EUCALYPT DISEASES IN SOUTHEAST ASIA, INDIA AND CHINA

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Chapter 3 Novel species of Celoporthe from Eucalyptus and Syzygium trees in China and Indonesia

ABSTRACT

Many species in the Cryphonectriaceae cause diseases of trees, including those in the genera Eucalyptus and Syzygium. During disease surveys on these trees in South China, fruiting structures typical of fungi in the Cryphonectriaceae and associated with dying branches and stems were observed. Morphological comparisons indicated that these fungi were distinct from the well-known Chrysoporthe deuterocubensis, also found on these trees in China. The aim of this study was to identify these fungi and evaluate their pathogenicity to Eucalyptus clones/species as well as Syzygium cumini. Three morphologicaly similar fungal isolates collected previously from Indonesia were also included in the study. Isolates were characterized based on comparisons of their morphology and DNA sequence data for the partial LSU and ITS nuclear ribosomal DNA, β-tubulin, and TEF-1α gene regions. Following glasshouse trials to select virulent isolates, field inoculations were undertaken to screen different commercial Eucalyptus clones/species and S. cumini trees for susceptibility to infection. Phylogenetic analyses showed that the Chinese isolates and those from Indonesia reside in a clade close to previously identified South African Celoporthe isolates. Based on morphology and DNA sequence comparisons, four new Celoporthe spp. were identified and they are described as C. syzygii, C. eucalypti, C. guangdongensis and C. indonesiensis. Field inoculations indicated that the three tested Chinese Celoporthe spp., namely C. syzygii, C. eucalypti and C. guangdongensis, are pathogenic to all tested Eucalyptus and S. cumini trees. Significant differences in the susceptibility of the inoculated Eucalyptus clones/species suggest that it will be possible to select disease tolerant planting stock for forestry operations in the future.
Keywords: Cryphonectriaceae, stem canker pathogens, Myrtales, plantation forestry, Southeast Asia

INTRODUCTION

The Cryphonectriaceae Gryzenh. & M.J. Wingf. (Diaporthales) represents a group of bark and/or wood-infecting fungi of trees and shrubs in various parts of the world (Gryzenhout et al. 2009). Species of Cryphonectriaceae exist naturally as virulent pathogens, facultative parasites or saprophytes on woody hosts. Some species have been introduced into new environments causing diseases on important trees such as those grown commercially in plantations or for their ornamental value, and include some of the most important tree pathogens in the world (Gryzenhout et al. 2009). Except for Cryphonectria (Sacc.) Sacc. & D. Sacc., which is the type genus, thirteen other genera have been described in this family (Nakabonge et al. 2006a; Cheewangkoon et al. 2009; Gryzenhout et al. 2009, 2010; Begoude et al. 2010; Vermeulen et al. 2010).
Several species of Cryphonectriaceae have been collected from trees in China in the past. Cryphonectria parasitica (Murrill) M.E. Barr, best known for causing a devastating canker disease of chestnuts (Castanea spp.) in the USA and Europe (Anagnostakis 1987, 1992) also causes canker and die-back on Chinese chestnut (Castanea mollissima Blume) trees in their native range (Fairchild 1913; Shear & Stevens 1913, 1916). Cryphonectria japonica (Tak. Kobay. & Kaz. Itô) Gryzenh. & M.J. Wingf. [ = Cryphonectria nitschkei (G.H. Otth) M.E. Barr], which was first collected in Japan, and Endothia gyrosa (Schwein.: Fr.) Fr., have been found on a Quercus sp. in China (Teng 1934; Kobayashi & Itô 1956; Myburg et al. 2004; Gryzenhout et al. 2009). Chrysoporthe deuterocubensis Gryzenh. & M.J. Wingf., previously treated as Chr. cubensis (Bruner) Gryzenh. & M.J. Wingf. (Van Der Merwe et al. 2010) has been reported from species of Eucalyptus and Syzygium in South China from a wide range of locations (Sharma et al. 1985; Hodges et al. 1986; Myburg et al. 2002; Zhou et al. 2008;Chen et al. 2010).
The genus Celoporthe Nakab., Gryzenh., Jol. Roux & M.J. Wingf., based on C. dispersa Nakab., Gryzenh., Jol. Roux & M.J. Wingf., is a recently described genus in the Cryphonectriaceae, described from both native and introduced Myrtales in South Africa (Nakabonge et al. 2006a). The fungus was associated with dying branches and stems on these trees. Currently, the genus is represented by a single species, despite DNA-based comparisons that showed the presence of three different but closely related phylogenetic sub-clades within the genus (Nakabonge et al. 2006a). C. dispersa is represented by isolates from native Syzygium cordatum Hochst.: C.Kraus, native Heteropyxis canescens Oliv. and non-native Tibouchina granulosa Cogn. (Nakabonge et al. 2006a) in South Africa. Based on DNA sequences comparisons, isolates previously collected from S. aromaticum (L.) Merr. & L.M.Perry in Indonesia (Myburg et al. 2003) also grouped closely with C. dispersa, but morphological evaluation was impossible due to an absence of specimens (Myburg et al. 2003; Nakabonge et al. 2006a). Inoculation trials showed that C. dispersa is more pathogenic on
Eucalyptus grandis W. Hill clone than T. granulosa (Nakabonge et al. 2006a). During surveys in South China for pathogens of trees in the Myrtaceae especially species of Eucalyptus and Syzygium, several pathogens affecting these trees were identified (Zhou et al. 2008). Besides Chr. deuterocubensis (Chen et al. 2010; Van Der Merwe et al. 2010), these surveys yielded a fungus on Eucalyptus and S. cumini trees resembling species of Celoporthe.
The aim of the present study was to characterize these isolates based on morphology and DNA sequence comparisons and to assess their pathogenicity to Eucalyptus and S. cumini using glasshouse and field inoculations.

MATERIALS AND METHODS

Sampling

Eucalyptus (Myrtales) plantations in the GuangDong Province of South China were investigated for the presence of fungal diseases during the periods November to December 2006, January 2007, and September to November 2008. Where present in these areas, S. cumini trees (Myrtales) were also examined for the presence of fungi in the Cryphonectriaceae, as these fungi are known to occur on Syzygium spp. (Heath et al. 2006; Gryzenhout et al. 2009). Sections of bark bearing fruiting structures (ascostromata and conidiomata) resembling the Cryphonectriaceae were collected from symptomatic trees and transported to the laboratory in order to make isolations.
Samples were incubated in moist chambers for 1–3 days to induce the production of spores from the fruiting bodies. Single tendrils of spores were transferred to 2% Malt Extract Agar (MEA) (Biolab, Merck, Midrand, South Africa) (20 g Biolab Malt Extract, 20 g Biolab Agar, 1 L water) and incubated at 25°C. From the resultant cultures, single hyphal tips were transferred to fresh 2% MEA to obtain pure cultures. Three unidentified isolates from Indonesia, originating from S. aromaticum and resembling fungi in the Cryphonectriaceae, obtained from the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa, were also inclued in this study (Table1). All cultures collected from China are maintained in the culture collection (CMW), and a duplicate set of isolates is maintained in a culture collection housed at the China Eucalypt Research Centre (CERC), Chinese Academy of Forestry (CAF), China. Representative isolates were also deposited with the Centraalbureau voor Schimmelcultures (CBS), Utrecht, the Netherlands (Table 1). The original bark material bearing fruiting structures was deposited in the National Collection of Fungi (PREM), Pretoria, South Africa.

DNA sequence comparisons

DNA extraction, PCR and sequencing reactions

Isolates originating from different Eucalyptus species/clones and S. cumini from the various areas sampled, as well as those showing differing culture and fruiting structure morphology (Table 1), were selected for DNA sequence comparisons. Prior to DNA extraction, isolates were grown in 2% MEA at 25°C for 5–7 days. For each isolate, actively growing mycelium from one MEA plate per isolate was scraped from the surface of the medium using a sterile scalpel and transferred to 1.5 mL Eppendorf tubes. DNA was extracted using the method described by Myburg et al. (1999). DNA was separated by electrophoresis on a 1% agarose gel, stained with ethidium bromide and visualised under ultraviolet (UV) light. Samples were treated with 3 mL RNase (1mg/mL) and left overnight at 37°C to degrade RNA. Gene regions amplified using the Polymerase Chain Reaction (PCR) included the conserved nuclear Large Subunit (LSU), the b-tubulin gene region 1 (BT1) and 2 (BT2), the Internal Transcribed Spacer (ITS) regions including the 5.8S gene of the ribosomal DNA operon (Gryzenhout et al. 2009) as well as the translation elongation factor 1-alpha (TEF-1α) gene region. Part of the LSU rDNA gene region was amplified using the primers LR0R and LR7 (Vilgalys & Hester 1990; Rehner & Samuels 1994), two regions within the BT gene were amplified using the primer pairs βt1a/βt1b and βt2a/βt2b, respectively (Glass & Donaldson 1995), the ITS regions including the 5.8S rDNA operon were amplified using the primer pairs ITS1 and ITS4 (White et al. 1990), and a fragment of the TEF-1α gene region was amplified using the primer pairs EF1-728F and EF1-986R (Carbone & Kohn 1999). PCR conditions for the LSU gene region were as outlined by Castlebury et al. (2002), those for the BT1/2 and ITS gene regions followed the protocols of Myburg et al. (2002), and the TEF-1α gene region was amplified using the method described by Slippers et al. (2004). PCR products were visualised with UV light on 1% agarose (ethidium bromide-stained) gels. Using 6% Sephadex G-50 columns (Steinheim, Germany), the amplified products were purified as suggested by the manufacturers.
Each PCR product was sequenced in both directions with the same primers used for PCR reactions. The ABI PRISMTM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Applied Biosystems, Foster City, California) was used to perform the sequencing reactions. Sephadex G-50 columns (6%) were used to purify the sequence products, wherafter electropherograms were generated on an ABI PRISM 3100 autosequencer (Perkin-Elmer Applied Biosystems, Foster City, California). Nucleotide sequences were edited using MEGA4 (Tamura et al. 2007). All sequences obtained in this study have been deposited in GenBank (Table 1).

Generic placement and species identification

To determine the generic placement of the isolates collected from Eucalyptus and S. cumini in South China, as well as those previously collected in Indonesia, sequences of the LSU gene region were analysed. These analyses were supplemented by analyses of the conserved 5.8S operon of the ITS region and the exon regions of the BT (including partial exon 4, exon 5, partial exon 6, and partial exon 7) gene regions for previously described species in the Cryphonectriaceae (Gryzenhout et al. 2009; Begoude et al. 2010; Gryzenhout et al. 2010; Vermeulen et al. 2010). For analyses of the LSU, the datasets of Gryzenhout et al. (2006a, 2009) were used as templates. Sequences were also supplemented with those of other recently published isolates of new genera in the Cryphonectriaceae, including Aurifilum Begoude, Gryzenh. & Jol. Roux, Cryptometrion Gryzenh. & M.J. Wingf., and Latruncella M. Verm., Gryzenh. & Jol. Roux (Begoude et al. 2010; Gryzenhout et al. 2010; Vermeulen et al. 2010). Togninia minima (Tul. & C. Tul.) Berl., T. fraxinopennsylvanica (T.E. Hinds) Georg Hausner, Eyjólfsd. & J. Reid, and Phaeoacremonium aleophilum W. Gams, Crous, M.J. Wingf. & Mugnai were used as outgroups (Gryzenhout et al. 2009). Two isolates of Diaporthe ambigua Nitschke were used as outgroups for analyses of the 5.8S gene and BT exon regions (Gryzenhout et al. 2009). A partition homogeneity test (PHT) was used to determine the congruence of the datasets for these two gene regions (Farris et al. 1995; Huelsenbeck et al.1996). After an outcome indicating congruence between the datasets, phylogenetic analyses were done in PAUP (Phylogenetic Analysis Using Parsimony) version 4.0b10 (Swofford 2002) of the individual and combined datasets.
To determine the species identities and phylogenetic relationships between the isolates from China and previously described species of Cryphonectriaceae, sequences of the BT1, BT2, ITS and TEF-1α gene regions were analysed separately and in combination. A partition homogeneity test (PHT) was used to determine the congruence of the datasets (Farris et al. 1995; Huelsenbeck et al. 1996). After an outcome indicating congruence between the datasets, phylogenetic analyses were done in PAUP (Phylogenetic Analysis Using Parsimony) version 4.0b10 (Swofford 2002). All sequences were aligned using the iterative refinement method (FFT-NS-i settings) of the online version of MAFFT v. 5.667 (Katoh et al. 2002), and adjusted and edited manually where necessary in MEGA 4 (Tamura et al. 2007). The sequence alignments for each of the datasets were deposited in TreeBASE (http://www.treebase.org). Three different phylogenetic analyses were conduced for each of the datasets. Maximum Parsimony (MP) analyses were done in PAUP 4.0b10 (Swofford 2002), Maximum Likelihood (ML) tests were conducted using PhyML 3.0 (Guindon & Gascuel 2003), and Bayesian inference was determined using the Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.1.2 (Ronquist & Huelsenbeck 2003).
For MP analyses, gaps were treated as a fifth character and the characters were all unordered and of equal weight with 1 000 random addition replicates. By using the heuristic search function and tree bisection and reconstruction (TBR) as branch swapping algorithms, the most parsimonious trees were obtained. Maxtrees were unlimited and branch lengths of zero were collapsed. A bootstrap analysis (50% majority rule, 1 000 replicates) was done to determine the confidence levels of the tree-branching points (Felsenstein 1985). Tree length (TL), consistency index (CI), retention index (RI) and the homoplasy index (HI) were used to assess the trees (Hillis & Huelsenbeck 1992). For ML and Bayesian analyses of each dataset, the best models of nucleotide substitution were established using Modeltest 3.7 (Posada & Crandall 1998) and MrModelTest version 2.3 (Nylander 2004), respectively.

Acknowledgements 
Preface 
Chapter 1 Literature Review: Diseases and their importance to eucalypt plantation forestry in China 
ABSTRACT 
1. INTRODUCTION
2. EUCALYPTS IN ASIA AND CHINA
2.1. Eucalypts in Asia
2.2. Eucalypts in China
2.2.1. History and current status
2.2.2. Distribution
2.2.3. Future development
3. EUCALYPT DISEASES IN SOUTHEAST ASIA, INDIA AND CHINA
3.1. Leaf and shoot diseases
3.1.1. Leaf and shoot diseases caused by fungi in the Mycosphaerellaceae and Teratosphaeriaceae
3.1.2. Cylindrocladium leaf blight
3.1.3. Quambalaria leaf and shoot blight
3.1.4. Other leaf diseases
3.2. Wilt diseases
3.2.1. Bacterial wilt
3.2.2. Ceratocystis wilt
3.3. Stem/branch cankers
3.3.1. Botryosphaeriaceae canker
3.3.2. Coniothyrium canker
3.3.3. Chrysoporthe canker
3.3.4. Cytospora canker
3.3.5. Pink disease
3.4. Root diseases
4. STATUS OF EUCALYPT DISEASES IN CHINA
5. PATHOGEN PATHWAYS
5.1. Air
5.2. Soil and media
5.3. Germplasm
5.4. Wood and wood products
5.5. Insects
5.6. Humans
6. EXAMPLES ILLUSTRATING THE ORIGINS AND GLOBAL MOVEMENT OF EUCALYPT PATHOGENS
6.1. The Chrysoporthe canker pathogens Chrysoporthe cubensis and Chr. austroafricana
6.2. The eucalypt leaf blight pathogen Teratosphaeria nubilosa
6.3. The eucalypt canker pathogen Teratosphaeria zuluensis
7. CONCLUSIONS
8. OBJECTIVES OF THIS DISSERTATION
9. REFERENCES
Chapter 2 Identification and pathogenicity of Chrysoporthe cubensis on Eucalyptus and Syzygium spp. in South China 
ABSTRACT
1. INTRODUCTION
2. MATERIALS AND METHODS
2.1. Sampling
2.2. Morphology
2.3. DNA sequence comparisons
2.4. Pathogenicity tests
2.4.1. Glasshouse trials
2.4.2. Field trials
3. RESULTS
3.1. Sampling
3.2 Morphology
3.3. DNA sequence comparisons
3.4. Pathogenicity tests
3.4.1 Glasshouse trials
3.4.2. Field trials in China
4. DISCUSSION
5. REFERENCES
Chapter 3 Novel species of Celoporthe from Eucalyptus and Syzygium trees in China and Indonesia 
ABSTRACT
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS
4. DISCUSSION
5. REFERENCES
Chapter 4 Characterization of Botryosphaeriaceae from plantation-grown Eucalyptus species in South China 
ABSTRACT
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS
4. DISCUSSION
5. REFERENCES
Chapter 5 Novel species of Calonectria associated with Eucalyptus leaf blight in Southeast China 
ABSTRACT
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS
4. DISCUSSION
5. REFERENCES
Chapter 6 High population diversity and increasing importance of the Eucalyptus stem canker pathogen, Teratosphaeria zuluensis, in South China 
ABSTRACT
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS
4. DISCUSSION
5. REFERENCES
Summary
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