Botryosphaeriaceae from tuart (Eucalyptus gomphocephala) woodland, including descriptions of four new species

mycological research 113 (2009) 337–353

journal homepage: www.elsevier.com/locate/mycres

Botryosphaeriaceae from tuart (Eucalyptus gomphocephala) woodland, including descriptions of four new species Katherine TAYLOR, Paul A. BARBER, Giles E. ST J. HARDY, Treena I. BURGESS* Faculty of Sustainability, Environmental and Life Sciences, Murdoch University, Murdoch 6150, WA, Australia

article info

abstract

Article history:

Eucalyptus gomphocephala (tuart) is a tree native to the southwest coast of Western Aus-

Received 7 May 2008

tralia, where, in some areas, there is a significant decline in the health of tuart. Botryos-

Received in revised form

phaeriaceous taxa have been isolated as endophytes and canker pathogens from

30 October 2008

numerous hosts in many parts of the world and have been implicated in the decline of

Accepted 13 November 2008

E. gomphocephala. In the present study, endophytic fungi were isolated from a wide variety

Published online 6 December 2008

of native woody plant species (Acacia cochlearis, A. rostellifera, Allocasuarina fraseriana, Agonis

Corresponding Editor: Kevin D. Hyde

flexuosa, Banksia grandis, E. gomphocephala, E. marginata and Santalum acuminatum), at two locations in native E. gomphocephala woodland; a site in decline at Yalgorup National Park and

Keywords:

a healthy site at Woodman Point Regional Park. Of the 226 isolates obtained, 154 were bo-

Endophytes

tryosphaeriaceous taxa, 80 % of which were found to be Neofusicoccum australe, isolated

Molecular taxonomy

from all hosts at both collection sites. Four new species are described, Dothiorella moneti,

Natural ecosystem

Dothiorella santali, Neofusicoccum pennatisporum, and a species belonging to a genus only

Neofusicoccum australe

recently included in the Botryosphaeriaceae, Aplosporella yalgorensis. The other species

Phylogenetics

isolated were Botryosphaeria dothidea on the new hosts A. rostellifera, A. cochlearis and E. marginata and Dichomera eucalypti, on the new host E. marginata. None of the new species formed lesions on excised stems of their host species, E. gomphocephala, or a common plantation species, E. globulus. However, Neofusicoccum australe formed lesions on excised stems of E. globulus and E. gomphocephala. ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction Eucalyptus gomphocephala (tuart) is a magnificent woodland tree endemic to the Swan Coastal Plain, with a natural distribution spanning a 400-km strip of land along the southwest coast of Western Australia from Jurien Bay in the north to the Sabina River in the south (Keighery et al. 2002). E. gomphocephala woodland covered approximately 111 600 hectares in area before the arrival of Europeans; however, less than 30 % of this original area remains largely due to agricultural and urban development (Government of Western Australia 2003) Since the mid-1990s there has also been a noticeable decline

in the health and vitality of E. gomphocephala in the Yalgorup region between Mandurah and Preston Beach (Government of Western Australia 2003) and to a lesser extent in other areas. The reasons for this decline are unknown, although many hypotheses have been proposed. An initial reason cited for the decline was that it was part of the natural disease cycle of Botryosphaeria, a fungal genus known to form cankers in woody hosts (Alves et al. 2006; Davison & Tay 1983; Farr et al. 2005; Phillips 1998; Phillips et al. 2006; Shearer et al. 1987; von Arx 1987). Chillcot (1992) hypothesised that, in conjunction with the native canker pathogens Endothia havensis (syn. Holocryphia eucalypti) and Cytospora eucalypticola, the introduced

* Corresponding author. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2008.11.010

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pathogen B. ribis might possibly play a role in the decline of E. gomphocephala in the Kwinana area. There have been numerous anamorph genera attached to Botryosphaeria. However, reviews of the genus combining morphological characteristics with molecular phylogenetics recognized only Fusicoccum and Diplodia, all others genera were viewed as synonyms of these (Denman et al. 2000; Smith & Stanosz 2001). Phillips et al. (2005) resurrected a further anamorph genus, Dothiorella, to accommodate two new species and the genus Lasiodiplodia was reinstated (Burgess et al. 2006a; Pavlic et al. 2004). Despite extensive revision of Botryosphaeria, its position at higher levels of classification was still confusing (Denman et al. 2000; Silvanesan 1984). Species from genera, such as Dichomera (Barber et al. 2005) and Scytalidium (Farr et al. 2005), not previously thought to be related to Botryosphaeria, were found to fall within this genus according to DNA-based phylogenetic analysis. This led Crous et al. (2006) to revise Botryosphaeria resulting in its separation into several new genera, in which the morphological characteristics of the anamorphic genera and their genetic relatedness played a strong role in the placement of species into a genus. The new genera are Neofusicoccum, Pseudofusicoccum, Neoscytalidium, Macrophomina, Dothiorella, and Saccharata. Botryosphaeria was only retained for B. dothidea and B. corticis. However, the phylogenetic placement of many dark-spored species could not be resolved and appropriate names were available for all former Botryosphaeria spp. For example, ‘Botrysophaeria’ obtusa was without an appropriate name until its description as Diplodia seriata (Phillips et al. 2007). Another genus, Aplosporella, has also recently been included in the Botryosphaeriaceae (Damm et al. 2007). The clade containing the dark-spored species with Diplodia or Lasiodiplodia anamorphs remained unresolved until the recent study of Phillips et al. (2008), where sequence data from SSU, LSU, ITS, b-tubulin, and EF1-a were combined. In addition to Lasiodiplodia and Diplodia, several new dark-spored genera have been described: Neodeightonia, Phaeobotryon, Barriopsis, Phaeobotryosphaeria, and Spensermartinsia. Species belonging to the Botryosphaeriaceae can also be isolated from dead tissue surviving as saprobes and from healthy tissue, surviving as endophytes (Burgess et al. 2005; Danti et al. 2002; Pavlic et al. 2004; Swart et al. 2000). They have previously been found on some of the most common Australian plant families including Proteaceae, Myrtaceae, and Mimosaceae, although most studies have been from commercial production systems; plantations, orchards, and vineyards. The causal agent of cankers in native forests in both eastern and Western Australia (Davison & Tay 1983; Old & Davison 2000; Old et al. 1990; Shearer et al. 1987) was reported to be B. ribis (Neofusicoccum ribis). However, these studies all predate significant revisions of the Botryosphaeriaceae, which resulted in some species being reduced to synonymy and new species being described (Slippers et al. 2004b, 2004c, 2004d). For this reason, reports of N. ribis pre-dating the year 2000 should be treated circumspectly and not regarded as confirmed reports of N. ribis. In Australia since 2000, N. parvum (Slippers et al. 2004b) and N. eucalypticola (Slippers et al. 2004c) have been reported from Eucalyptus, and N. australe was described and reported from Acacia, Eucalyptus, and Banksia spp. (Burgess et al. 2005; Slippers et al. 2004d). Burgess et al. (2005), in a study of plantations

K. Taylor et al.

and native forests in Western Australia, found four species. N. australe accounted for 94 % of the isolates, with Dichomera eucalypti, N. parvum, and N. macroclavatum being isolated infrequently. The predominance of N. australe led Burgess et al. (2005) to suggest that this species was endemic to Western Australia. Burgess et al. (2006b) observed gene flow of N. australe between plantation and native forests, and demonstrated the potential for the exchange of this pathogen between plantations and native forest. Species belonging to the Botryosphaeriaceae have also been identified in studies occurring in Australia on horticultural crops. Taylor et al. (2005) found N. australe and Diplodia seriata commonly on grapevines in Western Australia. N. parvum, N. mangiferae, Fusicoccum spp., and B. dothidea have also been isolated in Australia from Mangifera indica (Slippers et al. 2005a). Cunnington et al. (2007) sequenced the ITS region for 30 cultures isolated over the last 25 y from a number of horticultural crops and identified B. dothidea, D. seriata, N. australe, N. lutea, N. parvum, and. N. ribis Most Botryosphaeriaceae have been described from tree crops in the northern hemisphere and those studies concentrating on the southern hemisphere have generally been described from exotic tree species in plantations. This project aimed to identify endophytic fungi belonging to the Botryosphaeriaceae occurring on plant species within the E. gomphocephala woodland, and to determine their pathogenicity toward endemic E. gomphocephala, the introduced plantation species, E. globulus, and the host species they were isolated from. Based on their unique combination of cultural and morphological characters and sequence data, we formally describe four new species in the Botryosphaeriaceae, Dothiorella moneti sp. nov., D. santali sp. nov., N. pennatisporum sp. nov., and Aplosporella yalgorensis sp. nov.

Materials and methods Collection of host material Healthy tissue was collected from two sites, Yalgorup National Park (32 070 5800 S, 115 45 4400 E) and Woodman Point Regional Park (32 410 0700 S, 115 380 2400 E). All of the species chosen for the collection of material were common woody plants on the Swan Coastal Plain. The tuart woodland at the two sites had very different understorey complexes. There were three host species common to both localities. These were Eucalyptus gomphocephala, Santalum acuminatum, and Acacia rostellifera. The species specific to Yalgorup National Park were Banksia grandis, Allocasuarina fraseriana, Agonis flexuosa, and E. marginata. The species specific to Woodman Point Regional Park were Acacia cochlearis and Callitris preissii. One of the most significant differences in the vegetation between sites was the absence of Agonis flexuosa in Woodman Point Regional Park. This is important, as A. flexuosa has been implicated in the decline in the Yalgorup region (Bradshaw 2000). In contrast, C. preissii was present in Woodman Point Regional Park, but not Yalgorup National Park. This species is rare in other coastal areas around Perth and usually forms dense stands to the exclusion of other species in the area.

Botryosphaeriaceae from tuart (Eucalyptus gomphocephala) woodland

A single branch, 1–1.5 cm diam was collected from ten individual trees of each species. Individual trees and shrubs were chosen according to the accessibility of their branches and their GPS co-ordinates recorded. Material was collected and placed into plastic bags and transported back to the laboratory where it was stored at 4  C overnight.

Isolation of endophytes The stems were placed in jars containing full-strength bleach (37 % sodium hypochlorite) for 1 min, followed by 70 % ethanol for 1 min, and then washed in six changes of sterile water each for 1 min. The stems were then cut into small pieces using sterile (by wiping with 70 % ethanol) secateurs and plated onto half-strength potato–dextrose agar (1⁄2 PDA; Becton, Dickinson, Sparks, MD; 19.5 g PDA, 7.5 g agar, 1 l distilled water) with streptomycin (0.02 %) added. Cultures derived from the treated plates were sub-cultured onto fresh PDA plates. Isolates with culture morphology typical of botryosphaeriaceous taxa (fluffy, fast-growing, white turning olive green–grey within a few days) were retained for identification and incubated at 20  C in light. In total, 226 botryosphaeriaceous isolates were retained and stored for future analysis. A plug of mycelium and agar was removed using a cork borer (5 mm diam) from each of the putative botryosphaeriaceous isolates and plated onto the 1⁄2 PDA plates. These were maintained at 20  C for three weeks. Characteristics of the isolates recorded on days 4, 8, and 24 included colony diameter, colour of the culture at the centre and the edge according to the Munsell soil colour chart (Anon 2000), the absence or presence of staining in the agar, the mycelium type at the centre and edge (submerged, fluffy, dense, or appressed), and the margin shape (irregular or regular) and form (diffuse or not). Using these data, and visual assessments of the plates the isolates were sorted into groups. Representative isolates of all of the groups were then chosen for molecular identification and investigation. Cultures were maintained on 1⁄2 PDA at 20  C and stored in sterile water at 20  C. Voucher material is available from Perth Herbarium, and type cultures from the Western Australian Culture Collection.

Molecular characterisation For each isolate, approximately 50 mg fungal mycelium was scraped from the surface of 7-d-old cultures, frozen using liquid nitrogen, ground using a glass rod, suspended in 200 ml DNA extraction buffer [200 mM Tris–HCL (pH 8), 150 mM NaCl, 25 mM EDTA, 0.5 % sodium dodecyl sulphate (SDS)] and incubated for 1 h at 60  C. DNA was purified using the Ultrabind DNA purification kit following the manufacturer’s instructions (MO BIO Laboratories, Solana Beach, CA). First, a part of the ITS region of the rDNA operon was amplified for all isolates using the primers ITS-1F (50 CTT GGT CAT TTA GAG GAA GTA A) (Gardes & Bruns 1993) and ITS4 (50 TCC TCC GCT TAT TGA TAT GC) (White et al. 1990). The region was amplified using the following thermal cycling conditions, and initial denaturisation of 2 min at 94  C followed by ten cycles of 45 s at 94  C, 1 min at 50 s, 1 min at 72  C and then another 25 cycles of 30 s at 94  C, 1 min at 52  C, 1 min at 72  C, and a final extension of 5 min at 72  C.

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Three further gene regions were amplified for selected isolates including part of the LSU using primers LROR (50 ACC CGC TGA ACT TAA GC) and LR5 (50 TCC TGA GGG AAA CTT CG) and part of the elongation factor 1-a was amplified using primers EF1-728F (50 CAT CGA GAA GTT CGA GAA GG) and EF1-986R (50 TAC TTG AAG GAA CCC TTA CC) (Carbone & Kohn 1999). Isolates that did not amplify using these primers were amplified using EF-AF (50 - CAT CGA GAA GTT CGA GAA GG) and a degenerate primer EF-BR (50 CRA TGG TGA TAC CRC GCT C) or combinations of these and the aforementioned EF-1a primers (Sakaledis 2004). The ß-tubulin region was amplified using the primers Bt2a (50 GGT AAC CAA ATC GGT GCT GCT TTC) and Bt2b (50 ACC CTC AGT AGT GTA GTG ACC CTT GGC) (Glass & Donaldson 1995). The thermal cycling conditions were the same as those used in the ITS region. The clean up of products and sequencing were as described previously (Burgess et al. 2005).

Phylogenetic analysis In order to compare the botryosphaeriaceous isolates from this study with related species, sequences were obtained from GenBank (isolate code, identity and accession numbers for sequence data are given in TreeBASE SN3695). DNA sequences were automatically aligned using ClustalX (Jeanmougin et al. 1998) and the alignments adjusted manually in BioEdit 5.0.6 (Hall, 2001. Ibis Biosciences; http://www.mbio.ncsu.edu/ Bioedit/bioedit.html). Parsimony analysis was performed on individual datasets in PAUP (Phylogenetic Analysis Using Parsimony) v4.0b10 (Swofford 2003). Non-informative characters were removed prior to analysis and characters were unweighted and unordered, gaps were treated as a fifth character (newstate). The most parsimonious trees were obtained by using heuristic searches with random stepwise addition in 100 replicates, with the tree bisection–reconnection branch-swapping option on and the steepest-descent option off. Maxtrees were unlimited, branches of zero length were collapsed, and all multiple equally parsimonious trees were saved. Estimated levels of homoplasy and phylogenetic signal (retention and consistency indices) were determined (Hillis & Huelsenbeck 1992). Branch and branch node supports were determined using 1 K BS replicates (Felsenstein 1985). Two Diaporthe spp. were used as outgroup taxa for the LSU analysis and Saccharata proteae was used as the outgroup taxon for the combined ITSEF1-a analysis. Bayesian analysis was conducted on the same aligned dataset. First, MrModeltest v2.2 (Nylander 2004) was used to determine the best nucleotide substitution model. Phylogenetic analyses were performed with MrBayes v3.1 (Ronquist & Heuelsenbeck 2003) applying the best model. The MCMC analysis of four chains started from random tree topology and lasted 10 M generations. Trees were saved each 10 K generations, resulting in 10 K saved trees. Burn-in was set at 500 K generations after which the likelihood values were stationary leaving 9950 trees from which the consensus trees and PPs were calculated. PAUP 4.0b10 was used to reconstruct the consensus tree and maximum PP assigned to branches after a 50 % majority rule consensus tree was constructed from the 9950 sampled trees.

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K. Taylor et al.

Table 1 – Isolates used in this study Isolate

Identity

Host

Site

Genbank accession numbers ITS

MUCC476 MUCC477a MUCC478 MUCC479a MUCC480a MUCC481a MUCC482 MUCC483a MUCC484 MUCC485a MUCC486 MUCC487a MUCC488a MUCC489 MUCC490a MUCC491 MUCC492 MUCC493 MUCC494 MUCC495 MUCC496a MUCC497 MUCC498 MUCC499 MUCC500 MUCC501 MUCC502 MUCC503 MUCC504 WAC15154 MUCC506 MUCC507 MUCC508 WAC15155 WAC15156 WAC15157 MUCC512

Neofusicoccum australe N. australe N. australe N. australe N. australe N. australe N. australe N. australe N. australe N. australe N. australe N. australe N. australe N. australe N. australe N. australe N. australe N. australe N. australe N. australe N. australe N. protearum Dichomera eucalypti D. eucalypti Botryosphaeria dothidea B. dothidea B. dothidea B. dothidea B. dothidea Dothiorella moneti sp. nov. D. moneti sp. nov. D. moneti sp. nov. D. santali sp. nov. D. santali sp. nov. Neofusicoccum pennatisporum sp. nov. Aplosporella yalgorensis sp. nov. A. yalgorensis sp. nov.

Eucalyptus marginata E. marginata E. marginata Agonis flexuosa Allocasuarina fraseriana E. gomphocephala E. gomphocephala Santalum acuminatum S. acuminatum Acacia rostellifera A. rostellifera Banksia grandis B. grandis E. gomphocephala A. cochlearis E. gomphocephala E. gomphocephala E. gomphocephala A. rostellifera S. acuminatum Callitris preissii S. acuminatum E. marginata E. marginata E. marginata E. marginata A. rostellifera A. cochlearis A. rostellifera A. rostellifera A. rostellifera A. rostellifera S. acuminatum S. acuminatum Allocasuarina fraseriana Acacia cochlearis E. gomphocephala

Yalgorup Yalgorup Yalgorup Yalgorup Yalgorup Yalgorup Yalgorup Yalgorup Yalgorup Yalgorup Yalgorup Yalgorup Yalgorup Woodman Point Woodman Point Woodman Point Woodman Point Woodman Point Woodman Point Woodman Point Woodman Point Yalgorup Yalgorup Yalgorup Yalgorup Yalgorup Yalgorup Woodman Point Woodman Point Yalgorup Yalgorup Yalgorup Yalgorup Yalgorup Yalgorup Woodman Point Woodman Point

EF591891 EF591892 EF591893 EF591894 EF591895 EF591896 EF591897 EF591898 EF591899 EF591900 EF591901 EF591902 EF591903 EF591904 EF591905 EF591906 EF591907 EF591908 EF591909 EF591910 EF591911 EF591912 EF591913 EF591914 EF591915 EF591916 EF591917 EF591918 EF591919 EF591920 EF591921 EF591922 EF591923 EF591924 EF591925 EF591926 EF591927

LSU

b-tubulin

EF 1-a

EF591928

EF591945

EF591962

EF591929

EF591946

EF591963

EF591930 EF591931 EF591932 EF591933 EF591934 EF591935

EF591947 EF591948 EF591949 EF591950 EF591951 EF591952

EF591964 EF591965 EF591966 EF591967 EF591968 EF591969

EF591936

EF591953

EF591970

EF591937 EF591938 EF591939 EF591940 EF591941 EF591942 EF591943 EF591944

EF591954 EF591955 EF591956 EF591957 EF591958 EF591959 EF591960 EF591961

EF591971 EF591972 EF591973 EF591974 EF591975 EF591976 EF591977 EF591978

a Isolates used in the inoculation study.

Inoculation trial The pathogenicity of all selected isolates (Table 1) was tested on Eucalyptus globulus and E. gomphocephala, a common plantation species in Western Australia. The purported new species were also inoculated into excised stems from the host species from which they had been isolated. Isolates were grown on 1⁄2 PDA plates for 6 d at 20  C. The E. globulus stems (4 cm diam) were collected from one-year-old coppice at Channybearup plantation, Manjimup and were very straight, young, soft, and green. Young stems of E. gomphocephala (ca 4 cm diam) were collected from ash-bed regeneration in the native forest Ludlow, WA. These were more knarled, tougher, and older than the E. globulus stems. The excised stems of the other host species were collected from the original collection sites (Yalgorup National Park National Park and Woodman Point Regional Park). For Dothiorella moneti sp. nov., the host species were Acacia cochlearis and A. rostelifera, for Aplosporella yalgorensis sp. nov. the hosts were A. cochlearis and E. gomphocephala.

The host species for D. santali was Santalum acuminatum, and for Neofussicoccum pennatisporum sp. nov., the host species was Allocasuarina fraseriana. The stems of all these hosts were tougher and older compared with the stems of E. globulus as they had been collected from native stands. They were also smaller in diameter, mainly due to the natural growth habit of those species. The stems were cut into approximately 1 m sections and all of the leaves and small side stems removed and the sections stored in a cold room. The next day the 1 m lengths were cut into sections approximately 30 cm long. Each end was then dipped into melted candle wax in order to prevent the stem from drying and returned to the cold room. The next day the processed stems were inoculated. A scalpel (sterilized using 70 % ethanol) was used to cut a small section of the bark back, through the phloem leaving the central xylem untouched. A plug of agar covered with mycelium (approximately 1 cm2) was then placed mycelium down on the xylem, covered back again with the bark and wrapped tightly with

Botryosphaeriaceae from tuart (Eucalyptus gomphocephala) woodland

341

Table 2 – Number of isolates of each species collected from each host Yalgorup

Woodman point

E.g

S.a

E.m

Ag.f

Al.f

B.g

A.r

E.g

S.a

C.p

A.c

A.r

total

Neofusicoccum australe N. protearum Botryosphaeria dothidea Dichomera eucalypti Dothiorella moneti D. santali Aplosporella yalgorensis Neofusicoccum pennatisporum

20   1    

16 1    2  

10  2 3    

9       

2  1     1

9       

12  1 1 12   

30      1 

5       

1     

2  2  1



1 

7  1     

123 1 7 5 13 2 2 1

Total

21

19

15

9

4

9

26

5

31

1

6

8

154

E.m, Eucalyptus marginata; Ag.f, Agonis flexuosa; Al.f, Allocasuarina fraseriana; B.g, Banksia grandis; A.r, Acacia rostellifera; S.a, Santalum acuminatum; E.g, E. gomphocephala; C.p, Callitris preissii; A.c, Acacia cochlearis.

parafilm. Ten replicates were made for each of the isolates for each host species and ten controls (for both E. gomphocephala and E. globulus) or three controls (for other hosts) were made by inserting a plug of sterile agar. One replicate of each of the isolates was randomly placed in transparent ziplock bags (the E. globlulus stems were placed in different ziplock bags to that of the E. gomphocephala stems) and the stems were incubated in the dark at 24  C. The lesions on E. globulus inoculated with N. australe were measured on day 6 and those on E. gomphocephala on day 13. The bark was removed in order to accurately determine lesion length. Lesions caused by the new species on their hosts were measured on day 13. A oneway analysis of variance (ANOVA) was performed in the program STATISTICA (Statsoft, Tulsa, OK) to determine the variability in lesion length exhibited between the isolates.

Morphological characteristics Colony morphology, colour (Anon 2000) and growth rates between 5 and 35  C of representative isolates were determined on 1⁄2 PDA. To induce sporulation, plugs of mycelium were placed onto water agar (WA; Becton, Dickinson) between a pine needle and a eucalypt twig (3–7 mm diam and cut in half lengthways), which were previously autoclaved once a day on three consecutive days. The cultures were then placed under near UV-light to induce the formation of sporocarps. Sporocarps were mounted and squashed in 1 % analine blue and lactoglycerol beneath a coverslip and observed for shape, colour, size, and other distinguishing features using an Olympus RX51 Microscope with MicroPublisher 3.3 RTV Q imaging camera. The conidia were measured using the image analysis software Olysia BioReport 3.2 soft imaging system. The sporocarp measurements were taken separately from the eucalypt twig, and pine needle. Sporocarps and conidiogenous cells were measured manually on an Olympus BH-2 microscope. The 95 % confidence intervals of dimensions for sporocarps, conidia and conidiogenous cells were derived based on 50 observations where possible at x1000 magnification. Asci, ascospores, conidia, and conidiogenous cells were drawn using a drawing tube attached to an Olympus BH-2 microscope and following the method described by (Barber & Keane 2007).

Results Identification of cultures In total there were 42 groups of isolates based on culture morphology. Twelve groups had more than ten isolates, whereas the other groups contained few representative isolates, and several contained only one isolate. The ITS region was sequenced for 68 representative isolates (at least one isolate from each group). The resultant sequences were compared to sequences in GenBank using a BLAST search and isolates belonging to the Botryosphaeriaceae according to their ITS data were retained. Thirty-eight of the sequenced isolates from 22 groups belonged to the Botryosphaeriaceae (Table 2). Isolates were placed into eight species; four known species, and four apparently new species. Other fungi identified according to a BLAST search on GenBank were Alternaria spp., Paraconiothyrium sp., Colletotrichium sp., Embellisia sp., Bipolaris sp., Phoma sp., Paraphaeosphaeria sp., Preussia sp., Stemphylium sp., Pestalotiopsis sp., and Pleospora sp. The groups based on culture characteristics agreed well with the molecular data. Thus, of the 226 isolates originally kept for further study, 154 of the isolates were identified as botryosphaeriaceous taxa. The majority of isolates (123, 80 %) were identified as Neofusicoccum australe. These isolates had been placed into 15 groups during the sorting of cultures, including all isolates that stained the agar yellow and all those with a rosette growth pattern. Several culture groups that did not stain, or have rosette formation grouped with N. australe based on molecular analysis. N. australe was isolated from all the tree and shrub hosts studied. Some of the other known species were harder to distinguish based on culture morphology. Several isolates of Botryosphaeria dothidea, which were mostly found in one group based on culture characteristics, were found in another culture type group. Based on culture morphology, N. protearum was grouped with B. dothidea. Also isolates of Dichomera eucalypti were found in several groups based on cultural morphology. These species were infrequently isolated and a larger number of isolates may have resulted in more accurate classification. The high level of variation in these smaller groups was recognised and thus most of these isolates were

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sequenced to mitigate the known problems in categorising botryosphaeriaceous taxa based on culture morphology.

DNA sequence comparisons Four gene regions were sequenced for the selected isolates. The LSU sequence data were used to confirm the placement of isolates of the four purported new species in the correct genera as proposed by Crous et al. (2006). The aligned dataset consisted of 590 characters, of which 150 parsimony-informative characters were used in the analysis. These data contained significant phylogenetic signal (P < 0.01; gl ¼ 1.64). Heuristic searches of unweighted characters in PAUP resulted in 17 most parsimonious trees of 303 steps (CI ¼ 0.66, RI ¼ 0.87). Bayesian analysis resulted in a tree with similar topology and clades as those revealed in the parsimony tree (Fig 1, TreeBASE SN3695). This analysis resolved 12 clades corresponding to 11 genera. The clade containing Diplodia, Lasiodiplodia, and Phaeobotryosphaeria could not be resolved in this analysis. Two of the new species were resolved in clade 7 (Dothiorella). One of the new species was placed within the Neofusicoccum clade (clade 8) most closely aligned to N. protearum. The final species (clade 10) consisted of two isolates that formed a strongly supported clade within the Botryosphaeriaceae along with a new species Aplosporella prunicola recently described by (Damm et al. 2007). Individual gene trees were produced for aligned datasets for ITS, EF 1-a, and b-tubulin (TreeBASE SN3695). For each gene tree the new species resolved in the same clades with the same known species. Resolution (number of informative characters) varied between the different datasets and the number of species for which a background dataset existed on GenBank also varied. However, although the same species always resolved together in the same clade, the relationship of the genera in respect to each other differed. For example, Dothiorella was closest to the Diplodia clade in ITS and EF 1a analysis, but was closest to the Neofusicoccum clade in the b-tubulin analysis. As observed for many studies on the Botryosphaeriaceae, the combined ITS-EF 1-a dataset gave the best resolution of isolates and is presented here (Fig 2). The combined ITS-EF 1-a dataset consisted of 987 characters, resulting in 551 parsimony informative characters that were used in the analysis. These data contained significant phylogenetic signals (P < 0.01; gl ¼ 0.55). Heuristic searches of unweighted characters in PAUP resulted in 24 parsimonious trees of 1992 steps (CI ¼ 54, RI ¼ 0.84). Bayesian analysis resulted in a tree with the similar topology and clades as the parsimony tree (Fig 2, TreeBASE ¼ SN3695). This analysis resolved 12 clades corresponding to 12 genera in the Botryosphaeriaceae. Dothiorella santali sp. nov. and D. moneti sp. nov. resided in strongly supported terminal clades within the Dothiorella clades (clade 6). As observed with the LSU dataset, N. pennatisporum sp. nov., isolated from Allocasuarina remained phylogenetically closest to N. protearum. In clade 12, the two isolates representing Aplosporella yalgorensis, were distinct from A. prunicola. Phylogenetic analysis supports the description of four new species.

Innoculation trial All lesions caused by Neofussicoccum australe isolates were more visible on the Eucalyptus globulus stems than E.

K. Taylor et al.

gomphocephala stems. All N. australe isolates formed dark brown lesions on the surface of the E. globulus stems. When the bark was removed there was also brown discolouration of the cambium, appearing lighter at the edges. Lesions could not be observed on the surface of the inoculated E. gomphocephala stems, but could be observed and measured when the bark was removed. The lesion length formed in response to inoculation of N. australe isolates on E. globulus stems were longer and developed faster than those formed on stems of E. gomphocephala (Fig 3). All isolates of N. australe formed lesions significantly (P < 0.05) larger than those formed by the control. There was no significant difference between lesion length caused by the N. australe isolates collected from different hosts, indicating there was no significant relationship between pathogenicity and host. One isolate of N. australe, MUCC480 from Allocasuarina fraseriana, formed the longest lesions on both E. globulus and E. gomphocephala. Dothiorella moneti sp. nov., D. santali sp. nov., N. pennatisporum sp. nov. and Aplosporella yalgorensis sp. nov. did not form lesions on the stems of E. gomphocephala, E. globulus or the hosts from which they had been isolated.

Taxonomy Dothiorella moneti K.Taylor, Barber & T.I. Burgess, sp. nov. Figs 4, 9B MycoBank no.: MB511825 Etym.: named after the aboriginal word for host genus Acacia. Teleomorph: not seen but assumed to be a Dothidotthia sp. based on phylogenetic analysis. Conidiomata pyncidialia, superficialia, interdum furcata, cylindracea, mycelio cooperta, typice solitaria, aliquot paniculata, usque 0.5–1.5 mm longa, 50–650 mm diametro. Paraphyses infrequentes, cylindraceae, hyalinae, eseptatae. Cellulae conidiogenae holoblasticae, hyalinae, cylindraceae ad ampullaceae, (4–)6–12 (–16)  2–4(–5) mm (medianus 150 cellularum conidiogenarum mensarum 8.4  2.6 mm). Conidia primum hyalina, aseptata, postea fusco-brunnea, uniseptata (interdum ad cellulam conidiogenam affixa), ellipsoidea, obtusata, saepe ad basin truncata, et ad septum interdum valde constricta, plerumque ad medium cellulae apicalis latissima, (13–)17–22(–32)  (6–)7–10(–11) mm (medianus 300 conidiorum mensorum 19.8  8.4 mm). Typus: Australia: Western Australia: Yalgorup National Park, healthy stem of Acacia rostellifera, June 2005, K.M. Taylor (PERTH 07692978 dholotypus; culture ex-type WAC13154 ¼ MUCC505).

Conidiomata pycnidial, superficial, dark brown–grey, cylindrical, occasionally branched, mostly solitary, covered in mycelium, 0.5–1.5 mm in length and 0.1–0.5 mm in diameter. Paraphyses very rare, cylindrical, hyaline, aseptate. Conidiogenous cells holoblastic, hyaline, cylindrical to flask-shaped (4–)6–12(–16)  2–4(–5) (average of 150 conidiogenous cells 8.4  2.6 mm). Conidia initially hyaline and aseptate becoming dark brown and 1-septate sometimes while still attached to conidiogenous cell, ellipsoid, smooth-walled, obtuse apex, frequently truncate at base, often strongly constricted at the septum, usually widest at the middle of apical cell, (13–)17–22 (–32)  (6–)7–10(–11) mm (average of 300 conidia 19.8  8.4 mm), length:breadth (L:B) ratio ¼ 2.4.

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Diplodia seriata CBS112555 AY928050 Diplodia mutila CBS431.82 DQ377863 Phaeobotryosphaeria porosum CBS110496 DQ377894 Phaeobotryosphaeria porosum CBS110574 DQ377895 Diplodia pinea CBS393.84 DQ377893 Phaeobotryosphaeria visci CBS100163 DQ377870 Lasiodiplodia gonubiensis CBS115812 DQ377902

1

Lasiodiplodia theobromae CAA006 EU673254

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Phaeobotryon mamane CPC12445 EU673250 Phaeobotryon mamane CPC12443 EU673249

Botryosphaeria dothidea CBS115476 DQ377852 Botryosphaeria dothidea CBS331 33 DQ377849

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100/1.00

Botryosphaeria dothidea CBS110300 DQ377851 Botryosphaeria dothidea MUCC503 EF591936 Botryosphaeria dothidea MUCC501 EF591935 Botryosphaeria dothidea MUCC500 EF591934

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Macrophomina phaseolina CPC11070 DQ377908

88

Macrophomina phaseolina CPC11079 DQ377909

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Botryosphaeria mamane CBS117444 DQ377855 Neoscytalidium dimidiatum CBS251.49 DQ377923

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Neoscytalidium dimidiatum CBS499.66 DQ377925

Spencermartinsia viticola CBS117009 DQ377873 Spencermartinsia viticola CBS117006 EU673236

Dothiorella moneti WAC15154 EF591937 Dothiorella moneti MUCC507 EF591939 Dothiorella moneti MUCC506 EF591938

Dothiorella santala MUCC508 EF591940 Dothiorella santala WAC15155 EF591941 Dothiorella iberica CBS115041 DQ377853

7 0.88

Dothiorella sarmentorum CBS115038 DQ377860 Dothiorella sarmentorum IMI63581b AY928052 Dothiorella juglandis CBS188.87 DQ377891 Neofusicoccum ribis CMW7772 AY928044

61/0.69

Neofusicoccum parvum CBS110301 AY928046 Neofusicoccum luteum CBS110299 AY928043 Neofusicoccum australe MUCC485 EF591929

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Neofusicoccum australe MUCC496 EF591930

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Neofusicoccum australe MUCC479 EF591928

Neofusicoccum pennatuspora WAC15156 EF5919

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Neofusicoccum protearum MUCC497 EF591931

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Dichomera eucalypti MUCC499 EF591933 Dichomera eucalypti MUCC498 EF591932 Dichomera eucalypti CBS118099 DQ377886

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Pseudofusicoccum stromaticum CBS117448 DQ377931 Pseudofusicoccum stromaticum CBS117449 DQ377932

Aplosporella prunicola STE-U6327 EF564378

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Aplosporella prunicola STE-U6326 EF564377

Aplosporella yalgorupiae WAC15157 EF591943 Aplosporella yalgorupiae MUCC512 EF591944

11 100/1.00

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Saccharata proteae CBS115206 DQ377882 Saccharata proteae CBS119218 EU552145

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Guignardia bidwellii CBS111645 DQ377876 Guignardia philoprina CBS447.68 DQ377878 Diaporthe angelicae AR3776 AY196781 Diaporthe pustulata AR3535 AF408358

5 changes

Fig 1 – One of 31 most parsimonious trees of 340 steps resulting from the analysis of the LSU sequence data. BS values of the branch nodes are given in italics and the PPs resulting from Bayesian analysis are in brackets. Isolates from this study are in bold. Clades (genera) identified by Crous et al. (2006) are denoted by numbers in circles. The genus Aplosporella resides in clade 8.

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Lasiodiplodia theombromae CAA006

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Lasiodiplodia gonubiensis CBS115812

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Diplodia mutila 94-6 100/1.00

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Phaeobotryosphaeria porosum STE-U5132 Phaeobotryosphaeria porosum STE-U5046

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Phaeobotryosphaeria visci CBS100163 Phaeobotryon mamane CPC12443

100/1.00

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Diplodia seriata CMW7774 Diplodia pinea KJ94-07

100/1.00

Phaeobotryon mamane CPC12445 Spensermartinsia viticola CBS117006

100/1.00

Spensermartinsia viticola CBS117009 Dothiorella sp. CBS120688

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Dothiorella sp. CBS120689

99/0.99 73 1.00

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Dothiorella santala WAC15155 Dothiorella santala MUCC508

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Dothiorella moneti WAC15154

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Dothiorella juglandis CBS188.87

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Dothiorella sarmentorum CBS115038 Dothiorella sarmentorum IMI63581

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Dothiorella iberica CBS115041 94

Dothiorella iberica CBS115035

Neofusicoccum ribis CMW7054

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Neofusicoccum parvum CMW9081

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Neofusicoccum macroclavatum WAC12444 Neofusicoccum macroclavatum WAC12445

100 1.00

Neofusicoccum protearum MUCC497

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Neofusicoccum pennatuspora WAC15156

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98 1.00

Nefusicoccum viticlavatum STE-U5041 Nefusicoccum vitifusiforme STE-U5252 Dichomera eucalypti MUCC498

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Dichomera eucalypti MUCC499

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Dichomera eucalypti CMW15953

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Neofusicoccum australe CMW6387

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Neofusicoccum australe CMW9072

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Neofusicoccum australe MUCC479 Neofusicoccum australe MUCC485 Neofusicoccum australe MUCC496

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Neofusicoccum luteum CMW9076 Botryosphaeria corticis ATCC22928

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Botryosphaeria corticis ATCC22927 98 /0.98

Botryosphaeria dothidea MUCC500

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Botryosphaeria dothidea MUCC501

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Botryosphaeria dothidea MUCC503 Botryosphaeria dothidea CMW991

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Botryosphaeria dothidea CMW8000

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Macrophomina phaseolina MUCC531 100/1.00

Macrophomina phaseolina MUCC532

Neoscytalidium dimidiatum CBS204.33 Neoscytalidium dimidiatum CBS499.66 Pseudofusicoccum stromaticum CMW13434

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Pseudofusicoccum stromaticum CMW13435 100/1.00

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90 /0.88

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Aplosporella yalgorupiae WAC15157 Aplosporella yalgorupiae MUCC512 Aplosporella prunicola STE-U6326

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Saccharata proteae CBS114569 Saccharata proteae CBS119218 10 changes

Fig 2 – One of 18 most parsimonious trees of 1263 steps resulting from the analysis of the combined ITS and EF1-a sequence data. BS values of the branch nodes are given in italics and the PPs resulting from Bayesian analysis are in brackets. Isolates from this study are in bold.

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Lesion Length (mm)

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Neofusicoccum australe isolate Eucalyptus globulus

Eucalyptus gomphocephala

Fig 3 – Average lesion length (mm) of Eucalyptus globulus and E. gomphocephala inoculated with ten isolates of Neofusicoccum australe obtained from different host species (see Table 1). Lesions of E. globulus were measured after 6 d and those on E. gomphocephala after 13 d, thus the results of the two species cannot be directly compared.

Cultural characteristics: Appressed mycelial mat with diffuse irregular edges, initially white, edge remaining white, centre turning olive–grey to dark greenish grey and entire culture becoming dark olive–grey by day 8 and very dark greenish-grey with age. Pycnidia produced profusely in the centre of culture within 8 d. Optimal growth is at 25  C. Hosts: Acacia rostellifera, A. cochlearis Additional specimens examined: Australia: Western Australia: Yalgorup National Park, healthy stem of A. rostellifera, June 2005, K.M. Taylor (PERTH 07692986, culture MUCC506); Yalgorup National Park, A. rostellifera, K.M. Taylor (PERTH 07692994, culture MUCC507). Dothiorella santali K.Taylor, Barber & T.I. Burgess, sp. nov. Figs 5, 9C–D MycoBank no.: MB511828 Etym.: named after the host genus Santalum Teleomorph: not seen, but assumed to be a Dothidotthia sp. based on phylogenetic analysis Conidiomata pycnidialia, superficialia, furcata, globosa, interdum mycelio cooperta, solitaria, 100–600 mm longa, 50–650 mm diametro. Paraphyses cylindraceae, hyalinae, infrequentes, eseptatae. Cellulae conidiogenae holoblasticae, hyalinae, cylindraceae ad ampullaceae, (4–)6–12(–17)  3–3(–4) mm (medianus 50 cellularum conidiogenarum mensarum 8.6  2.4 mm). Conidia primum hyalina, aseptata, postea brunnea, uniseptata (interdum ad cellulam conidiogenam affixa), ellipsoidea, obtusata, interdum ad basin truncata, septo interdum exigue constricto, plerumque ad medium cellulae apicalis latissima, (15–)16–20(–22)  7–11(–13) mm (medianus 100 conidiorum mensorum 18.2  9.0 mm). Typus: Australia: Western Australia: Yalgorup National Park, from healthy stem of S. acuminatum, June 2005, K.M. Taylor (PERTH 07693028 d holotypus; culture ex-type WAC13155 ¼ MUCC509).

Conidiomata pycnidial, mostly superficial, dark brown to black, globose, solitary, occasionally covered in mycelium,

100–600 mm in height and 50–650 mm diam. Paraphyses cylindrical, hyaline, very rare, aseptate. Conidiogenous cells holoblastic, hyaline, cylindrical to flask shaped (4–)6–12(–17)  2–3(–4). (average of 50 conidiogenous cells 8.6  2.4 mm). Conidia initially hyaline and aseptate becoming pigmented brown and 1-septate, often while still attached to conidiogenous cell, ellipsoid, obtuse at apex, sometimes truncate at the base, sometimes slightly constricted at the septum, usually widest at the middle of apical cell, (15–)16–20(–22)  7–11(–13) mm (average of 100 conidia 18.2  9.0 mm, L:B ratio ¼ 2.0). Cultural characteristics: Initially white, appressed mycelial mat, within 8 d turning greenish to dark greenish-grey and fluffy, becoming very dark greenish-grey to black with age. Pycnidia produced on the agar. Optimal growth is at 25  C Host: Santalum acuminatum Additional specimens examined: Australia, Western Australia: Yalgorup National Park, S. acuminatum, K.M. Taylor (PERTH 07693001, culture MUCC508). Notes: Dothiorella is characterised by having pigmented 1septate ascospores, although ascospores were not observed in the present study. A distinguishing feature of Dothiorella conidia is that they become septate and pigmented within the pycnidial cavity and often while still attached to the conidiogenous cells (Phillips et al. 2005, 2008). This was clearly observed for D. moneti and D. santali. The two newly described species D. moneti and D. santali were closely related but consistently separated into distinct groups for all gene regions examined. Both species were phylogenetically closest to the recently described D. iberica, D. sarmentorum (Phillips et al. 2005). Another species, formerly known as D. viticola (Luque et al. 2005) has been moved to a new genus Spensermartinsia (Phillips et al. 2008). Morphologically D. moneti and D. santali have shorter and narrower

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Fig 4 – Dothiorella moneti (A) conidia and (B) conidiogenous cells producing conidia. Bar [ 10 mm.

conidia on average (19.8  8.4 mm and 18.2  9 mm for D. moneti and D. santali, respectively, compared with 23.2  10.9 mm and 21.6  9.8 mm for D. iberica and D. sarmetorum, respectively) although ranges of all species overlap. Conidia of the two isolates of D. santali (MUCC508 and MUCC509) from Santalum acuminatum differed from each other in size 17.7  8.5 mm and 19.2  10.9 mm, respectively. In addition, MUCC509 producing considerably less and smaller pycnidia than MUCC508 on pine needles and eucalypt twigs and these pycnidia also contained fewer spores. They also had different culture morphology and were originally placed into separate groups and only identified as the same species after molecular characterisation. MUCC508 was fluffier and had a distinctive rosette growth pattern, which was completely absent from MUCC509. Although the rosette pattern of growth of MUCC508 was never lost, it became less distinguished with repeated sub-culturing. Both cultures produced more fruiting bodies on agar than on pine needles. All isolates of D. moneti were derived from material collected from Acacia species, mainly A. rostellifera. Hansford

(1954) reported Physalospora acaciae as the casual agent of cankers in some Acacia spp. in Australia. Dingley (1970) termed the causal agent for this disease, and that of a similar disease in New Zealand as B. acaciae. Hansford (1954) reported a Diplodia–like anamorph forming pycnidia on cankers of Botryosphaeria acacia from material collected in the eastern states of Australia, although this was not observed in New Zealand (Dingley 1970). Conidial characteristics were not provided with the description of B. acaciae (Hansford 1954); however, recent molecular studies of B. acaciae have determined its placement in the N. parvum complex (Priest & Cunnington, pers. comm.), and thus, it would not be synonymous with D. moneti. D. moneti had distinctive culture morphology and was only isolated from Acacia spp. The frequency of isolation and the lack of disease symptoms suggest this species is a common endophyte, particularly of A. rostellifera. D. santali was isolated twice and only from S. acuminatum. Neofusicoccum pennatisporum K.Taylor, Barber & T.I. Burgess, sp. nov. Figs 6,7 MycoBank no.: MB511826

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Fig 5 – Dothiorella santali (A) conidia and (B) conidiogenous cells producing conidia. Bar [ 10 mm.

Etym.: named for the protrusions observed on the ascospore, giving the appearance of small wings. Teleomorph: Botryosphaeria-like. Produced only once, some asci and ascospores were observed but there was too little material available for description. Drawings provided. Conidiomata pycnidialia, superficialia, fusco-brunnea ad nigra, cylindracea–triangularia vel irregularia, plerumque solitaria, aliquot mycelio asperula, in ligno pineo 0.3–1 mm longa sed in agaro ad 2 mm longa, 0.1–0.5 mm diametro. Paraphyses infrequentes, cylindraceae, hyalinae, eseptatae. Cellulae conidiogenae holoblasticae, hyalinae, cylindraceae ad ampullaceae, 4–10 (–12)  (1–)2–3 (–4) mm (medianus 100 cellularum conidiogenarum mensarum 6.5  2.1 mm). Conidia hyalina, typice eseptata, frequenter uniseptata (16 % sporarum mensarum) sed aetate ad 5-septata (1 % sporarum mensarum), typice fusiformia, obtusa, ad basin frequenter truncata sed aliquando rotundatus, parietibus laevibus, (31–) 40–50 (–64)  6–10 (–12) mm, (medianus 100 conidiarum mensarum 45.4  9.7 mm). Microconidia hyalina, eseptata, fusiformia, ad extremitates rotundata vel truncata, (2–) 3–6 (–7)  1–2 (medianus 100 microconidiorum mensorum 4.4  1.5). Typus: Australia: Western Australia: Yalgorup National Park, from healthy stem of Allocasuarina fraseriana, June 2005, K.M. Taylor (PERTH 07693044 dholotypus; culture ex-type WAC13153 ¼ MUCC510).

Conidiomata pycnidial, superficial, dark-brown to black, cylindrical to triangular to irregular, mostly solitary, rough with some mycelium, 300–1000 mm in length and 100–500 mm diam

on pine but up to 2 mm in length on agar. Paraphyses very rare, cylindrical, hyaline, aseptate. Conidiogenous cells holoblastic, hyaline, cylindrical to flask shaped, 4–10(–12)  (1–)2–3(–4) mm (average of 100 conidiogenous cells 6.5  2.1 mm). Conidia hyaline, usually aseptate, often with one septum but can have up to five septa with age, typically fusiform, smooth-walled, apex obtuse, base frequently truncate but sometimes rounded, (31–)40– 50(–64)  6–10 (–12) mm, (average of 100 conidia 45.4  9.7 mm). L:B ratio ¼ 0.46. Microconidia hyaline, aseptate, fusiform, either rounded or truncate at both ends, (2–)3–6 (–7)  1–2 mm (average of 100 microconidia 4.4  1.5 mm). Optimal growth is from 25–30  C. Cultural characteristics. Appressed mycelial mat with diffuse irregular edges, white centre, darkening slightly with age, pycnidia produced profusely. Host: Allocasuarina fraseriana. Notes: The conidia of Neofusicoccum pennatisporum were unusually long (40–50  6–10 mm), when compared with other Neofusicoccum spp., including N. macroclavatum (25–35  6–8 mm), which is also found in Western Australia, and N. protearum (25–30  7–8 mm), which is phylogenetically the most closely related species to N. pennatisporum according to DNAbased sequence data. N. pennatisporum was also observed to produce the teleomorph (observed once on pine needles), which had distinctive protrusions on either end of the

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Fig 6 – Neofusicoccum pennatisporum (A) conidia; (B) conidiogenous cells producing conidia, and (C) microconidia. Bar [ 10 mm.

ascospore unlike ascospores from other species in the clade. Many species in this genus produce a Dichomera synanamorph with pigmented, muriform conidia (Barber et al. 2005). Although microconidia were observed for this species in the present study, no pigmented spores of any kind were observed. Aplosporella yalgorensis K.Taylor, Barber & T. Burgess, sp. nov. Figs 8,9A MycoBank no.: MB511827 Etym.: named after Yalgorup National Park in which it was found. Teleomorph: unknown. Conidiomata pyncnidialia, plerumque immersa sed ad maturitatem erumpentia, globosa–subconica, solitaria, laevia, botryosa, nigra, 300–650 mm longa, 150–1000 mm diametro. Paraphyses vulgares, hyalinae, cylindraceae, aliquando septatae. Cellulae conidiogenae holoblasticae, hyalinae, cylindraceae ad ampullaceae, (2–)6–12(–30)  2–6 mm (medianus 100 cellularum conidiogenarum mensarum 8.8  2.7 mm). Conidia initium hyalina, ad maturitatem fusco-brunnea, aseptata, ellipsoidea, ad extremitates obtusa,

(16–)18–22(–26)  (7–)8–13(–14) mm (medianus 100 conidiorum mensorum 19.9  10.7 mm), parietibus sub magnificatione magna foveatis. Typus: Australia: Western Australia: Woodman Point Regional Park, from a healthy stem of A. cochlearis, June 2005, K.M. Taylor (PERTH 07693060 dholotypus; culture ex-type WAC13156 ¼ MUCC511).

Conidiomata pycnidial, immersed but erumpent at maturity, black, globose to conical, mostly solitary, smooth, botryose, 300–650 mm in height and 150–1000 mm diam. Single or multi-loculate. Paraphyses very common, hyaline, cylindrical, sometimes septate. Conidiogenous cells holoblastic, hyaline, cylindrical to flask-shaped (2–)6–12(–30)  2–6 (average of 100 conidiogenous cells 8.8  2.7 mm). Conidia initially hyaline becoming pigmented dark brown at maturity, aseptate, cell wall pitted at high magnification, ellipsoid obtuse at both ends (16–)18–22(–26)  (7–)8–13(–14) (average of 100 conidia 19.9  10.7 mm, L:B ratio ¼ 1.9). Optimal growth is at 25  C.

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host species or other species tested. (Damm et al. 2007) speculated that some species within this genus might prove to be enodophytic, as this species appears to be. Cultures of A. yalgorensis (MUCC 511 and MUCC 512) were initially considered as having different morphology and were placed into separate groups, although these differences diminished over time and cultures eventually became indistinguishable. Conidial dimensions of these two isolates were very similar (18–22  8–13 mm) and overlapped that of the most recently described species, A. prunicola (19–22  10– 12 mm) (Damm et al. 2007). A. yalgorensis keys’ out to A. embeliae, if it is considered a saprophyte, in the key provided by Pande & Rao (1995) who described several Aplosporella spp. in India. However, although the conidia of A. embeliae are minutely pitted, they are wider (18–22  12–16) than that of the species described in this paper.

Discussion

Fig 7 – Neofusicoccum pennatisporum (A) asci and (B) ascospore. Bar [ 10 mm.

Cultural characteristics. Initially white, submerged mycelium, within 8 d becoming greenish-grey, fluffy, dark greenish-grey with age, producing some pycnidia on the agar. Hosts: Acacia cochlearis, Eucalyptus gomphocephala. Additional specimens examined: Australia: Western Australia: Yalgorup National Park., from healthy stem of E. gomphocephala, June, 2005, K.M. Taylor (PERTH 07693052, culture MUCC512). Notes: Aplosporella has only recently been included in the Botryosphaeriaceae with the molecular analysis of a newly described species, A. prunicola (Damm et al. 2007). There have been over 300 species included within Aplosporella, which appears to be heterogeneous, and therefore, not all are likely to belong to the Botryosphaeriaceae. Like other genera in the Botryosphaeriacae, many of the morphological features of the species overlap and most have been described according to new host records (Sutton 1980). Pande & Rao (1995) revised the genus for those species found in India and provided a key that failed to classify A. yalgorensis. Without a complete revision of the genus, A. yalgorensis appears to be a novel species, isolated from endemic hosts in Western Australia. Other species that have been isolated from Acacia include A. beaumontiana (13–20  10–11.5 mm), A. clerodendri (12–16  8–10 mm) and A. subhyaline (18–22  4–6 mm), although none of these species have pitted conidial walls, which were so distinctive in A. yalgorensis (Pande & Rao 1995). No Aplosporella species have been isolated from Eucalyptus, as far as the authors are aware. A. yalgorensis did not appear to be pathogenic on its

Eight species of botryosphaeriaceous fungi were obtained from nine host species in the Eucalyptus gomphocephala woodlands. Neofusicoccum australe was very common and recovered from all hosts; Dichomera eucalypti, Botryosphaeria dothidea and N. protearum were found in low frequency. The other species have been described here as Dothiorella moneti, D. santali, N. pennatisporum, and Aplosporella yalgorensis. The most common species was N. australe, accounting for 80 % of isolations. The high frequency with which N. australe was isolated agreed with similar results in other investigations on endophytes of woody hosts conducted in Western Australia, particularly in native forest (Burgess et al. 2005; Scott 2003; Taylor et al. 2005). The large variation in culture morphology, including non-staining isolates, suggests a high level of genetic variation within the population. The frequency, variability and host range of N. australe isolated supports a Western Australian origin of this species as previously proposed (Burgess et al. 2006b). The description of N. australe includes the production of a yellow pigment in culture (Slippers et al. 2004d). However, in the current study many of the isolates failed to produce this distinctive stain. Dichomera eucalypti was isolated from E. marginata, a new host record. This species has been previously isolated from E. diversicolor in WA (Burgess et al. 2005) and from E. pauciflora and E. camaldulensis in Victoria (Barber et al. 2005). In a recent investigation, several species of Neofusicoccum were found to have Dichomera synanamorphs (Barber et al. 2005) and although no Neofusicoccum synanamorph has been observed for D. eucalypti in Australia, phylogenetic analysis places this species firmly within the Neofusicoccum clade. Lazzizera et al. (2008) found only 2 bp differing between D. eucalypti and N. vitifusiforme in a combined ITS-EF1-a phylogeny and have suggested that D. eucalypti is the syanamorph of N. vitifusiforme. A single isolate very closely related to N. protearum was recovered, but not as might be expected from the proteaceous species, Banksia grandis, examined in this study, but rather from Santalum acuminatum, which belongs to the family Santalaceae. Neofusicoccum protearum has previously been isolated from introduced Protea spp. in Australia (Denman et al. 2003), but not from other host genera.

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Fig 8 – Aplosporella yalgorensis (A) conidia and (B) paraphyses and conidiogenous cells producing conidia. Bar [ 10 mm.

Several isolates of Botryosphaeria dothidea were collected from Acacia rostellifera, A. cochlearis, and E. marginata and confirmed through sequencing of ITS region as new host records. B. dothidea has previously been isolated from cankers in South Africa (Smith et al. 1994, 1996) and reported on E. grandis in Florida (Barnard et al. 1987), although neither identification has been confirmed by molecular analysis. It has also been isolated occasionally in eastern Australia (Slippers et al. 2004a) The distribution of the species isolated in the present study did vary between the collection sites, Yalgorup National Park and Woodman Point Regional Park. Neofusicoccum australe was the most frequently isolated species at both sites, particularly on E. gomphocephala. It was isolated even more frequently from healthy E. gomphocephala at Woodman Point Regional Park than from declining E. gomphocephala in Yalgorup National Park. The ITS region of representative isolates were sequenced and the confirmed new host records are Callitris preissii, E. gomphocephala, S. acuminatum, Banksia grandis, Allocasuarina fraseriana, E. marginata, Acacia rostellifera, A. cochlearis, and Agonis flexuosa. Dothiorella moneti, the second most frequently isolated species, was isolated most from Acacia rostellifera in Yalgorup National Park. There was one isolate collected from Woodman Point Regional Park; however, it was from Acacia cochlearis and not A. rostellifera, although material

from A. rostellifera was also collected. All other species were isolated infrequently making it difficult to determine any strong affinity to site or host. N. australe was shown to form lesions on excised stems of both E. gomphocephala and E. globulus. The rapid colonisation and spread of N. australe following inoculation of E. globulus excised stems has been shown previously (Burgess et al. 2005). The development of lesions on E. gomphocephala was more variable than on E. globulus. This was most likely due to the variability in the age and physiological status of the stems, which were collected in the mixed-age native woodland, in comparison to the stems of E. globulus, which were collected from an evenaged plantation. D. moneti, D. santali, N. pennatisporum and Aplosporella yalgorensis were not pathogenic on the hosts they were isolated from or on E. globulus and E. gomphocephala. The cosmopolitan nature of N. australe in Western Australia suggests unhampered movement within the environment. The isolation of this species more frequently from asymptomatic tissue of confirms it is a common endophyte and the formation of lesions on all hosts so far tested suggests it is also latent pathogen capable of causing disease in stressed or damaged trees, rather than an aggressive primary pathogen. The E. gomphocephala ecosystem is one of several woodland ecosystems in Western Australia undergoing

Botryosphaeriaceae from tuart (Eucalyptus gomphocephala) woodland

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Fig 9 – Aplosporella yalgorensis. (A) Scanning election micrograph showing surface features of conidia. (B) Light micrograph of conidia of Dothiorella moneti. (C) Light micrograph of conidia of D. santali. (D) Light micrograph of conidiogenous cells and conidia of D. santali. Bars [ 10 mm

severe decline (Barber & Hardy 2006). Stem borers have been previously suggested as a cause of the decline of E. gomphocephala (Longman & Keighery 2002); however, destructive sampling of declining E. gomphocephala have shown numerous trees suffering decline do not exhibit borer damage (P.A.B., unpubl. data). The prevalence of cankers or disease associated with borer damage is common on E. gomphocephala. These cankers are likely to contribute to the decline rather than be the primary cause. The incidence of cankers is likely to increase if the primary cause of the decline inducing stress on E. gomphocephala is not halted. All forest trees are considered to have fungi existing endophytically, the majority of which are probably undescribed (Sieber 2007). The actual life cycle of endophytes and net consequence to the host is difficult to determine, and in most cases unknown (Sieber 2007), although the number and diversity of endophytes does change with the season and the age of the host tissue (Saikkonen 2007; Slippers & Wingfield 2007). Many endophytic fungi are potentially pathogenic when environmental circumstances are detrimental to the host and advantageous to the growth of the fungus, while others are considered mutualistic because they deter herbivores. Even pathogenic fungi in stressed tissue may be considered mutualistic in some respects, as the colonisation and removal of weakened tissue and competition with potentially more invasive fungi could be considered beneficial to the host (Slippers & Wingfield 2007). Fungi belonging to the Botryosphaeriaceae appear to be successful as opportunistic endophytic colonists and are horizontally transmitted between hosts (Saikkonen 2007; Slippers & Wingfield 2007). Interestingly, it appears that species with the more extensive host and geographical range (e.g. Diplodia pinea, D. seriata,

Neofusicoccum australe, N. ribis, Lasiodiplodia theobromae) are frequently the most pathogenic (Burgess et al. 2005; Pavlic et al. 2007; Slippers & Wingfield 2007; Taylor et al. 2005; van Niekerk et al. 2004). In this study the most common species found on all host, N. australe, was the only species that appeared to be pathogenic on any host. It was not unexpected to find new fungal species in the present study. Our knowledge of micro-fungi, particularly endophytes, associated with vegetation in native forests and woodlands is limited and most investigations in Australia have resulted in the description of new fungal species (Burgess et al. 2005; Pavlic et al. 2008; Slippers et al. 2004c, 2004d, 2005b). There are several reasons why investigation into the natural microbial communities is important. First, it is difficult to understand how ecosystems are changing in response to human and environmental pressures without knowledge of the ecosystem prior to change (Saikkonen 2007). Many microbial communities are in danger of changing, or have already done so, without us being aware of it. In the case of declining woodlands, we are likely to lose a great deal of our microbial biodiversity with the loss of our woodland species. It is also important to gain knowledge of native and introduced pathogens to help us understand, conserve and protect both environmental and human interests.

Acknowledgements This research was partly funded by the Australian Research Council (LP0346931). We acknowledge the assistance of members of the Tuart Health Research Group (THRG), Diane White for technical support, and Alex George for the Latin translations.

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