Green and brown bridges between weeds and crops reveal novel Diaporthe species in Australia

Abstract

Diaporthe (syn. Phomopsis) species are well-known saprobes, endophytes or pathogens on a range of plants. Several species have wide host ranges and multiple species may sometimes colonise the same host species. This study describes eight novel Diaporthe species isolated from live and/or dead tissue from the broad acre crops lupin, maize, mungbean, soybean and sunflower, and associated weed species in Queensland and New South Wales, as well as the environmental weed bitou bush (Chrysanthemoides monilifera subsp. rotundata) in eastern Australia. The new taxa are differentiated on the basis of morphology and DNA sequence analyses based on the nuclear ribosomal internal transcribed spacer region, and part of the translation elongation factor-1α and ß-tubulin genes. The possible agricultural significance of live weeds and crop residues (‘green bridges’) as well as dead weeds and crop residues (‘brown bridges’) in aiding survival of the newly described Diaporthe species is discussed.

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Persoonia 35, 2015: 39 – 49
www.ingentaconnect.com/content/nhn/pimj http://dx.doi.org/10.3767/003158515X687506
RESEARCH ARTICLE
INTRODUCTION
Diaporthe (syn. Phomopsis) species have been recorded on a
wide range of hosts. Species in this genus are well-known in
the plant pathology literature as the cause of many significant
plant diseases worldwide, including stem cankers, leaf and
pod blights, and seed decay (Rehner & Uecker 1994, Santos
et al. 2011, Udayanga et al. 2011). Further, Diaporthe species
have been recorded as opportunistic saprobes on decaying
leaves, twigs and stem residues, as well as endophytes on
healthy leaves, stems, seeds and roots (Muralli et al. 2006,
Gomes et al. 2013).
The recent use of DNA sequence-based methods and the appli-
cation of the Genealogical Concordance Phylogenetic Species
Recognition (GCPSR) criteria have resulted in a rapid increase
in the discovery of cryptic species in several large genera of
plant pathogenic fungi, such as Colletotrichum (Damm et al.
2012a, b, Weir et al. 2012), Diaporthe (Shivas & Cai 2012,
Udayanga et al. 2014) and Fusarium (O’Donnell et al. 2009,
2012). This approach also provides a more stable taxonomy
for Diaporthe, from which a clearer understanding about the
host range of particular species is emerging. It is known that
many species of Diaporthe have wide host ranges (Mengistu
et al. 2007, Santos et al. 2011, Udayanga et al. 2011, Gomes
et al. 2013) and multiple species can colonise the same host
(Farr et al. 2002, Crous & Groenewald 2005, van Niekerk et
al. 2005, Thompson et al. 2011).
It is well documented in plant pathology literature that live weeds
and volunteer crop plants serve as alternative hosts for a range
of pathogens, including Diaporthe species, by providing a ‘green
bridge’ that facilitates pathogen survival between crop phases.
Following the first outbreaks of Diaporthe helianthi (syn. Pho-
mopsis helianthi) on sunflower in the former Yugoslavia (now
Serbia), Mihaljcevic & Muntañola-Cvetković (1985) recovered
Diaporthe species from 15 plant species, including the weeds
Xanthium italicum and X. strumarium. Subsequent studies by
Vrandečić et al. (2010) confirmed Arctium lappa, X. italicum
and X. strumarium as weed hosts for D. helianthi.
Alternative weed hosts have been suspected to play a role in
the epidemiology of three species, D. gulyae, D. kochmanii and
D. kongii, recently found associated with sunflower stem canker
in Australia (Thompson et al. 2011). During recent investiga-
tions to identify alternative hosts of the Diaporthe species that
cause sunflower canker in eastern Australia, eight novel species
were identified based on GCPSR criteria, from both live crop
and weed hosts as well as crop stubble and weed residues in
Queensland (Qld) and New South Wales (NSW). Dead stand-
ing weeds and residues are common amongst crop stubble in
Australian broad acre and low tillage cropping systems, where
herbicides are often used for weed control. Additionally, one
of the new Diaporthe species was also identified from a study
into the cause of dieback of the coastal environmental weed
Chrysanthemoides monilifera subsp. rotundata (bitou bush)
in northern NSW. All eight species of Diaporthe are described
and illustrated here.
MATERIALS AND METHODS
Isolates
Isolates from broad acre cropping regions
Plant material was collected from a range of summer crops in-
cluding lupin, maize, mungbean, soybean and sunflower, as well
as major weed species and plant residues on the soil surface
Green and brown bridges between weeds and crops reveal
novel Diaporthe species in Australia
S.M. Thompson1,2, Y.P. Tan 3, R.G. Shivas3, S.M. Neate1, L. Morin 4, A. Bissett5,
E.A.B. Aitken2
1 Centre for Crop Health, University of Southern Queensland, West Street,
Toowoomba. Queensland 4350 Australia;
corresponding author e-mail: sue.thompson@usq.edu.au.
2 School of Agriculture and Food Science, The University of Queensland, St
Lucia, Queensland 4072, Australia.
3 Plant Pathology Herbarium, Department of Agriculture, Fisheries and
Forestry, Ecosciences Precinct, Dutton Park, Queensland 4102, Australia.
4 CSIRO Ecosystem Sciences and Biosecurity Flagship, G.P.O. Box 1700,
Canberra, Australian Capital Territory 2601, Australia.
5 CSIRO Plant Industry, G.P.O. Box 1600, Canberra, Australian Capital Ter-
ritory 2601, Australia.
Key words
alternate weed hosts
multi-locus
Phomopsis
phylogeny
taxonomy
Abstract Diaporthe (syn. Phomopsis) species are well-known saprobes, endophytes or pathogens on a range of
plants. Several species have wide host ranges and multiple species may sometimes colonise the same host species.
This study describes eight novel Diaporthe species isolated from live and/ or dead tissue from the broad acre crops
lupin, maize, mungbean, soybean and sunflower, and associated weed species in Queensland and New South
Wales, as well as the environmental weed bitou bush (Chrysanthemoides monilifera subsp. rotundata) in eastern
Australia. The new taxa are differentiated on the basis of morphology and DNA sequence analyses based on the
nuclear ribosomal internal transcribed spacer region, and part of the translation elongation factor-1α and ß-tubulin
genes. The possible agricultural significance of live weeds and crop residues (‘green bridges’) as well as dead
weeds and crop residues (‘brown bridges’) in aiding survival of the newly described Diaporthe species is discussed.
Article info Received: 2 May 2014; Accepted: 30 October 2014; Published: 10 February 2015.

40 Persoonia – Volume 35, 2015
across the broad acre cropping regions of Qld and NSW (Table
1). The material included necrotic lesions or visible pycnidia
on stems, leaves, petioles, heads and seeds from live plants
and/ or dead plants. Specimens from plant residues were only
selected from material for which the inflorescence was present
so that the plant species could be identified.
Small pieces (10–30 mm) of tissue or entire seeds were surface
sterilised in 1 % sodium hypochlorite solution for 3 min, then
rinsed with sterile distilled water. The surface sterilised tissue
was placed onto 9 cm diam Petri plates containing water agar
amended with 100 µg/ mL streptomycin sulphate (WAS), and
incubated at 23–25 °C under ambient light. After 2–21 d, co-
nidial ooze from individual pycnidia characteristic of Diaporthe
species was streaked onto potato dextrose agar (PDA) (Oxoid)
amended with 100 µg/mL streptomycin sulphate (PDAS) and
incubated as above. After 12– 24 h, single germinating conidia
or hyphal tips were removed aseptically with a fine needle and
placed on the surface of fresh plates of PDAS and incubated
as above.
Isolates from bitou bush
Stems of live bitou bush plants affected by dieback were col-
lected from Bongil Bongil National Park and Bellingen Head
State Park in northern NSW (Table 1). Pieces of stem tips with
necrotic symptoms were cut in 1–2 cm long sections, including
the margin between healthy and dead tissue, immersed in 70 %
ethanol for 30 s followed by 2 % sodium hypochlorite for 2– 4
min. Tissue pieces were rinsed three times in sterile distilled
water, blotted dry with paper towel, then cut longitudinally with
a sterile scalpel and placed on 1/2 strength PDA amended with
200 µg/mL streptomycin sulphate (1/ 2 PDAS) or on acidified
PDA (one drop of 25 % lactic acid added per plate when pour-
ing) in 9 cm diam Petri dishes. Plates were incubated at room
temperature under 24 h fluorescent lights.
The bark of stem pieces (c. 1–3 cm diam and 12–15 cm long),
cut near the base of wilting bitou bush plants was removed,
using a sharp, surface-sterilised knife, over more than half
of the circumference of the pieces and for c. 7–8 cm long in
the middle of the pieces. A surface-sterilised wood chisel was
then used to remove thin slices (up to c. 30, each c. 1–3 cm
long) from the wood (xylem) of each of the stem pieces. Small
pieces (c. 0.5 cm2) were cut from each slice of wood tissue
and surface sterilised either by: i) immersing in 2 % NaOCl
for 1 min, followed by 1 min in 70 % ETOH, then rinsed three
times in sterile distilled water; or ii) by spraying 70 % ETOH
onto the pieces surface, and then blotting dry with paper towel
and plating onto 1/2 PDAS. Plates were incubated as above.
Pieces of hyphae at the margin of colonies that grew from the
bitou bush pieces were transferred onto fresh 1/2 PDAS and
PDA plates, and incubated as above. Plates were examined
for pycnidia characteristic of Diaporthe species at weekly
intervals over a 2 mo period with a stereoscopic microscope.
Single-conidium isolates were produced as described above
for isolates from broad acre cropping regions and grown on
Species Isolate no.a Host Localityb GenBank accession no.c
ITS TEF BT
Diaporthe ambigua CBS 114015* Pyrus communis South Africa AF230767 GQ250299 KC343978
Diaporthe anacardii CBS 720.97* Anacardium occidentale East Africa KC343024 KC343750 KC343992
Diaporthe batatas CBS 122.21 Ipomea batatas USA KC343040 KC343766 KC344008
Diaporthe beilharziae BRIP 54792* Indigofera australis NSW, Australia JX862529 JX862535 KF170921
Diaporthe charlesworthii BRIP 54884m* Rapistrum rugostrum Qld, Australia KJ197288 KJ197250 KJ197268
Diaporthe cinerascens CBS 719.96 Ficus carica Bulgaria KC343050 KC343776 KC344018
Diaporthe cuppatea CBS 117499* Aspalathus linearis South Africa AY339322 AY339354 KC344025
Diaporthe elaeagni CBS 504.72 Elaeagnus sp. Netherlands KC343064 KC343790 KC344032
Diaporthe endophytica CBS 133811* Schinus terebinthifolius Brazil KC343065 KC343791 KC344033
Diaporthe foeniculacea CBS 123208* Foeniculum vulgare Portugal KC343104 KC343830 KC344072
Diaporthe goulteri BRIP 55657a* Helianthus annuus Qld, Australia KJ197289 KJ197252 KJ197270
Diaporthe gulyae BRIP 54025* Helianthus annuus Qld, Australia JF431299 JN645803 KJ197271
Diaporthe helianthi CBS 592.81* Helianthus annuus Serbia KC343115 GQ250308 KC343841
Diaporthe hordei CBS 481.92 Hordeum vulgare Norway KC343120 KC343846 KC344088
Diaporthe infecunda CBS 133812 Schinus terebinthifolius Brazil KC343126 KC343852 KC344094
Diaporthe kongii BRIP 54031* Helianthus annuus Qld, Australia JF431301 JN645797 KJ197272
Diaporthe macinthoshii BRIP 55064a* Rapistrum rugostrum Qld, Australia KJ197290 KJ197251 KJ197269
Diaporthe masirevicii BRIP 57330 Chrysanthemoides monilifera subsp. rotundata NSW, Australia KJ197275 KJ197237 KJ197255
BRIP 54256 Glycine max Qld, Australia KJ197276 KJ197238 KJ197256
BRIP 57892a* Helianthus annuus Qld, Australia KJ197277 KJ197239 KJ197257
BRIP 54120c Zea mays Qld, Australia KJ197278 KJ197240 KJ197258
Diaporthe melonis CBS 507.78* Cucumis melo USA KC343141 KC343867 KC344109
Diaporthe middletonii BRIP 57329 Chrysanthemoides monilifera subsp. rotundata NSW, Australia KJ197285 KJ197247 KJ197265
BRIP 54884e* Rapistrum rugostrum Qld, Australia KJ197286 KJ197248 KJ197266
Diaporthe miriciae BRIP 55662c Glycine max Qld, Australia KJ197283 KJ197245 KJ197263
BRIP 54736j* Helianthus annuus NSW, Australia KJ197282 KJ197244 KJ197262
BRIP 56918a Vigna radiata Qld, Australia KJ197284 KJ197246 KJ197264
Diaporthe neoarctii CBS 109490* Ambrosia trida USA KC343145 KC343871 KC344113
Diaporthe phaseolorum CBS 116019 Caperonia palustris USA KC343175 KC343901 KC344143
Diaporthe raonikayaporum CBS 133182* Spondias mombin Brazil KC343188 KC343914 KC344156
Diaporthe sackstonii BRIP 54669b* Helianthus annuus Qld, Australia KJ197287 KJ197249 KJ197267
Diaporthe seraniae BRIP 55665a* Helianthus annuus Qld, Australia KJ197274 KJ197236 KJ197254
BRIP 54136 Lupinus albus ‘Rosetta’ NSW, Australia KJ197273 KJ197235 KJ197253
Diaporthe sojae CBS 180.55 Glycine soja KC343200 KC343926 KC344168
Diaporthe stitica CBS 370.54 Buxus sempervirens Italy KC343212 KC343938 KC344180
Diaporthella corylina CBS 121124 Corylus sp. China KC343004 KC343730 KC343972
a BRIP: Plant Pathology Herbarium, Dutton Park, Queensland, Australia; CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.
b NSW, New South Wales; Qld, Queensland; USA, United States of America.
c Other than those in bold, all sequences were downloaded from GenBank and published in van Rensberg et al. (2006), Santos et al. (2010), Udayanga et al. (2011, 2012), Gomes et al. (2013)
and Tan et al. (2013).
* Ex-type or ex-epitype culture.
Table 1 Diaporthe spp., and the outgroup taxon Diaporthella corylina, included in the phylogenetic analysis of this study. Newly described taxa and deposited
sequences are in bold.

41
S.M. Thompson et al.: Diaporthe species in Australia
1/2 PDAS under the same conditions as above. All isolates
recovered were deposited in the Plant Pathology Herbarium
(BRIP), Brisbane, Australia.
Morphology
To determine morphological characteristics, isolates were grown
on water agar with pieces of sterilised wheat stems placed on
the surface (WSA) and incubated under a 12 h photoperiod
with near ultraviolet light (NUV) (Smith 2002) at 23 °C. Fungal
structures were mounted on glass slides in lactic acid (100 %
v/ v) for microscopic examination after 28 d of incubation. At
least 20 measurements of selected structures were made and,
means and standard deviations (SD) calculated. Ranges were
expressed as (min–) mean-SD – mean+SD (– max) with values
rounded to 0.5 μm. Images were captured with a Leica DFC 500
camera attached to a Leica DM5500B compound microscope
with Nomarski differential interference contrast.
For colony morphology, 3-d-old cultures on 9 cm diam plates
of PDA and oatmeal agar (OMA) (Oxoid) that had been grown
in the dark at 23 °C were grown for a further 7 d under 12 h
photoperiod with NUV light at 23 °C (Thompson et al. 2011).
Colony colours (surface and reverse) were described according
to the colour charts of Rayner (1970). Nomenclatural novel-
ties were deposited in MycoBank (Crous et al. 2004) (www.
mycobank.org).
DNA isolation, amplification and analyses
For isolates from broad acre cropping regions, mycelia were
scraped off PDA cultures and macerated with 0.5 mm glass
beads (Daintree Scientific) in a Tissue Lyser (QIAGEN). Ge no-
mic DNA was then extracted with the Gentra Puregene DNA
Extraction kit (QIAGEN) according to the manufacturer’s in-
structions. For isolates from bitou bush, genomic DNA was
extracted from mycelia scraped off 1/2 PDAS cultures using
Mo-Bio Ultraclean Microbial DNA Isolation Kit.
The internal transcribed spacer (ITS) region of the nuclear ri-
bosomal genes was amplified with the primers ITS4 (White et
al. 1990), and V9G (de Hoog & Gerrits van den Ende 1998)
or ITS1F (Gardes & Bruns 1993) for the isolates from broad
acre cropping regions and bitou bush, respectively. For all
isolates, the primers EF1-728 F (Carbone & Kohn 1999) and
EF2 (O’Donnell et al. 1998) were used to amplify part of the
translation elongation factor 1-α (TEF) gene, and the primers
T1 (O’Donnell & Cigelnik 1997) and Bt2b (Glass & Donaldson
1995) were used to amplify part of the ß-tubulin (BT) gene.
The ITS region of the bitou bush isolates was amplified with
Platinum Taq (Invitrogen) according to manufacturer’s instruc-
tions and the PCR conditions were 95 °C for 30 s, 55 °C for
30 s, 72 °C for 1 min ×25 cycles. PCR products were purified
with the Agencourt AMPure XP system (Beckman Coulter).
The ITS region of the broad acre cropping isolates and the BT
and TEF loci of all isolates in this study were amplified with
the Phusion High-Fidelity PCR Master Mix (Finnzymes) and
the PCR conditions were 98 °C for 30 s, followed by 30 cycles
of 98 °C for 10 s, 55 °C (ITS and TEF) or 60 °C (BT) for 30 s,
72 °C for 30 s, and a final extension at 72 °C for 5 min. The PCR
products were purified with the QIAquick PCR Purification Kit
(QIAGEN), and sequenced by Macrogen Incorporated (Seoul,
Korea) using the amplification primers.
All unique sequences from different host-isolate combinations
generated in this study were assembled using Vector NTi
Advance 11.0 (Invitrogen). The ITS sequences were initially
aligned with representative Diaporthe species from recent
studies (Thompson et al. 2011, Udayanga et al. 2012, Gomes
et al. 2013) using MAFFT alignment algorithm (Katoh et al.
2009) in the software Geneious (Biomatters Ltd). Diaporthella
corylina (CBS 121124) was selected as outgroup taxon in the
phylogenetic analyses based on its position as sister genus in
Diaporthales (Vasilyeva et al. 2007).
A Neighbour-Joining (NJ) analysis using the Kimura-2 para-
meter with Gamma distribution was applied (data not shown),
and the closest phylogenetic neighbours were selected for a
combined analyses using BT, ITS and TEF genes. The se-
quences of each gene were aligned separately and manually
adjusted where needed. Alignment gaps were treated as miss-
ing character states, and all characters were unordered and of
equal weight. Bayesian analysis was performed with MrBayes
v. 3.2.1 (Huelsenbeck & Ronquist 2001, Ronquist & Huelsenbeck
2001) in Geneious. The Markov Chain Monte Carlo (MCMC)
analysis used four chains and started from a random tree topo-
logy. The sample frequency was set at 200 and the temperature
of the heated chain was 0.3. Burn-in was set at 25 % after which
the likelihood values were stationary. Maximum Likelihood (ML)
analysis, including 1 000 bootstrap replicates, were run using
RAxML v. 7.2.8 (Stamatakis & Alchiotis 2010) in Geneious.
The nucleotide substitution model chosen was General Time
Reversible (GTR) with a gamma-distributed rate of variation.
The concatenated alignment and resulting tree were deposited
in TreeBASE (study S15707). Unique fixed nucleotides posi-
tions are used to characterise and differentiate two species
from closely related phylogenetic species. For each species
that was described, the closest phylogenetic neighbour was
selected and this focused dataset was subjected to single
nucleotide polymorphisms (SNPs) analyses. These SNPs
were determined for each aligned data partition using DnaSP
v. 5.10.01 (Librado & Rozas 2009).
RESULTS
Isolates
More than 500 Diaporthe isolates were recovered from live or
dead plant tissues or seeds, from the crops sunflower, soybean,
mungbean, lupin, maize, as well as from a range of weed spe-
cies in the broad acre cropping regions of Qld and NSW (Table
2). Of these isolates, 147 could not be assigned to known taxa
based on ITS sequence BLASTn search results against the
GenBank database. Many of the remaining isolates recovered
from a number of crop and weed hosts were identified as one
of three recently described species from sunflower, namely,
D. gulyae, D. kochmanii and D. kongii (Thompson et al. 2011)
(data not shown). Fifteen Diaporthe isolates were recovered
from the bitou bush material, including eight isolates of D. kongii
(data not shown).
Phylogenetic analyses
Approximately 600 bases of the ITS region were sequenced
from the isolates investigated in this study and initially aligned
against 116 sequences from 106 Diaporthe species, most of
which were from ex-type cultures. The evolutionary relation-
ships of these sequences were analysed using the NJ method
(data not shown; TreeBASE study S15707). From this NJ
phylogenetic tree, 19 Diaporthe taxa closest to the isolates in
this study were selected for a combined analyses using the ITS,
TEF and BT sequences. The combined sequence (ITS, TEF
and BT) alignment for the Bayesian and ML analyses contained
1 642 characters from 35 isolates (including the outgroup taxon)
(Table 1). The Bayesian analysis lasted 1 100 000 generations,
and the consensus tree with posterior probability was calculated
from 4 951 trees left after 110 000 trees were discarded at the
burn-in phase. The tree topology and bootstrap values of the
ML analysis supported the trees obtained from the Bayesian
analysis. The multilocus phylogenetic tree (Fig. 1), along with mor-

42 Persoonia – Volume 35, 2015
Plant host1 Host family Diaporthe spp.
charlesworthii goulteri macintoshii middletonii masirevicii miriciae sackstonii serafiniae weieri
Crop
Glycine max Fabaceae – – – – L L – – L
Helianthus annuus Asteraceae – S – – L, S L, S L L, S L, D
Lupinus alba Fabaceae – – – – – – – D –
Vigna radiata Fabaceae – – – – L L – – –
Zea mays Poaceae – – – – L – – – L
Weed
Bidens pilosa Asteraceae – – – – – – – L –
Chrysanthemoides monilifera Asteraceae – – – L L – – – L
subsp. rotundata
Datura ferox Solanaceae – – – – – – – D –
Gaura parviflora Onagraceae – D – – – – – – –
Malva paraflora Malvaceae – – – – – – – L –
Rapistrum rugosum Brassicaceae D – D D L, D D – – D
Sesbania cannabina Fabaceae – – – – L – – – –
Solanum nigrum Solonaceae – – – – – – – L –
1 Material from which the fungi were isolated is indicated in table: L = live stem (including leaf or petiole) tissue; D = dead stem (including petiole) tissue; S = seeds.
Table 2 Crops and weeds from which the novel Diaporthe spp. species described in this paper were isolated.
Diaporthella corylina CBS 121124
Diaporthe raonikayaporum CBS 133182
BRIP 55657a* Helianthus annuus
Diaporthe ambigua CBS 114015*
Diaporthe batatas CBS 122.21
Diaporthe helianthi CBS 592.81*
Diaporthe hordei CBS 481.92
Diaporthe beilharziae BRIP 54792*
Diaporthe cuppatea CBS 117499*
BRIP 54884m* Rapistrum rugosum
Diaporthe melonis CBS 507.78
Diaporthe gulyae BRIP 54025*
Diaporthe neoarctii CBS 109490*
Diaporthe infecunda CBS 133812*
BRIP 54669b* Helianthus annuus
Diaporthe anacardii CBS 720.97
Diaporthe foeniculacea CBS 123208
Diaporthe cinerascens CBS 719.96
Diaporthe phaseolorum CBS 116019
Diaporthe sojae CBS 180.55*
BRIP 54136 Lupinus albus ‘Rosetta’
BRIP 55665a* Helianthus annuus
BRIP 57329 Chrysanthemioides monilifera subsp. rotundata
BRIP 54884e* Rapistrum rugosum
BRIP 55064a* Rapistrum rugosum
Diaporthe endophytica CBS 133811*
BRIP 55662c Glycine max
Diaporthe stictica CBS 370.54
Diaporthe elaeagni CBS 504.72
Diaporthe kongii BRIP 54031*
BRIP 56918a Vigna radiata
BRIP 54736j* Helianthus annuus
BRIP 54120c Zea mays
BRIP 54256 Glycine max
BRIP 57330 Chrysanthemoides monilifera subsp. rotundata
BRIP 57892a* Helianthus annuus
3.0
Diaporthe masirevicii



Diaporthe miriciae





Diaporthe serafiniae


Diaporthe middletonii
Diaporthe sackstonii





 Diaporthe macintoshii
Diaporthe charlesworthii
Diaporthe goulteri





Fig. 1 Phylogenetic tree based on the combined multilocus (ITS, TEF and BT) alignment. The tree with the highest log likelihood (-8570) is shown. Bayesian
posterior probabilities (pp) and RAxML bootstrap values (bs) are given at the nodes (pp/bs). Only those with bs percentage of greater than 60 are shown.
A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.4745)). The tree is drawn to
scale, with branch lengths measured in the number of substitutions per site. Ex-type cultures are indicated by an asterisk (*).

43
S.M. Thompson et al.: Diaporthe species in Australia
phological examinations (see below), support the establishment
of eight novel Diaporthe species, which are described below.
Taxonomy
Diaporthe charlesworthii R.G. Shivas, S.M. Thomps. & Y.P.
Tan, sp. nov. — MycoBank MB808668; Fig. 2a–f
Etymology. In recognition of Australian sunflower grower Kevin Charles-
worth (Ryeford Qld), for his contributions to the sunflower industry and
passionate advocate of research.
Conidiomata pycnidial and multilocular, scattered, abundant on
PDA, OMA and WSA after 4 wk, subglobose, up to 1 mm diam,
ostiolate, necks absent or up to 1 mm. Conidiophores formed
from the inner layer of the locular wall, 0–2-septate, branched
at septa, hyaline to subhyaline, cylindrical, 15–35 × 1.5–3 μm.
Conidiogenous cells cylindrical to flexuous, tapered towards the
apex, hyaline, 10–25 × 1.5–3.0 μm. Alpha conidia abundant,
fusiform to cylindrical, rounded at the apex, narrowed towards
the base, hyaline, (6–)7–9.5(–11) × 2 – 2.5 μm. Beta conidia
abundant amongst the alpha conidia, flexuous to J-shaped,
hyaline, 25–35 × 1.0–1.5 μm. Perithecia not seen.
Cultural characteristics — Colonies on PDA after 10 d reach-
ing the edge of the plate, margin coralloid with feathery branch-
es, adpressed, without aerial mycelium, with numerous irregu-
larly zonate dark stromata up to 2 mm diam, isabelline becom-
ing lighter towards the margin; reverse similar to the surface
with zonations more apparent. On OMA covering entire plate
after 10 d, with little aerial mycelium and numerous scattered
pale mouse grey irregular stromata up to 1.5 cm diam, pale
isabelline between the stromata; reverse irregularly mottled,
cinnamon to isabelline.
Specimen examined. AustrAliA, Queensland, Gatton, from stem of Rapis-
trum rugosum, 24 Nov. 2011, S.M. Thompson (T12757Z), holotype BRIP
54884m (includes ex-type culture).
Notes ― The multigene analysis of isolate BRIP 54884m
was not significantly homologous to any sequences in Gen-
Bank. No morphologically similar isolates are known from
Rapistrum rugosum. Therefore, this isolate is designated as
representative of a new taxon. Diaporthe charlesworthii is one
of three novel species isolated in this study from dead stems
of R. rugosum (Brassicaceae), a widely distributed weed in
eastern Australia.
Diaporthe goulteri R.G. Shivas, S.M. Thomps. & Y.P. Tan, sp.
nov. — MycoBank MB808669; Fig. 2g–j
Etymology. In recognition of Australian scientist Ken Goulter, for his
significant contributions to Australian sunflower pathology including the
differentiation of sunflower rust races and early studies on the diversity of
Diaporthe species.
Conidiomata multilocular, rare on PDA after 4 wk, abundant
on OMA and WSA after 4 wk and often on a thin layer of dark
textura angularis 50 –100 μm thick with sharp margins on ir-
regularly patches up to 1 cm diam, ostiolate, necks absent or
less than 250 μm on PDA and OMA after 4 wk, necks up to 1.5
mm on wheat straw pieces on WA after 4 wk, abundant pale
yellow conidial droplets exude from ostioles, sienna coloured
droplets form on thin dark patches of textura angularis. Conidi-
ophores formed from the inner layer of the locular wall, reduced
to conidiogenous cells or 1-septate, hyaline to pale yellowish
brown, filiform, 10 – 30 × 1.5–3 μm. Conidiogenous cells cy-
lindrical to flexuous, tapered towards the apex, hyaline, 5–15
× 1.5– 2.5 μm. Alpha conidia abundant, fusiform to cylindrical,
Fig. 2 Diaporthe spp. — a – f: Diaporthe charlesworthii (ex-type BRIP 54884m) after 4 wk. a. Culture on PDA (top) and OMA (bottom); b. conidiomata on
OMA; c. conidiomata on PDA; d. conidiophores; e. alpha conidia and beta conidia; f. beta conidia. — g– j: Diaporthe goulteri (ex-type BRIP 55657a) after 4 wk.
g. Culture on PDA (top) and OMA (bottom); h. conidiomata on sterilised wheat straw; i. conidiomata on OMA; j. alpha conidia. –– Scale bars: a, g = 1 cm;
b, c, h, i = 1 mm; d– f, j = 10 µm.

44 Persoonia – Volume 35, 2015
Fig. 3 Diaporthe spp. — a– e: Diaporthe macintoshii (ex-type BRIP 55064a) after 4 wk. a. Culture on PDA; b. pycnidia on sterilised wheat straw; c. pycnidia on
OMA; d. conidiophores; e. alpha conidia and beta conidia. — f– j: Diaporthe masirevicii (ex-type BRIP 57892a) after 4 wk. f. Culture on PDA; g. conidiomatum
on OMA; h. alpha conidia; i. conidiophores; j. alpha conidia and beta conidia. — k– p: Diaporthe middletonii (ex-type BRIP 54884e) after 4 wk. k. Culture on
PDA (top) and OMA (bottom); l. pycnidia on sterilised wheat straw; m. conidiophores; n. alpha conidia; o. pycnidia on OMA; p. beta conidia. — q–u: Diaporthe
miriciae (ex-type BRIP 54736j) after 4 wk. q. Culture on PDA; r. conidiomata on sterilised wheat straw; s. conidiophores; t. section across conidiomatum;
u. alpha and beta conidia. — Scale bars: a, f, k, q = 1 cm; b, c, g, l, o, r = 1 mm; d, e, h–j, m, n, p, s, u = 10 µm; t = 100 µm.

45
S.M. Thompson et al.: Diaporthe species in Australia
rounded at the apex, slightly narrowed towards the base, hya-
line, (6–)6.5 – 8(–9) × 2 – 2.5(–3) μm. Beta conidia not seen.
Perithecia not seen.
Cultural characteristics — Colonies on PDA covering entire
plate after 10 d, adpressed, white to buff; reverse buff. On
OMA covering entire plate after 10 d, white tinged with pale
vinaceous, with several scattered circular mouse grey patches
up to 1 cm diam, these patches are sometimes confluent and
at the centres have olivaceous mycelium with droplets of cinna-
mon coloured exudate and one or a few funiculose columns of
white mycelium up to 3 mm high; reverse uniformly buff.
Specimen examined. AustrAliA, Queensland, Ryeford, from a seed of
Helianthus annuus, 15 Feb. 2011, S.M. Thompson (T12996A); holotype BRIP
55657a (includes ex-type culture).
Notes ― Cultures of D. goulteri produced a cinnamon col-
oured exudate under the conditions described here. It is not
known if this phenotypic characteristic is taxonomically useful.
A BLASTn search with the ITS sequence showed the closest
match was to HQ44993 from Solidago canadensis in China,
with 99 % identity (2 bp difference).
Diaporthe macintoshii R.G. Shivas, S.M. Thomps. & Y.P. Tan,
sp. nov. — MycoBank MB808670; Fig. 3a–e
Etymology. In recognition of Australian agronomist Paul McIntosh, for his
indefatigable and gregarious service to the Australian sunflower industry over
30 years.
Conidiomata pycnidial, solitary or aggregated in small groups,
scattered, abundant on PDA, OMA and WSA after 4 wk, sub-
globose, up to 0.5 mm diam, ostiolate, necks absent, cream
conidial droplets exuded from some ostioles. Conidiophores
formed from the inner layer of the locular wall, 0–2-septate,
hyaline to subhyaline, cylindrical, 10–20 × 1.5–3.5 μm. Coni-
diogenous cells cylindrical to flexuous, tapered towards the
apex, hyaline, 10–15 × 1.5–2.5 μm. Alpha conidia abundant,
fusiform to oval, narrowed towards apex and base, hyaline,
(6.5–)8 –11(–15) × 2 – 3(–3.5) μm. Beta conidia abundant
amongst the alpha conidia, flexuous to hamate, hyaline, 15–30
× 1.0–1.5 μm. Perithecia not seen.
Cultural characteristics — Colonies on PDA after 10 d reach-
ing the edge of the plate, margin coralloid, adpressed, with
scattered dark stromata up to 1 mm diam, isabelline with low
tufts of off white mycelium; reverse mottled buff to isabelline
with darker patches corresponding to stromata. On OMA cover-
ing entire plate after 10 d, adpressed with some funiculose
mycelium towards the margin, ropey, dark mouse grey, with
numerous scattered dark stromata up to 2 mm diam; reverse
mottled buff with irregular dark patches.
Specimen examined. AustrAliA, Queensland, Toowoomba, from stem
of Rapistrum rugosum, 6 Dec. 2011, S.M. Thompson (T12768A); holotype
BRIP 55064a (includes ex-type culture).
Notes ― Diaporthe macintoshii is one of three novel species
isolated in this study from dead stems of R. rugosum in south-
east Qld. A BLASTn search with the ITS sequence showed the
closest match was to HQ130721 from Warburgia ugandensis,
with 99 % identity (6 bp difference).
Diaporthe masirevicii R.G. Shivas, L. Morin, S.M. Thomps. &
Y.P. Tan, sp. nov. — MycoBank MB808671; Fig. 3f – j
Etymology. Named after the eminent Serbian plant pathologist Stevan
Maširević, a distinguished member of the Yugoslavian research team who
investigated the first outbreaks of D. helianthi and developed many of the
techniques that are currently used to evaluate and screen sunflowers for
resistance to Diaporthe.
Conidiomata pycnidial, very scarce, scattered on PDA, OMA
and WSA after 4 wk, solitary, subglobose, up to 250 μm diam,
ostiolate, without necks, abundant subhyaline to pale yellow
conidial droplets exuded from ostioles. Conidiophores formed
from the inner layer of the locular wall, 1–3-septate, hyaline to
pale yellowish brown, filiform, 20–40 × 1.5 –3.5 μm. Conidio-
genous cells cylindrical to flexuous, tapered towards the apex,
hyaline, 10–25 × 1.5–3.0 μm. Alpha conidia abundant, cylindri-
cal, rounded at the ends, biguttulate, hyaline, (5.5–)6 –7.5(– 8)
× 2– 3 μm. Beta conidia flexuous to hamate, hyaline, 15 – 30 ×
1.0–1.5 μm. Perithecia not seen.
Cultural characteristics — Colonies on PDA covering entire
plate after 10 d, adpressed, with patches of floccose mycelium,
white, sometimes with ropey hazel sectors; reverse similar to
the surface. On OMA covering entire plate after 10 d, white with
abundant funiculose and floccose mycelium; reverse mottled
isabelline.
Specimens examined. AustrAliA, Queensland, Glenore Grove, from the
stem of Helianthus annuus, 15 Aug. 2012, S.M. Thompson (T13228C), holo-
type BRIP 57892a (includes ex-type culture); Gatton, from leaf of Zea mays
31 Jan. 2011, J. McIntosh (T12539D), BRIP 54120c; unknown Queensland,
from leaf of Glycine max, 20 Jan. 2011, S.M. Thompson (T12523A), BRIP
54256; New South Wales, South Bellinger Head State Park, from stem of
Chrysanthemoides monilifera subsp. rotundata, 1 June 2011, L. Morin (019),
BRIP 57330.
Notes — The phylogenetic inference from combined se-
quence data shows D. masirevicii clustered closely with D. en-
dophytica and D. kongii (Fig. 1). Diaporthe masirevicii produced
pycnidia scattered on PDA, OMA and WSA after 4 wk, com-
pared to D. endophytica, which was sterile. Diaporthe masire-
vicii is distinguished from D. endophytica and D. kongii based
on either ITS, TEF or BT sequences. The type of D. masirevicii
was isolated from a lodged crop of sunflower, together with
D. gulyae, which causes sunflower stem canker (Thompson et
al. 2011). Additionally, D. masirevii is one of three novel spe-
cies isolated in this study from dead stems of R. rugosum.
This species was also found on Chrysanthemoides monilifera
subsp. rotundata, which is an important weed of coastal dune
vegetation in eastern Australia.
Diaporthe middletonii R.G. Shivas, L. Morin, S.M. Thomps.
& Y.P. Tan, sp. nov. — MycoBank MB808672; Fig. 3k – p
Etymology. In recognition of Australian plant pathologist Keith Middleton,
for his innovative contributions to plant pathology of summer crops, especially
his early studies of sunflower rust (Puccinia helianthi) and Rhizopus sp.
infection in sunflower.
Conidiomata pycnidial, up to 300 μm diam on PDA and WSA
after 4 wk, aggregated in scattered groups or multilocular on a
50–100 μm thick layer of dark textura angularis with sharp mar-
gins that irregularly covers most of the agar surface on OMA af-
ter 4 wk, subglobose, ostiolate, necks absent or about 200 μm,
cream conidial droplets exuded from a few ostioles. Conidio-
phores formed from the inner layer of the locular wall, reduced
to conidiogenous cells or 1-septate, hyaline to pale yellowish
brown, cylindrical, 10–25 × 1.5–3.5 μm. Conidiogenous cells
cylindrical, hyaline, 5–20 × 1.5–2.5 μm. Alpha conidia abun-
dant, fusiform to cylindrical, rounded at the apex, obconically
truncate at base, mostly biguttulate, hyaline, (5–)6.0 –7.5(–8) ×
2–2.5(– 3) μm. Beta conidia scarce abundant, flexuous, mostly
J-shaped, hyaline, 20–35 × 1.0–1.5 μm. Perithecia not seen.
Cultural characteristics — Colonies on PDA covering entire
plate after 10 d, with scant aerial mycelium and numerous
scattered dark stromata visible as black dots, buff; reverse
similar to the surface. On OMA covering entire plate after 10 d,
with scattered funiculose mycelium up to 1 cm high, surface
mostly leaden black with irregular faintly pale vinaceous patches

46 Persoonia – Volume 35, 2015
towards the edge of the plate; reverse buff. Rosy vinaceous
pigment produced in WA around colonised wheat straw pieces
after 4 wk.
Specimens examined. AustrAliA, Queensland, Gatton, from stem of Rapi-
strum rugosum, 24 Nov. 2011, S.M. Thompson (T12757H), holotype BRIP
54884e (includes ex-type culture); New South Wales, Bongil Bongil National
Park, from stem of Chrysanthemoides monilifera subsp. rotundata, 1 June
2011, L. Morin (056), BRIP 57329.
Notes ― Diaporthe middletonii is one of three novel species
found on R. rugosum, as well as one of three novel species
found on Chrysanthemoides monilifera subsp. rotundata, which
is an important weed of coastal dune vegetation in eastern
Australia. A BLASTn search with the ITS sequence of the type
isolate, BRIP 54884e, showed 100 % match to EF68935 from
Coffea arabica in Hawaii, USA; 99 % identity (3– 5 bp differ-
ence) to EU878434 from Luehea divaricata in Brazil; 99 %
identity to JQ936257 from Glycine max cv. Conquista; and 99 %
identity to KF467129 from Centrolobium ochroxylum in Ecuador.
Diaporthe miriciae R.G. Shivas, S.M. Thomps. & Y.P. Tan, sp.
nov. — MycoBank MB808673; Fig. 3q–u
Etymology. Named after Australian scientist Elizabeth Miric, who first
recognised diversity in the Australian isolates of Diaporthe (Phomopsis) on
sunflower in her PhD thesis entitled: ‘Pathological, morphological and molecu-
lar studies of a worldwide collection of the sunflower pathogens Phomopsis
helianthi and Phoma macdonaldii’ (University of Queensland, 2002).
Conidiomata pycnidial or multilocular, scattered or aggregated
on PDA, OMA and WSA after 4 wk, solitary, ostiolate with necks
up to 1 mm, pale yellow conidial droplets exuded from some
ostioles. Conidiophores formed from the inner layer of the locu-
lar wall, reduced to conidiogenous cells or 1–2-septate, hyaline
to subhyaline, cylindrical to obclavate, 10–20 × 1.5–3 μm.
Conidiogenous cells cylindrical to obclavate, tapered towards
the apex, hyaline, 5–12 × 1.5– 3 μm. Alpha conidia abundant,
fusiform to oval, rounded at the apex, narrowed at the base,
hyaline, 6–7.5(– 9) × 2–2.5(– 3) μm. Beta conidia scattered
or in groups amongst the alpha conidia, flexuous to hamate,
hyaline, 20–35 × 1.0–1.5 μm. Perithecia not seen.
Cultural characteristics — Colonies on PDA covering entire
plate after 10 d, adpressed, with a few scattered dark stromata
up to 2 mm diam, buff; reverse rosy buff. On OMA covering
entire plate after 10 d, ropey with a few scattered funiculose
columns, white tinged with pale vinaceous with irregular pale
mouse grey patches up to several cm diam associated with
stromata; reverse uniformly rosy buff.
Specimens examined. AustrAliA, New South Wales, Premer, from stubble
of Helianthus annuus, 11 Aug. 2011, S.M. Thompson (T12711M), holotype
BRIP 54736j (includes ex-type culture); Queensland, Warra, from Vigna
radiata, 19 Apr. 2012, S.M. Thompson (T13081F), BRIP 56918a; central
Queensland, from stem of Glycine max, 28 Mar. 2012, S.M. Thompson
(T13001C), BRIP 55662c.
Notes ― A BLASTn search with the ITS sequence of the type
isolate, BRIP 54736j, showed 100 % identity to AY148440 from
Gossypium hirsutum in Australia; FJ785447 and FJ785451 from
Glycine max in Mississippi, USA; KJ471541 from Melocactus
ernestii in Brazil; and HF586483 from a strain identified as
D. phaseolorum from a human granulomatous lesion in Brazil,
although the identity of this isolate is doubtful, as the current
precedent (van Rensburg et al. 2006, Mengistu et al. 2007,
Santos et al. 2011, Gomes et al. 2013) is to accept strain CBS
116019 (= ATCC 64802 = FAU458) from Stokesia laevis in Mis-
sissippi, USA, as authentic for the name. Diaporthe miriciae can
be easily differentiated from D. phaseolorum based on either
ITS, TEF or BT loci. Diaporthe miriciae has been found on three
hosts from two families and may be a widespread endophyte or
saprobe in eastern Australia. Diaporthe miriciae also clusters
with D. sojae, a pathogen of Glycine species, which indicates
it may also be a pathogen (Fig. 1).
Diaporthe sackstonii R.G. Shivas, S.M. Thomps. & Y.P. Tan,
sp. nov. — MycoBank MB808674; Fig. 4a–e
Etymology. Named after the eminent Canadian plant pathologist Walde-
mar E. Sackston, for his pioneering contribution to sunflower disease research
on an international scale from the 1950s to the 1990s.
Conidiomata pycnidial or multilocular, solitary, scattered, scarce
on PDA after 4 wk, abundant on OMA after 4 wk on a thin 50 –
100 μm thick layer of dark textura angularis with sharp margins
that irregularly covers much of the agar surface, abundant on
WSA after 4 wk, up to 1 mm diam, ostiolate, necks up to 0.5 mm,
cream conidial droplets exuded from some ostioles. Conidi-
ophores formed from the inner layer of the locular wall, reduced
to conidiogenous cells or septate, filiform, 15–40 × 1.5–3 μm,
hyaline to pale yellowish brown. Conidiogenous cells cylindri-
cal to lageniform, tapered towards the apex, hyaline, 10–15 ×
1.5–3.0 μm. Alpha conidia abundant, fusiform, rounded at the
apex, obconically truncate at base, hyaline, 6–7(–8) × 2–2.5
μm. Beta conidia not seen. Perithecia not seen.
Cultural characteristics — Colonies on PDA covering entire
plate after 10 d, adpressed, with a few scattered dark stromata
up to 1 mm diam surrounded by patches of white sparse myce-
lium, buff; reverse isabelline with a few dark scattered stromata
up to 3 mm diam. On OMA covering entire plate after 10 d, white
tinged with pale vinaceous with pale mouse grey patches, with
many scattered dark stromata mostly up to 4 mm diam; reverse
uniformly cinnamon.
Specimen examined. AustrAliA, Queensland, Clermont, from a petiole
of Helianthus annuus, 10 June 2011, S.M. Thompson (T12667B); holotype
BRIP 54669b (includes ex-type culture).
Notes ― The phylogenetic inference from the combined se-
quence data showed D. sackstonii clustered next to D. infe-
cunda (Gomes et al. 2013), as well as the newly described
D. serafiniae. In culture, D. sackstonii produced abundant pyc-
nidia on PDA and OMA, compared to D. infecunda, which was
sterile. Diaporthe sackstonii differs from D. serafiniae in three
loci: ITS positions 40 (C), 78 (C) and 85 (G); TEF 91 % match
(Identities 263/290, Gaps 8 / 290); BT 98 % match (Identities
635/649, Gaps 3/649).
Diaporthe serafiniae R.G. Shivas, S.M. Thomps. & Y.P. Tan,
sp. nov. — MycoBank MB808675; Fig. 4f–j
Etymology. Named after the dedicated Australian agronomist Loretta
Serafin, for her research on sunflower crop production and who provided
the samples from which this species was isolated.
Conidiomata multilocular, scattered, abundant on PDA, OMA
and WSA after 4 wk, up to 2 mm diam, ostiolate, with necks up
to 1.5 mm, cream conidial droplets exuded from most ostioles.
Conidiophores formed from the inner layer of the locular wall,
1-septate, hyaline to pale yellowish brown, fusiform, 15–25
× 1.5 –3.5 μm. Conidiogenous cells cylindrical to flexuous,
tapered towards the apex, hyaline, 5–20 × 1.5– 2.5 μm. Alpha
conidia abundant, fusiform, rounded at the apex, narrowed to-
wards the base, biguttulate, hyaline, 5.5–7(–8) × 1.5–2.5 (– 3)
μm. Beta conidia not seen. Perithecia not seen.
Cultural characteristics — Colonies on PDA covering entire
plate after 10 d, adpressed, white numerous scattered dark
stromata up to 2 mm diam; reverse uniformly mottled white to
buff. On OMA covering entire plate after 10 d, adpressed, with
numerous scattered dark stromata up to 4 mm diam; reverse
uniformly isabelline.

47
S.M. Thompson et al.: Diaporthe species in Australia
Specimens examined. AustrAliA, Queensland, Glenore Grove, from seed
of an ornamental variety of Helianthus annuus, 1 Apr. 2012, S.M. Thompson
(T13010A), holotype BRIP 55665b (includes ex-type culture); New South
Wales, from stem of Lupinus albus ‘Rosetta’, L. Serafin (T12568A), BRIP
54136.
Notes ― The phylogenetic inference from the combined se-
quence data showed D. serafiniae clustered close to D. infe-
cunda (Gomes et al. 2013) (Fig. 1). In culture, D. serafiniae
produced abundant pycnidia on PDA and OMA, compared to
D. infecunda, which was sterile.
DISCUSSION
The application of principles of genealogical concordance
species concepts based on multigene phylogenetic analysis
has led, in recent years, to the discovery of many new cryptic
species in some important genera of plant pathogenic fungi,
e.g. Colletotrichum (Damm et al. 2012a, b, Weir et al. 2012),
Phyllosticta (Wikee et al. 2013) and Diaporthe (Gomes et al.
2013, Tan et al. 2013). There are about 2 000 names for Dia-
porthe (including Phomopsis) species in the literature (Gomes
et al. 2013). Many epitypes have been recently designated
for species of Diaporthe (Udayanga et al. 2012, Gomes et
al. 2013), which has helped to stabilise the taxonomy of this
genus. However, many Diaporthe species still lack ex-type
(including epitype and neotype) cultures from which DNA is
easily extracted for molecular phylogenetic analysis. Gomes et
al. (2013) proposed two approaches to resolve the taxonomy
of Diaporthe species – either recollect and redescribe all the
existing species (which is impractical) or start again. A new
start is not as daunting as it seems as the nomenclatural code
that governs the naming of fungi has a tool that facilitates this
approach in provision for lists of rejected as well as protected
names (McNeill et al. 2012). In reality, plant pathologists and
mycologists seem to have embraced a new start, as since 2010
there have been approximately 40 new species of Diaporthe
described (see MycoBank, www.mycobank.org), including 12
from Australia (Thompson et al. 2011, Crous et al. 2011, 2012,
Tan et al. 2013).
Colonisation of the same host plant by multiple Diaporthe
species has been reported before (Farr et al. 2002, Crous &
Groenewald 2005, van Niekerk et al. 2005, Thompson et al.
2011) and appears to be quite common in nature. Five of our
new species were isolated from live sunflower stems. Of these
five species, D. masirevicii and D. miriciae were also associ-
ated with cankers on live soybean and mungbean plants. Some
new species appeared to be endophytic such as the species
found on asymptomatic live maize plants and some may play
a role in the dieback disease of bitou bush and tip dieback
symptoms on hosts such as Sesbania cannabina and Bidens
pilosa. Another group, which includes D. charlesworthi and
D. macintoshii, may be primarily saprophytic, having only been
isolated from decaying plant material. Detailed investigations
of the pathogenicity and host range of all species are required
to shed light on their ecology.
The presence of D. goulteri, D. masirevicii, D. miriciae and
D. serafiniae in live crops as well as crop stubble and weed
residues, highlights the potential of decaying plant material on
the soil surface to act as a reservoir of inoculum for subsequent
crops. It is well recognised that crop stubble aids the survival
of Diaporthe species, such as D. toxica on lupins (Cowling et
al. 1987), D. phaseolorum var. caulivora on soybeans (Kmetz
et al. 1979), and D. helianthi on sunflower (Maširević & Gulya
1992). The role of broadleaf weed residues as an aid to survival
Fig. 4 Diaporthe spp. — a– e: Diaporthe sackstonii (ex-type BRIP 54669b) after 4 wk. a. Culture on OMA; b. conidiomata on sterilised wheat straw;
c. conidio mata on OMA; d. conidiophores; e. alpha conidia. — f– j: Diaporthe serafiniae (ex-type BRIP 55665b) after 4 wk. f. Culture on PDA; g. conidiomata
on sterilised wheat straw; h. conidiomata on OMA; i. conidiophores; j. alpha conidia. –– Scale bars: a, f = 1 cm; b, g, h = 1 mm; c = 100 µm; d, e, i, j = 10 µm.

48 Persoonia – Volume 35, 2015
is not well documented for many pathogenic fungal species.
Our results indicate that dead weeds at the edges of cultivated
fields and waterways as well as unburied weed residues, on
the soil surface and amongst crop plants in low tillage systems,
create a ‘brown bridge’ of dead plant material that may harbour
multiple pathogenic, saprobic or endophytic species of Dia-
porthe. We suggest that the ‘brown bridge’ of weed residues
plays as significant a role in aiding the survival of Diaporthe
species. This is comparable to the ‘green bridge’ of alternative
live weed hosts, such as those that facilitate survival of patho-
genic Diaporthe species between cropping phases (Mihaljcevic
& Muntañola-Cvetković 1985, Roy et al. 1997, Li et al. 2001,
2010, Vrandečić et al. 2010).
Of added significance for disease management is the isolation
from maize of D. gulyae, a highly virulent pathogen on sunflower
(Thompson et al. 2011). Diaporthe gulyae was isolated from
asymptomatic maize plants, indicating endophytic colonisa-
tion. Maize is often recommended as a rotation crop to follow
broadleaf crops such as sunflower, soybean and mungbean,
which are susceptible to a number of damaging stem and pod
cankers caused by Diaporthe species. More sampling of maize
is required to confirm its possible role in the epidemiology of
Diaporthe species that are pathogens of broadleaf rotational
crop species. These findings support the observation by Delaye
et al. (2013) and Malcolm et al. (2013) that the complex infection
and survival associations between fungi and plants, including
endophytic associations are poorly known.
Two species of Diaporthe isolated from sunflower, D. kongii
(Thompson et al. 2011) and D. masirevicii, were also recovered
from bitou bush, which is invasive in coastal dune vegetation
(Vranjic et al. 2012) away from the inland broad acre crop-
ping regions in Qld and NSW. This provides evidence that the
distribution, life style and host range of many Diaporthe spe-
cies may be broader than expected and more complex than
currently known. Both sunflower and bitou bush belong to the
Asteraceae, and whether this is significant with respect to the
possible hosts and distribution of these fungi is not known.
There have been 20 species, including those from this study, of
Diaporthe described from Australia since 2010 (Thompson et al.
2011, Crous et al. 2011, 2012, Tan et al. 2013). Some have been
identified as significant plant pathogens although the ecological
significance of most is not known. This study starts to address
the case that Hyde et al. (2010) made to reassess and revise
plant-associated pathogens, especially Diaporthe, in order to
preserve the effective role that biosecurity agencies play in
keeping unwanted plant pathogens out of Australia. Although
the host range and pathogenicity of these eight newly described
Diaporthe species is largely unknown, our study highlights the
importance of both ‘green bridges’ and ‘brown bridges’ in the
epidemiology of Diaporthe species.
Acknowledgements Broad acre component: The authors would like to
acknowledge the Queensland Department of Agriculture, Fisheries and
Forestry (DAFFQ), the Grains Research and Development Corporation
(GRDC), the University of Queensland (UQ), as well as the generous as-
sistance of growers and advisors. Additionally, we acknowledge Drs Tom
Gulya (USDA-ARS), Alistair McTaggart (UQ), Vu Tuan Nguyen (DAFFQ),
Malcolm Ryley (DAFFQ), and Ms Ella Trembizki (DAFFQ) for their technical
and philosophical support of this study. Bitou bush component: This study
was supported by CSIRO, NSW National Parks and Wildlife Service, and the
Australian Government National Weeds and Productivity Research Program
administered by the Rural Industries Research and Development Corpora-
tion. We thank Mr Shamsul Hoque (CSIRO Plant Industry) and Ms Ruth
Aveyard (CSIRO Ecosystem Sciences) for technical assistance, and also
acknowledge the range of stakeholders for their support and/or permission
to collect samples on their land.
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