in the culture collection of the China Eucalypt Research Centre, Chinese Academy of Forestry, Zhanjiang, China. Representative isolates were also deposited at the China Forestry Culture Collection Centre, Beijing, China (Table 1).

Tab.1 Isolates of Quambalaria species collected from eucalypt trees in southern China in 2015 and 2016 and used for phylogenetic and morphological analysis

Identity	Isolate No.a	GenBank accession No.b			Host	Location	Collector	Reference
		ITS	LSU
Quambalaria coyrecup	WAC12947cde	DQ823431	DQ823444		Corymbia calophylla	Western Australia, Australia	T Paap	Paap et al.[23]
Q. coyrecup	WAC12948de	DQ823433	DQ823446		C. calophylla	Western Australia, Australia	T Paap	Paap et al.[23]
Q. coyrecup	WAC12949e	DQ823432	DQ823445		C. calophylla	Western Australia, Australia	T Paap	Paap et al.[23]
Q. coyrecup	WAC12950de	DQ823429	DQ823447		C. calophylla	Western Australia, Australia	T Paap	Paap et al.[23]
Q. coyrecup	WAC12951de	DQ823430	DQ823448		C. calophylla	Western Australia, Australia	T Paap	Paap et al.[23]
Q. coyrecup	BRIP48338d	EF444877	N/Ag		C. polycarpa	Darwin, Northern Territory, Australia	R Pitkethley	Pegg et al.[24]
Q. coyrecup	BRIP48339d	EF444878	N/A		C. polycarpa	Darwin, Northern Territory, Australia	R Pitkethley	Pegg et al.[24]
Q. cyanescens	CBS357.73cde = CMW5583	DQ317622	DQ317615		skin of man	Netherlands	TF Visser	de Beer et al.[22]
Q. cyanescens	CBS876.73de = CMW5584	DQ317623	DQ317616		Eucalyptus pauciflora	New South Wales, Australia	VF Brown	de Beer et al.[22]
Q. cyanescens	WAC12952de	DQ823419	DQ823440		C. calophylla	Western Australia, Australia	T Paap	Paap et al.[23]
Q. cyanescens	WAC12953de	DQ823422	DQ823443		C. ficifolia	Western Australia, Australia	T Paap	Paap et al.[23]
Q. cyanescens	WAC12954e	DQ823420	DQ823442		C. calophylla	Western Australia, Australia	T Paap	Paap et al.[23]
Q. cyanescens	WAC12955de	DQ823421	DQ823441		C. calophylla	Western Australia, Australia	T Paap	Paap et al.[23]
Q. cyanescens	BRIP48396d	EF444874	N/A		Native C. citriodora	Beaudesert, Queensland, Australia	GS Pegg	Pegg et al.[24]
Q. cyanescens	BRIP48398d	EF444875	N/A		Native C. citriodora	Beaudesert, Queensland, Australia	GS Pegg	Pegg et al.[24]
Q. cyanescens	BRIP48403d	EF444876	N/A		Native C. citriodora	Beaudesert, Queensland, Australia	GS Pegg	Pegg et al.[24]
Q. eucalypti	CBS118844cde = CMW1101	DQ317625	DQ317618		Eucalyptus grandis	Kwambonambi, South Africa	MJ Wingfield	de Beer et al.[22]
Q. eucalypti	CBS 119680de = CMW11678	DQ317626	DQ317619		E. grandis clone NH58	Kwambonambi, South Africa	L Lombard	de Beer et al.[22]
Q. eucalypti	CMW14329	DQ317614	N/A		E. grandis× E. camaldulensis clone	Kwambonambi, South Africa	J Roux	Roux et al.[30]
Q. eucalypti	CBS118615= CMW17252	DQ317609	N/A		E. nitens	Rooihoogte, South Africa	ZL Mthalane & J Roux	Roux et al.[30]
Q. eucalypti	CMW17253	DQ317610	N/A		E. nitens	Rooihoogte, South Africa	ZL Mthalane & J Roux	Roux et al.[30]
Q. eucalypti	CMW17254	DQ317611	N/A		E. nitens	Rooihoogte, South Africa	ZL Mthalane & J Roux	Roux et al.[30]
Q. eucalypti	CMW17255	DQ317612	N/A		E. nitens	Rooihoogte, South Africa	ZL Mthalane & J Roux	Roux et al.[30]
Q. eucalypti	CBS118616= CMW17771	DQ317613	N/A		E. grandis clone	Kwambonambi, South Africa	J Roux	Roux et al.[30]
Q. eucalypti	UY1036	EU439922	N/A		Myrceugenia glaucescens	Uruguay	C. A. Pérez	Pérez et al.[28]
Q. eucalypti	UY1718	EU439923	N/A		M. glaucescens	Uruguay	C. A. Pérez	Pérez et al.[28]
Q. eucalypti	PE3/MEAN 996	JX297605	N/A		E. globulus	Portugal	N/A	Braganca et al.[27]
Q. eucalypti	PE6/MEAN 997	JX297603	N/A		E. globulus	Portugal	N/A	Braganca et al.[27]
Q. eucalypti	PE27/MEAN 998	JX297604	N/A		E. globulus	Portugal	N/A	Braganca et al.[27]
Q. eucalypti	PE28/MEAN 999	JX297600	N/A		E. globulus	Portugal	N/A	Braganca et al.[27]
Q. eucalypti	PE29/MEAN 1000	JX297602	N/A		E. globulus	Portugal	N/A	Braganca et al.[27]
Q. eucalypti	PE30/MEAN 1001	JX297601	N/A		E. globulus	Portugal	N/A	Braganca et al.[27]
Q. eucalypti	PE52/MEAN 1002	JX297606	N/A		E. globulus	Portugal	N/A	Braganca et al.[27]
Q. eucalypti	PE53/MEAN 1003	JX297598	N/A		E. globulus	Portugal	N/A	Braganca et al.[27]
Q. eucalypti	PE54/MEAN 1004	JX297599	N/A		E. globulus	Portugal	N/A	Braganca et al.[27]
Q. eucalypti	PE93/MEAN 1006	KR336802	N/A		E. globulus	Portugal	N/A	Braganca et al.[27]
Q. eucalypti	PE96/MEAN 1009	KR336803	N/A		E. globulus	Portugal	N/A	Braganca et al.[27]
Q. eucalypti	PE151/MEAN 1012	KR336804	N/A		E. globulus	Portugal	N/A	Braganca et al.[27]
Q. eucalypti	PE152/MEAN 1013	KR336805	N/A		E. globulus	Portugal	N/A	Braganca et al.[27]
Q. eucalypti	PE153/MEAN 1014	KR336806	N/A		E. globulus	Portugal	N/A	Braganca et al.[27]
Q. eucalypti	PE154/MEAN 1015	KR336807	N/A		E. globulus	Portugal	N/A	Braganca et al.[27]
Q. eucalypti	BRIP48367	EF444823	N/A		C. torelliana× C. citriodora subsp. variegata	Walkamin, Queensland, Australia	GS Pegg	Pegg et al.[24]
Q. eucalypti	BRIP48422d	EF444832	N/A		E. dunnii	New South Wales, Australia	AJ Carnegie	Pegg et al.[24]
Q. eucalypti	BRIP48498d	EF444844	N/A		E. grandis	New South Wales, Australia	AJ Carnegie	Pegg et al.[24]
Q. eucalypti	BRIP48507d	EF444822	N/A		E. grandis	Moggill, Queensland, Australia	GS Pegg	Pegg et al.[24]
Q. eucalypti	CERC8476d	KY615009	N/A		E. grandis	Guangdong, China	SF Chen & JQ Li	This study
Q. eucalypti	CERC8477g	KY615010	N/A		E. grandis	Guangdong, China	SF Chen & JQ Li	This study
Q. eucalypti	CERC8478	KY615011	N/A		E. grandis	Guangdong, China	SF Chen & JQ Li	This study
Q. eucalypti	CERC8479g	KY615012	KY615050		E. grandis	Guangdong, China	SF Chen & JQ Li	This study
Q. eucalypti	CERC8480g	KY615013	N/A		E. grandis	Guangdong, China	SF Chen & JQ Li	This study
Q. eucalypti	CERC8481	KY615014	KY615051		E. grandis	Guangdong, China	SF Chen & JQ Li	This study
Q. eucalypti	CERC8482g	KY615015	N/A		E. grandis	Guangdong, China	SF Chen & JQ Li	This study
Q. eucalypti	CERC8483	KY615016	N/A		E. grandis	Guangdong, China	SF Chen & JQ Li	This study
Q. pitereka	DAR19773cde	DQ823423	DQ823438		C. eximia	New South Wales, Australia	J Walker & AL Bertus	Paap et al.[23]
Q. pitereka	CMW 6707de	DQ317627	DQ317620		Corymbia maculata	New South Wales, Australia	MJ Wingfield	de Beer et al.[22]
Q. pitereka	CBS118828de = CMW5318	DQ317628	DQ317621		C. citriodora subsp. variegata	Queensland, Australia	M Ivory	de Beer et al.[22]
Q. pitereka	CMW23610	EF427372	N/A		C. citriodora	Guangdong, China	YJ Xie	Zhou et al.[17]
Q. pitereka	CMW23611	EF427373	N/A		C. citriodora	Guangdong, China	YJ Xie	Zhou et al.[17]
Q. pitereka	CMW23612	EF427374	N/A		C. citriodora	Guangdong, China	YJ Xie	Zhou et al.[17]
Q. pitereka	CMW23613d	EF427375	N/A		C. citriodora	Guangdong, China	YJ Xie	Zhou et al.[17]
Q. pitereka	BRIP48325	EF427366	N/A		C. citriodora subsp. variegata	Queensland, Australia	GS Pegg	Zhou et al.[17]
Q. pitereka	BRIP48361d	EF427367	N/A		C. citriodora subsp. variegata	Queensland, Australia	GS Pegg	Zhou et al.[17]
Q. pitereka	BRIP48370d	EF427368	N/A		C. citriodora subsp. variegata	Queensland, Australia	GS Pegg	Zhou et al.[17]
Q. pitereka	BRIP48384d	EF427369	N/A		C. citriodora subsp. variegata	Queensland, Australia	GS Pegg	Zhou et al.[17]
Q. pitereka	BRIP48386ed	EF427370	N/A		C. citriodora subsp. variegata	Queensland, Australia	GS Pegg	Zhou et al.[17]
Q. pitereka	BRIP48531d	EF427371	N/A		C. citriodora subsp. variegata	Queensland, Australia	GS Pegg	Zhou et al.[17]
Q. pitereka	WAC12957e	DQ823426	DQ823437		C. ficifolia	Western Australia, Australia	T Paap	Paap et al.[23]
Q. pitereka	WAC12958e	DQ823427	DQ823436		C. calophylla	Western Australia, Australia	T Paap	Paap et al.[23]
Q. pitereka	QP26e	DQ823424	DQ823434		C. citriodora subsp. variegata	Queensland, Australia	GS Pegg	Paap et al.[23]
Q. pitereka	QP45de	DQ823425	DQ823439		C. citriodora subsp. variegata	Queensland, Australia	GS Pegg	Paap et al.[23]
Q. pitereka	BRIP48346d	EF444845	N/A		C. citriodora subsp. citriodora	Davies Creek, Queensland, Australia	GS Pegg	Pegg et al.[24]
Q. pitereka	BRIP48317	EF444854	N/A		C. henryi	Coolabunia, Queensland, Australia	GS Pegg	Pegg et al.[24]
Q. pitereka	BRIP48381d	EF444858	N/A		C. citriodora subsp. citriodora	Silkwood, Queensland, Australia	GS Pegg	Pegg et al.[24]
Q. pitereka	BRIP48383d	EF444859	N/A		C. citriodora subsp. variegata	Beaudesert, Queensland, Australia	GS Pegg	Pegg et al.[24]
Q. pitereka	WAC12956d	DQ823428	N/A		C. ficifolia	Western Australia, Australia	T Paap	Paap et al.[23], Pegg et al.[24]
Q. pitereka	BRIP48349d	EF444860	N/A		C. torelliana × C. citriodora subsp. variegata	Mareeba, Queensland, Australia	GS Pegg	Pegg et al.[24]
Q. pitereka	BRIP48325d	EF427366	N/A		C. citriodora subsp. variegata	Binjour, Queensland, Australia	GS Pegg	Pegg et al.[24]
Q. pitereka	BRIP48328d	EF444872	N/A		Native C. citriodora subsp. variegata	Dilkoon, New South Wales, Australia	GS Pegg	Pegg et al.[24]
Q. pitereka	BRIP48432d	EF444873	N/A		C. citriodora subsp. variegata	Grafton, New South Wales, Australia	GS Pegg	Pegg et al.[24]
Q. pitereka	CERC8486de	KY615017	KY615052		C. citriodora provenance CERC10	Guangdong, China	SF Chen & GQ Li	This study
Q. pitereka	CERC8488e	KY615018	KY615053		C. citriodora provenance CERC12	Guangdong, China	SF Chen & GQ Li	This study
Q. pitereka	CERC8489	KY615019	N/A		C. citriodora provenance CERC13	Guangdong, China	SF Chen & GQ Li	This study
Q. pitereka	CERC8491	KY615020	N/A		C. citriodora provenance CERC15	Guangdong, China	SF Chen & GQ Li	This study
Q. pitereka	CERC8494eg	KY615021	KY615054		C. citriodora provenance CERC17	Guangdong, China	SF Chen & GQ Li	This study
Q. pitereka	CERC9093	KY615022	N/A		C. citriodora provenance CR76	Guangdong, China	SF Chen & Y Lin	This study
Q. pitereka	CERC9094	KY615023	N/A		C. citriodora provenance N371	Guangdong, China	SF Chen & Y Lin	This study
Q. pitereka	CERC9095	KY615024	N/A		C. citriodora provenance N28	Guangdong, China	SF Chen & Y Lin	This study
Q. pitereka	CERC9096	KY615025	N/A		C. citriodora provenance N411	Guangdong, China	SF Chen & Y Lin	This study
Q. pitereka	CERC9097eg	KY615026	KY615055		C. citriodora provenance N223	Guangdong, China	SF Chen & Y Lin	This study
Q. pitereka	CERC9098g	KY615027	N/A		C. citriodora provenance N322	Guangdong, China	SF Chen & Y Lin	This study
Q. pitereka	CERC9099eg	KY615028	KY615056		C. citriodora provenance CR033	Guangdong, China	SF Chen & Y Lin	This study
Q. pitereka	CERC9100	KY615029	N/A		C. citriodora provenance CR039	Guangdong, China	SF Chen & Y Lin	This study
Q. pitereka	CERC9101	KY615030	N/A		C. citriodora provenance CR92	Guangdong, China	SF Chen & Y Lin	This study
Q. pitereka	CERC9102	KY615031	N/A		C. citriodora provenance CR36	Guangdong, China	SF Chen & Y Lin	This study
Q. pitereka	CERC9103e	KY615032	KY615057		C. citriodora provenance N601	Guangdong, China	SF Chen & Y Lin	This study
Q. pitereka	CERC9104	KY615033	N/A		C. citriodora provenance N28	Guangdong, China	SF Chen & Y Lin	This study
Q. simpsonii	CBS 124772de	GQ303290	GQ303321		Eucalyptus tintinnans	Edith Falls, Australia	BA Summerell	Cheewangkoon et al.[26]
Q. simpsonii	CBS 124773de	GQ303291	GQ303322		Eucalyptus sp.	Lamphoon, Thailand	R Cheewangkoon	Cheewangkoon et al.[26]
Q. simpsonii	CERC8496dg	KY615034	N/A		E. urophylla × E. grandis	Hainan, China	SF Chen & QL Liu	This study
Q. simpsonii	CERC8499	KY615035	N/A		E. urophylla × E. grandis	Hainan, China	SF Chen & QL Liu	This study
Q. simpsonii	CERC8505d	KY615036	N/A		E. urophylla × E. grandis	Hainan, China	SF Chen & QL Liu	This study
Q. simpsonii	CERC8507de	KY615037	KY615058		E. urophylla × E. grandis	Hainan, China	SF Chen & QL Liu	This study
Q. simpsonii	CERC8512d	KY615038	N/A		E. urophylla × E. grandis	Hainan, China	SF Chen & QL Liu	This study
Q. simpsonii	CERC8514	KY615039	N/A		E. urophylla × E. grandis	Hainan, China	SF Chen & QL Liu	This study
Q. simpsonii	CERC8516	KY615040	N/A		E. urophylla × E. grandis	Hainan, China	SF Chen & QL Liu	This study
Q. simpsonii	CERC8517e	KY615041	KY615059		E. urophylla × E. grandis	Hainan, China	SF Chen & QL Liu	This study
Q. simpsonii	CERC8519dg	KY615042	N/A		E. urophylla × E. grandis	Hainan, China	SF Chen & QL Liu	This study
Q. simpsonii	CERC8526	KY615043	N/A		E. urophylla × E. grandis	Hainan, China	SF Chen & QL Liu	This study
Q. simpsonii	CERC8532	KY615044	N/A		E. urophylla × E. grandis	Guangdong, China	SF Chen & JQ Li	This study
Q. simpsonii	CERC8534deg	KY615045	KY615060		E. urophylla × E. grandis	Guangdong, China	SF Chen & JQ Li	This study
Q. simpsonii	CERC8536e	KY615046	KY615061		E. urophylla × E. grandis	Guangdong, China	SF Chen & JQ Li	This study
Q. simpsonii	CERC8539eg	KY615047	KY615062		E. urophylla × E. grandis	Guangdong, China	SF Chen & JQ Li	This study
Q. simpsonii	CERC8541d	KY615048	N/A		E. urophylla × E. grandis	Guangdong, China	SF Chen & JQ Li	This study
Q. simpsonii	CERC8543d	KY615049	N/A		E. urophylla × E. grandis	Guangdong, China	SF Chen & JQ Li	This study
Microstroma juglandis	R.B. 2042de	DQ317634	DQ317617		Juglans regia	Germany	R Bauer	de Beer et al.[22]

Note: a Designation of isolates and culture collections: WAC, Department of Agriculture Western Australia Plant Pathogen Collection, Perth, Australia; BRIP, the plant pathology herbarium for Queensland Department of Primary Industries and Fisheries, Australia; CBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CMW, Tree Protection Co-operative Program, Forestry and Agricultural Biotechnology Institute, University of Pretoria, South Africa; CERC, China Eucalypt Research Centre (CERC), Chinese Academy of Forestry (CAF), ZhanJiang, GuangDong, China; DAR, the plant pathology herbarium for the Department of Agriculture in NSW, Australia; MEAN, fungal collection of Instituto Nacional de Investigação Agrária e Veterinária – INIAV, Oeiras, Portugal; R.B., Herbarium R. Bauer, Tübingen, Germany; Isolate numbers in boldface were collected in this study; b GenBank numbers in boldface were sequenced in this study; c Holotype specimens or ex-type isolates; d Isolates used in phylogenetic analyses by ITS sequence; e Isolates used in phylogenetic analyses by LSU sequence; f N/A= not available; g Isolates used in morphological studies.

2.2 DNA extraction, PCR and sequence reactions

Isolates collected from eucalypt trees in this study were identified based on DNA sequence comparisons (Table 1). For DNA extraction, isolates were grown on 2% MEA at 25°C for 10 d after which actively growing mycelium for each isolate was scraped from the surface of the medium using sterile scalpel blades and transferred to 1.5-mL Eppendorf tubes. DNA was extracted using “method 5” described by Van Burik et al.^[31^]. The concentration of resulting DNA was checked using a Nano-Drop 2000 Spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Two gene regions, the internal transcribed spacer (ITS) regions including the 5.8S gene of the rDNA operon and the conserved nuclear large subunit (LSU) rDNA were amplified as described by Chen et al.^[32^]. Nucleotide sequences were edited using MEGA version 4 software^[33^]. All sequences obtained in this study were deposited in GenBank (Table 1).

2.3 Phylogenetic analyses

To identify the isolates, sequences of ITS and LSU gene regions were compared to sequences of all described Quambalaria species, including the ex-type cultures of all the identified species from GenBank (Table 1). Also, in order to examine the diversity of the Quambalaria species, the haplotypes were determined from the polymorphic nucleotides within the aligned sequence data of ITS and LSU regions for isolates collected in this and previous studies.

To characterize the haplotypes from ITS sequences, all haplotypes designated by Pegg et al.^[24^] were determined for all isolates of Quambalaria spp. from this and previous studies (Table 1). For phylogenetic analyses, two isolates representing each haplotype were used. Where only one isolate was available for a particular haplotype, this isolate was duplicated in the phylogenetic analyses to determine whether it would reside in an independent clade. Microstroma juglandis was used as the outgroup taxon (Table 1).

For haplotype determination using LSU sequences, representative Chinese isolates which included all the haplotypes determined based on the ITS sequences, and all isolates for which the LSU had been sequenced in previous studies were included (Table 1). All isolates used for haplotype determination by LSU sequences were used in the phylogenetic analyses. Where only one isolate was available for a particular haplotype, the isolates were duplicated in the phylogenetic analyses. M. juglandis was also used as the outgroup taxon for the LSU analyses (Table 1).

Sequences in ITS and LSU data sets were aligned using the iterative refinement method (FFT-NS-i settings) of the online platform of MAFFT v. 5.667^[34^]. The alignments were further edited manually in MEGA version 4 software^[33^]. All alignments were deposited in TreeBASE.

The phylogenetic analyses were conducted using the maximum likelihood (ML) method, the ML tests were conducted with PHYML v. 3.0^[35^] and the best models of nucleotide substitution were established with MODELTEST v. 3.7^[36^]. The analyses were conducted using PHYML v. 3.0^[35^]. Additional ML parameters in PHYML included retention of the maximum number of 1000 trees and the determination of nodal support by nonparametric bootstrapping with 1000 replicates. The phylogenetic trees were viewed using MEGA version 4 software^[33^].

2.4 Morphology

Single hyphal tip cultures of each Quambalaria sp. identified using DNA sequence data were subculture on 2% MEA media for 2 weeks at 25°C for morphological analysis. Four isolates for each identified Quambalaria sp. were used for comparisons of colony morphology. Conidiogenous cells and conidia were mounted in sterile water on microscope slides for measurements to be made using a Zeiss Axio Imager A1 microscope and a Zeiss AxioCam MRc digital camera with Zeiss Axio Vision Rel. 4.8 software (Carl Zeiss Ltd., Munchen, Germany). For each isolate, 25 measurements were made of conidia and ten of conidiophores. These measurements were compared with those published for species of Quambalaria. Results are presented as (minimum–) (mean – standard deviation) – (mean+ standard deviation) (–maximum).

3 Results

3.1 Collections of fungal isolates

A total of 41 fungal isolates showing typical morphology of Quambalaria species were isolated. Seventeen isolates were from leaves or shoots on 17 C. citriodora trees of 16 provenances in two experimental plantations in Guangdong Province, eight isolates were from leaves of one E. urophylla× E. grandis clone in one plantation in Guangdong Province, and 16 isolates were from cankers caused by T. zuluensis on the stems of E. urophylla× E. grandis clones in four plantations in Guangdong and Guangxi. Each of the 41 isolates was from a single tree and all were included in the DNA sequence comparisons and phylogenetic analyses (Table 1).

3.2 Phylogenetic analyses

The aligned ITS sequence data set consisted of 65 taxa and 634 characters (TreeBASE No. 20574). For the ML analyses, the Model Test analysis recommended a HKY+ I+ G model [Lset Base= (0.2639, 0.2186, 0.2071); Nst (number of substitution rate categories) = 2; Transition/transversion ratio= 2.6045; Rate matrix= (1.0000, 4.3151, 2.9747, 2.9747, 8.1747); Rates= gamma; Shape= 0.7544]. The phylogenetic analyses showed that isolates sequenced in this study resided in three clades that represent Q. pitereka, Q. eucalypti and Q. simpsonii (Fig. 2).

Fig.2 Phylogenetic tree based on maximum likelihood analysis of ITS sequence data of haplotypes of five Quambalaria species, Q. coyrecup (QCO), Q. cyanescens (QCY), Q. eucalypti (QE), Q. pitereka (QP) and Q. simpsonii (QS). Bootstrap values>60% are presented at branches, bootstrap values<60% or absent values are not shown. Haplotypes and isolates from eucalypts in this study are in boldface and highlighted. Isolates representing ex-type are marked with T, isolates repeated are marked with R. The tree is rooted to Microstroma juglandis.

Full size|PPT slide

For the ITS sequences, all Chinese and all those from previous studies represented 32 haplotypes. These included three, seven, four, 12 and six haplotypes of Q. coyrecup, Q. cyanescens, Q. eucalypti, Q. pitereka Q. simpsonii, respectively (Tables 2–4, S1). The Chinese isolates collected in this study represented six haplotypes including one of Q. pitereka, one of Q. eucalypti, and four newly designated haplotypes of Q. simpsonii (Table S1).

Tab.2 Four haplotypes of Q. eucalypti as determined from the polymorphic nucleotides within the aligned sequence data of ITS region for isolates collected from species of Eucalyptus, C. torelliana× C. citriodora subsp. variegate and M. glaucescens

Haplotype	121a	158	159	160	161	162	558
QE1	T	–	–	–	–	–	Tb
QE2	T	–	–	–	–	–	C
QE3	C	–	–	–	–	–	C
QE4	T	T	T	A	T	A	C

Note: a Base pair (bp) positions in aligned data; b Nucleotides that are different from the majority consensus sequence are underlined and highlighted in bold.

Tab.3 Twelve haplotypes of Q. pitereka as determined from the polymorphic nucleotides within the aligned sequence data of ITS region for isolates collected from species of Corymbia

Haplotype	24a	54	107	112	214	219	233	236	390	451	606	614
QP1	T	A	G	G	T	Gb	T	C	C	C	C	A
QP2	T	A	G	A	T	G	T	C	C	C	C	A
QP3	T	A	G	A	T	A	T	C	C	C	C	A
QP4	T	A	G	G	T	A	T	C	T	C	C	A
QP5	T	A	G	G	T	A	T	C	C	A	C	A
QP6	T	A	G	G	T	A	T	C	C	C	T	G
QP7	A	G	G	G	G	A	T	C	C	C	C	G
QP8	A	G	G	G	T	A	T	C	C	C	C	G
QP9	T	G	G	G	G	A	T	C	C	C	C	G
QP10	A	G	A	G	T	A	T	C	C	C	C	G
QP11	T	G	G	G	T	A	T	C	C	C	C	G
QP12	T	G	G	A	T	G	C	G	C	C	C	A

Note: a Base pair (bp) positions in aligned data; b Nucleotides that are different from the majority consensus sequence are underlined and highlighted in bold.

Tab.4 Six haplotypes of Q. simpsonii as determined from the polymorphic nucleotides within the aligned sequence data of ITS region for isolates collected from species of Eucalyptus

Haplotype	4a	171	553	605	621
QS1	Aa	A	T	T	–
QS2	A	A	C	T	–
QS3	G	A	C	T	T
QS4	G	A	C	T	–
QS5	G	A	C	C	–
QS6	G	G	C	C	–

Note: a Base pair (bp) positions in aligned data; b Nucleotides that are different from the majority consensus sequence are underlined and highlighted in bold.

The aligned LSU sequence data set consisted of 37 taxa and 561 characters (TreeBASE No. 20574). For ML analyses, model test analysis recommended a TrN+ G model [Lset Base= (0.2492, 0.1916, 0.3025); Nst= 6; Rate matrix= (1.0000, 7.7487, 1.0000, 1.0000, 31.1002); Rates= equal]. The phylogenetic analyses showed that isolates sequenced in this study resided in three clades of Q. pitereka, Q. eucalypti and Q. simpsonii, respectively (Fig. 3).

Fig.3 Phylogenetic tree based on Maximum Likelihood analysis of large subunit sequence data of haplotypes of five Quambalaria species, Q. coyrecup (QCO), Q. cyanescens (QCY), Q. eucalypti (QE), Q. pitereka (QP) and Q. simpsonii (QS), respectively. Bootstrap values>60% are presented at branches, bootstrap values<60% or absent values are not shown. Haplotypes and isolates from eucalypts in this study are in boldface and highlighted. Isolates representing ex-type are marked with T, isolates repeated are marked with R. The tree is rooted to Microstroma juglandis.

Full size|PPT slide

For the LSU sequences, 13 Chinese isolates which included all six haplotypes determined based on ITS sequences were used for phylogenetic analyses. These isolates and all of those sequenced in previous studies represented six haplotypes. These included two haplotypes of Q. pitereka and one each of Q. coyrecup, Q. cyanescens, Q. eucalypti and Q. simpsonii (Table S1). The Chinese isolates included in this study represented three haplotypes including one newly designated haplotype of Q. pitereka, and one haplotype for each of Q. eucalypti and Q. simpsonii (Table S1).

3.3 Morphology

Four isolates of Q. pitereka (CERC8494, CERC9097, CERC9098 and CERC9099), Q. eucalypti (CERC8477, CERC8479, CERC8480 and CERC8482) and Q. simpsonii (CERC8496, CERC8519, CERC8534 and CERC8539) were used in the morphological analysis. Colonies of these species were finely floccose becoming powdery and white (Figs. 4a, 4c and 4e). The morphological characteristics of the fruiting structures of these species are summarized in Table 5 and illustrated in Figs. 4b, 4d and 4f. Conidiogenous cells of Q. pitereka, Q. eucalypti and Q. simpsonii were (7.4–89.6)mm × (1.4–2.6) mm (av. 46.0 mm × 2.0 mm), (8.4–77.1)mm × (1.3–2.8) mm (av. 37.4 mm × 2.2 mm), and (7.0–82.1) mm × (1.5–2.9) mm (av. 25.6 mm × 2.4 mm), respectively. The conidia of Q. pitereka (primary conidia narrow fusiform, av. 10.9 mm × 3.4 mm, length/width= 3.2; secondary conidia narrow fusiform, av. 6.0 mm × 2.7 mm, length/width= 2.2) are longer and narrower (by length/width) than that of Q. eucalypti (primary conidia ellipsoid, av. 6.2 mm × 3.8 mm, length/width= 1.6; secondary conidia obovoid, av. 3.3 mm × 2.6 mm, length/width= 1.3) and Q. simpsonii (primary conidia fusiform, av. 7.9 mm × 3.3 mm, length/width= 2.4; secondary conidia obovoid to ellipsoid, av. 3.7 mm × 2.4 mm, length/width= 1.5), the conidia of Q. simpsonii are slight longer and narrower than that of Q. eucalypti. The morphology of Q. pitereka, Q. eucalypti and Q. simpsonii identified in this study is similar to the results of previous studies^[20,23,26^].

Fig.4 Cultures grown on malt extract agar at 25°C after 2 weeks and the primary and secondary conidia. (a,b) Quambalaria pitereka; (c,d) Q. eucalypti; (e,f) Q. simpsonii.

Full size|PPT slide

Tab.5 Primary conidial and secondary conidia measurements of three Quambalaria species identified in this study

Species	Isolate No.	Primary conidia				Secondary conidia
		(L × W) sizea/mm	(L × W) meanb/mm	L/Wc		(L × W) sizea/mm	(L × W) meanb/mm	L/Wc
Q. pitereka	CERC8494	(7.0–)7.5–13.0(–20.5) × (2.5–)3.0–3.5(–4.0)	10.2 × 3.3	3.1		(4.0–)4.5–6.0(–7.0) × 2.0–2.5(–3.0)	5.2 × 2.5	2.1
	CERC9097	(8.0–)8.5–12.5(–15.0) × (2.5–)2.5–3.5(–4.0)	10.5 × 3.0	3.5		(4.5–)5.0–6.5(–7.0) × 2.0–2.5(–3.0)	5.7 × 2.3	2.5
	CERC9098	(8.0–)10.5–14.0(–16.0) × (2.0–)3.0–4.5(–5.0)	12.2 × 3.6	3.4		6.0–7.5(–8.0) × (2.0–)2.5–3.5(–4.0)	6.8 × 3.0	2.3
	CERC9099	(8.0–)9.0–12.5(–15.0) × (3.0–)3.5–4.5(–5.5)	10.7 × 4.0	2.7		5.5–7.0(–7.5) × 2.5–3.5(–4.0)	6.3 × 3.1	2.2
	Averaged	(7.0–)8.5–13.0(–20.5) × (2.0–)2.5–4.0(–5.5)	10.9 × 3.4	3.2		(4.0–)5.0–7.0(–78.0) × 2.0–3.0(–4.0)	6.0 × 2.7	2.2
Q. eucalypti	CERC8477	(4.5–)5.0–7.5(–8.0) × (3.0–)3.5–4.5(–4.5)	6.2 × 3.9	1.6		2.5–3.0(–3.5) × 2.5–3.0	3.0 × 2.5	1.2
	CERC8479	(5.5–)6.0–7.0(–7.5) × (2.5–)3.0–4.0(–4.5)	6.3 × 3.7	1.7		2.5–3.5(–4.0) × (2.0–)2.5–3.0	3.0 × 2.5	1.2
	CERC8482	(4.5–)5.0–6.5(–7.0) × (3.0–)3.5–4.0(–4.5)	5.7 × 3.9	1.5		3.0–4.0(–5.0) × 2.5–3.0	3.6 × 2.8	1.3
	CERC8480	(5.5–)6.0–7.0(–8.0) × (3.0–)3.5–4.0(–4.5)	6.4 × 3.8	1.7		3.0–3.6(–4.0) × 2.5–3.0	3.4 × 2.7	1.3
	Averaged	(4.5–)5.5–7.0(–8.0) × (2.5–)3.5–4.0(–4.5)	6.2 × 3.8	1.6		(2.5–)3.0–4.0(–5.0) × (2.0–)2.5–3.0	3.3 × 2.6	1.3
Q. simpsonii	CERC8496	(6.0–)6.5–9.5(–11.0) × (2.0–)2.5–3.5(–4.0)	8.3 × 3.1	2.7		(3.0–)3.5–4.5(–5.0) × 2.0–2.5(–3.0)	4.0 × 2.4	1.7
	CERC8519	(5.5–)6.0–8.0(–9.0) × 3.0–4.0(–4.5)	7.1 × 3.6	2.0		(2.5–)3.0–3.5(–4.0) × 2.0–2.5	3.3 × 2.3	1.4
	CERC8534	(6.0–)7.0–9.0(–10.0) × (2.0–)3.0–3.5(–4.0)	7.9 × 3.2	2.5		(3.0–)3.5–4.5(–5.0) × 2.0–3.0	4.0 × 2.5	1.6
	CERC8539	(5.5–)6.5–10.5(–12.5) × (2.5–)3.0–4.0(–4.5)	8.4 × 3.4	2.5		3.0–4.0(–4.5) × 2.0–3.0	3.6 × 2.5	1.4
	Averaged	(5.5–)6.5–9.5(–12.5) × (2.0–)3.0–4.0(–4.5)	7.9 × 3.3	2.4		(2.5–)3.0–4.0(–5.0) × 2.0–2.5(–3.0)	3.7 × 2.4	1.5

Note: a L × W= length × width, minimum–(average–standard deviation)–(average+ standard deviation)–maximum; b L × W= length × width; c L/W= average length/average width; d average measurements of the Quambalaria species.

4 Discussion

In this study, three species of Quambalaria, Q. pitereka, Q. eucalypti and Q. simpsonii, were identified from Eucalyptus and Corymbia plantations in Guangdong and Hainan Provinces in southern China. These Quambalaria spp. were identified and characterized based on phylogenetic analysis of sequence data for LSU and ITS regions, and morphology. This is the first report of Q. eucalypti in Asia and the first report of Q. simpsonii on eucalypts in China.

Quambalaria pitereka is specific to eucalypts in the genus Corymbia. This fungus is widely distributed in different regions/sites on different species of Corymbia in Australia^[24,25^]. Outside Australia, Q. pitereka has previously been reported only on C. citriodora in one plantation in Guangdong Province^[17^]. The results of this study showed that the sequenced isolates of Q. pitereka include 12 haplotypes, only one of these was found in China and the remaining haplotypes were known only from Australia. This high level of genetic diversity for isolates from Australia supported the view^[24^] that Q. pitereka was native to that country. In the present study, Q. pitereka was isolated from 17 C. citriodora provenances in two experimental plantations. These are relatively distant from the site where Q. pitereka was first reported in 2007^[17^] and the ITS haplotype was the same as that found in the study of Zhou et al.^[17^]. These results suggest that Q. pitereka could spread actively between different regions and C. citriodora provenances in China.

Quambalaria eucalypti is considered to be one of the most important pathogens of eucalypts. Outside Australia, this fungus was first reported on Eucalyptus in nurseries in South Africa^[20^] and it was later found in Brazil^[37^] and Portugal^[27^] where it causes leaf spots, shoot infections and lesions on seedling stems. Q. eucalypti has also been recorded in Eucalyptus plantations in Brazil^[38^], South Africa^[30^], Australia^[24^] and Portugal^[27^] where it can result in severe shoot and leaf blight and stem cankers^[24,27,30^]. Other than on Eucalyptus, Q. eucalypti has been isolated from leaf lesions on native M. glaucescens trees in Uruguay^[28^] and Corymbia species in Australia^[24^]. In this study, Q. eucalypti was isolated from a diseased E. urophylla× E. grandis clone. It appears to be a pathogen of emerging importance in China.

The ITS haplotype determination showed that all four haplotypes of Q. eucalypti determined in this study are found in Australia. Only two of the four haplotypes have been found in other countries including China, Portugal, South Africa and Uruguay. Portugal, South Africa and Uruguay share the same haplotype, the other haplotype apart from Australia was only found in China. Results in this study support the view that Q. eucalypti is native to Australia and that this is the source of introductions to new areas^[24^].

Quambalaria simpsonii was first reported from species of Eucalyptus in Australia and Thailand, but it is unknown whether this is a pathogen^[26^]. In the present study, Q. simpsonii was consistently isolated with T. zuluensis from cankered E. urophylla× E. grandis stems in four sites in Guangdong and Guangxi, China. Whether Q. simpsonii is pathogenic to Eucalyptus trees, and the ecological interaction between Q. simpsonii and T. zuluensis remains to be clarified.

5 Conclusions

The genus Quambalaria presently includes six species. Most of these are pathogens that cause leaf and shoot blight, and cankers on Eucalyptus and Corymbia. They are considered native to Australia but have been inadvertently introduced into countries of Africa, Asia, Europe and South America. This has most likely occurred via the trade in eucalypt germplasm^[39^]. In the present study, three Quambalaria spp. were identified in China; Q. pitereka on C. citriodora, Q. eucalypti on clones E. urophylla × E. grandis and Q. simpsonii isolated from stem cankers of E. urophylla× E. grandis caused by T. zuluensis. These are widespread in areas of China where eucalypts are grown and they are likely to become more important to commercial forestry in the future.

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Supplementary materials

The online version of this articale at https:// doi.org/10.15302/J-FASE-2017173 contains supplementary material (Table S1).

Acknowledgements

The research was financially supported by the Fundamental Research Funds for the Central Non-profit Research Institution of CAF (CAFYBB2014MA018), and the Overseas Outstanding Scholars Lecture Program, the Fundamental Research Funds for the Central Non-profit Research Institution of CAF (CAFYBB2017ZF005). The authors thank Ms. Jieqiong Li, Ms. Yan Lin and Prof. Jianzhong Luo for their assistance in collecting disease samples in the Guangdong and Hainan Provinces.

Compliance with ethics guidelines

Shuaifei Chen, Qianli Liu, Guoqing Li, and Michael J. Wingfield declare they have no conflicts of interest or financial conflicts to disclose.

This article does not contain any studies with human or animal subjects performed by any of the authors.

RIGHTS & PERMISSIONS

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)