Calonectria spp. causing leaf spot, crown and root rot of ... - Persoonia

Persoonia 27, 2011: 73–79 

www.ingentaconnect.com/content/nhn/pimj 

RESEARCH ARTICLE 

http://dx.doi.org/10.3767/003158511X615086 

Calonectria spp. causing leaf spot, crown and root rot of 

ornamental plants in Tunisia 

L. Lombard 1 , G. Polizzi 2* , V. Guarnaccia 2 , A. Vitale 2 , P.W. Crous 1 

Key words 

Calonectria 

crown and root rot 

DNA phylogeny 

leaf spot 

pathogenicity 

systematics 

Abstract Calonectria spp. are important pathogens of ornamental plants in nurseries, especially in the Northern 

Hemisphere. They are commonly associated with a wide range of disease symptoms of roots, leaves and shoots. 

During a recent survey in Tunisia, a number of Calonectria spp. were isolated from tissues of ornamental plants 

showing symptoms of leaf spot, crown and root rot. The aim of this study was to identify these Calonectria spp. using 

morphological and DNA sequence comparisons. Two previously undescribed Calonectria spp., C. pseudomexicana 

sp. nov. and C. tunisiana sp. nov., were recognised. Calonectria mexicana and C. polizzii are newly reported for the 

African continent. Pathogenicity tests with all four Calonectria spp. showed that they are able to cause disease on 

seedlings of Callistemon spp., Dodonaea viscosa, Metrosideros spp. and Myrtus communis. 

Article info Received: 5 September 2011; Accepted: 15 October 2011; Published: 18 November 2011. 

INTRODUCTION 

Species of Calonectria are common pathogens of a wide 

range of plant hosts in nurseries cultivated through seedings 

or vegetative propagation (Crous 2002, Lombard et al. 2010a). 

Nursery disease symptoms associated with these fungi include 

crown, collar and root rot, leaf spots and cutting rot (Crous 2002, 

Vitale & Polizzi 2008, Polizzi et al. 2009, Lombard et al. 2010a). 

Calonectria spp. have been reported worldwide from agricultural 

and forestry nurseries (Crous et al. 1991, Crous 2002, Lombard 

et al. 2009, 2010a, b, d), whereas in Europe, they have only 

been reported from commercial ornamental nurseries (Polizzi 

& Crous 1999, Polizzi 2000, Crous 2002, Henricot & Culham 

2002, Polizzi et al. 2007a, b, Vitale & Polizzi 2008, Polizzi et 

al. 2009, Lombard et al. 2010a). 

Past reports have shown that C. morganii and C. pauciramosa 

are the most common Calonectria spp. found in ornamental 

nurseries in the Northern Hemisphere (Polizzi & Crous 1999, 

Polizzi 2000, Polizzi & Catara 2001, Polizzi et al. 2006a, b, 

2007a, b). Based on phylogenetic studies, C. morganii appears 

to be restricted to Brazil, Europe and the USA (Crous et al. 1993, 

Overmeyer et al. 1996, Schoch et al. 2000), whereas C. pauciramosa 

has a more global distribution and has been shown 

to better adapt to different environmental conditions (Crous 

2002, Lombard et al. 2010b, Chen et al. 2011). Calonectria 

pauciramosa was also regarded as the dominant pathogen in 

nurseries in Australia and South Africa (Crous 2002, Schoch 

et al. 2001, Lombard et al. 2010b). 

In November 2010, a survey was conducted in an ornamental 

nursery in Carthage, Tunis, Tunisia. Various plant species were 

collected showing symptoms of leaf spots, crown and root rot. 

Isolations consistently yielded Calonectria spp. and the aim 

of this study was to identify these species, and confirm their 

pathogenicity. 

1 

Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Uppsalalaan 

8, 3584 CT Utrecht, the Netherlands 

2 

Dipartimento di Gestione dei Sistemi Agroalimentari e Ambientali, sezione di 

Patologia Vegetale, University of Catania, Via S. Sofia 100, 95123 Catania, 

Italy; corresponding author e-mail: gpolizzi@unict.it. 

MATERIALS AND METHODS 

Disease survey and isolates 

During November 2010, an ornamental nursery located in 

Carthage, Tunis, Tunisia was surveyed for diseased plants. 

Several samples of Callistemon spp., Dodonaea viscosa, 

Myrtus communis and Metrosideros spp. showing leaf spots, 

crown and root rot were randomly collected for analysis (Fig. 1, 

Table 1). Infected tissues collected from symptomatic plants 

were superficially disinfected with 1.0 % sodium hypochlorite 

for 2 min, rinsed with sterile water, placed on potato-dextrose 

agar (PDA, Oxoid) and incubated in the dark at 24 °C. Representative 

isolates of Calonectria from each ornamental species 

were obtained from single-spore colonies made from 14 d old 

cultures grown on PDA. Representative isolates have been 

deposited at the CBS-KNAW Fungal Biodiversity Centre (CBS), 

Utrecht, The Netherlands (Table 1). 

DNA sequence comparisons 

Total genomic DNA was extracted from single-conidial isolates 

grown on 2 % malt extract agar (MEA) for 7 d, using the Ultra 

Clean Microbial DNA isolation kits (Mo Bio Laboratories, 

Inc., California, USA) according to the manufacturer’s protocol. 

Partial gene sequences were determined for β-tubulin (BT), 

histone H3 (HIS3) and translation elongation factor-1α (TEF- 

1α) using the primers and protocols described by Lombard et 

al. (2010c). 

To ensure the integrity of the sequences, the amplicons were 

sequenced in both directions using the same primer pairs used 

for amplification. Sequence data from Lombard et al. (2010b, d) 

were used as reference data and subsequent alignments were 

generated using MAFFT v. 6 (Katoh & Toh 2010) and manually 

corrected where necessary. 

Congruency of the sequence datasets for the separate loci was 

determined using tree topologies of 70 % reciprocal Neighbour- 

Joining bootstrap trees with Maximum Likelihood distances that 

were compared visually to identify conflicts between partitions 

(Gueidan et al. 2007). Molecular evolution models for the 

separate gene regions were determined in Modeltest v. 3.7 

© 2011 Nationaal Herbarium Nederland & Centraalbureau voor Schimmelcultures 

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74 Persoonia – Volume 27, 2011 

a 

b 

d 

e 

c 

f 

g h i 

Fig. 1 Symptoms of leaf spot caused by Calonectria spp. on several ornamental plants. a. Myrtus communis; b. Metrosideros thomasii; c. Callistemon sp.; 

d. Callistemon sp.; e. Dodonaea viscosa; f. Callistemon viminalis; g. Metrosideros excelsa; h. Metrosideros excelsa cv. Aurea; i. Metrosideros sp. 

(Posada & Crandall 1998) and bootstrap analyses were run 

for 10 000 replicates. 

PAUP (Phylogenetic Analysis Using Parsimony, v. 4.0b10, 

Swofford 2002) was used to analyse the DNA sequence datasets. 

Phylogenetic relationships were estimated by heuristic 

searches with 1 000 random addition sequences and tree bisection-reconnection 

was used, with the branch swapping option 

set on ‘best trees’ only. All characters were weighted equally 

and alignment gaps were treated as missing data. Measures 

calculated for parsimony included tree length (TL), consistency 

index (CI), retention index (RI) and rescaled consistence 

index (RC). Bootstrap analysis (Hillis & Bull 1993) was based 

on 1 000 replications. 

A second phylogenetic analysis using a Markov Chain Monte 

Carlo (MCMC) algorithm was done to generate trees with Bayesian 

probabilities in MrBayes v. 3.1.1 (Ronquist & Huelsenbeck 

2003). Nucleotide substitution models were determined using 

MrModeltest (Nylander 2004) for each gene region and included 

in the analyses. Two analyses of four MCMC chains were run 

from random trees for one million generations and sampled 

every 100 generations. All runs converged on the same likelihood 

score and tree topology and therefore the first 800 trees 

were discarded as the burn-in phase of each analysis and 

posterior probabilities determined from the remaining trees. 

The phylogenetic analyses included 46 partial gene sequences 

for each gene region, representing 20 Calonectria spp. (Table 

1). Calonectria colombiensis (CBS 112221) and C. chinensis 

(CBS 112744) were used as outgroup taxa in both analyses 

(Lombard et al. 2009). All novel sequences were deposited 

in GenBank and the alignments in TreeBASE (http://www. 

treebase.org). 

Taxonomy 

Morphological characterisation of the Calonectria isolates was 

done using single conidial cultures prepared on MEA and synthetic 

nutrient-poor agar (SNA; Nirenburg 1981, Lombard et al. 

2009). Inoculated plates were incubated at room temperature 

and examined after 7 d. Gross morphological characteristics 

of the anamorph state were determined by mounting fungal 

structures in clear lactic acid and 30 measurements at ×1 000 

magnification were made for each isolate using a Zeiss Axioscope 

2 microscope with interference contrast (DIC) illumination. 

The 95 % confidence levels were determined and extremes 

of conidial measurements are given in parentheses. For other 

structures, only extremes are presented. Colony characteristics 

were noted after 7 d of growth on MEA at 24 °C and colony 

colours determined using the colour charts of Rayner (1970). 

Descriptions, nomenclature and illustrations were deposited in 

MycoBank (Crous et al. 2004). 

Pathogenicity 

In order to test the pathogenicity of the Calonectria spp. 

collected in this study, seven isolates representing different 

Calonectria species identified by morphology and DNA sequence 

comparisons were selected for inoculation trials (Table 

1). A conidial suspension (1.0 × 10 5 conidia/mL) was prepared 

for each isolate by adding sterile water to plates of carnation leaf 

agar (CLA; Fisher et al. 1982) 7 d after inoculation and dislodging 

the conidia. The conidial suspension was sprayed onto the 

canopy (until run-off) of potted 2–6 mo old plants of Callistemon 

citrinus cv. Splendens, C. laevis, C. viminalis, Dodonaea viscose, 

Metrosideros excelsa, M. excelsa cv. Aurea, M. thomasii, 

Myrtus communis, M. communis subsp. tarentina. The conidial 

suspension of the isolate CBS 130351 was also applied to the 

crown of M. communis plants (10 mL/plant). All plants were 

subsequently covered with plastic bags for 48 h and maintained

L. Lombard et al.: Calonectria in Tunisia 

75 

Table 1 Calonectria isolates used in the phylogenetic analyses and pathogenicity trials. 

Species Isolate number 1 β-tubulin 2 Histone H3 2 TEF-1α 2 Host /substrate 

Calonectria brasiliensis CBS 230.30 4 GQ267241 GQ267259 GQ267328 Eucalyptus sp. 

CBS 114257 GQ267242 GQ267260 GQ267329 Leaf litter 

C. cerciana CBS 123693 4 FJ918510 FJ918528 FJ918559 E. grandis × urophylla 

CBS 123695 FJ918511 FJ918529 FJ918560 E. grandis × urophylla 

C. chinensis CBS 112744 AY725618 AY725660 AY725709 Soil 

C. colombiana CBS 115127 4 FJ972423 FJ972442 FJ972492 Soil 

CBS 115638 FJ972422 FJ972441 FJ972491 Soil 

C. colombiensis CBS 112220 4 GQ267207 AY725662 AY725711 Soil 

C. hawksworthii CBS 111870 4 AF333407 DQ190649 FJ918558 Nelumbo nucifera 

C. insularis CBS 114558 4 AF210861 FJ918526 FJ918556 Soil 

CBS 114559 AF210862 FJ918525 FJ918555 Soil 

C. leucothoës CBS 109166 FJ918508 FJ918523 FJ918553 Leucothoë axillaris 

C. mexicana CBS 110918 4 AF210863 FJ972460 FJ972526 Soil 

CBS 130353 3 JN607280 JN607265 JN607295 Dodonaea viscosa 

C. morganii CBS 110666 FJ918509 FJ918527 FJ918557 Ilex vomitoria 

CBS 119669 DQ521599 DQ521601 GQ421796 Pistacia lentiscus 

C. pauciramosa CPC 971 FJ918514 FJ918531 FJ918565 E. grandis 

CPC 416 FJ918515 FJ918532 FJ918566 E. grandis 

C. polizzii CBS 123402 4 FJ972419 FJ972438 FJ972488 Arbutus unedo 

CBS 125270 FJ972417 FJ972436 FJ972486 Callistemon citrinus 

CBS 130351 3 JN607270 JN607255 JN607285 Myrtus communis 

CBS 130352 3 JN607275 JN607260 JN607290 Metrosideros thomasii 

DISTEF-TMC2 JN607269 JN607254 JN607284 Myrtus communis 

DISTEF-TMEA1 JN607272 JN607257 JN607287 Metrosideros excelsa cv. Aurea 

DISTEF-TMN3 JN607274 JN607259 JN607289 Metrosideros sp. 

C. pseudomexicana CBS 130354 3,4 JN607281 JN607266 JN607496 Callistemon sp. (rouge) 

CBS 130355 3 JN607282 JN607267 JN607497 Callistemon sp. (rouge) 

DISTEF-TCROU4 JN607283 JN607268 JN607498 Callistemon sp. (rouge) 

C. pseudoscoparia CBS 125256 GQ267228 GQ267277 GQ267348 E. grandis 

CBS 125257 4 GQ267229 GQ267278 GQ267349 E. grandis 

C. scoparia CPC 1675 FJ972426 FJ972476 FJ972525 Eucalyptus sp. 

CPC 1679 FJ972427 GQ267246 GQ267298 Eucalyptus sp. 

C. spathulata CBS 112689 AF308463 FJ918524 FJ918554 E. viminalis 

CBS 555.92 4 GQ267215 GQ267261 GQ267331 Araucaria angustifolia 

C. sulawesiensis CBS 125248 GQ267223 GQ267272 GQ267343 Eucalyptus sp. 

CBS 125253 GQ267220 GQ267269 GQ267340 Eucalyptus sp. 

C. tunisiana CBS 130356 3 JN607277 JN607262 JN607292 Callistemon sp. (rouge) 

CBS 130357 3,4 JN607276 JN607261 JN607291 Callistemon laevis 

DISTEF-TCV1 JN607278 JN607263 JN607293 Callistemon viminalis 

DISTEF-TCROS4 JN607279 JN607264 JN607294 Callistemon sp. (rosè) 

DISTEF-TME1 JN607271 JN607256 JN607286 Metrosideros excelsa 

DISTEF-TMN1 JN607273 JN607258 JN607288 Metrosideros sp. 

C. variabilis CBS 112691 GQ267240 GQ267264 GQ267335 Eucalyptus sp. 

CBS 114677 AF333424 GQ267263 GQ267334 Eucalyptus sp. 

C. zuluensis CBS 125268 4 FJ972414 FJ972433 FJ972483 Eucalyptus sp. 

CBS 125272 FJ972415 FJ972434 FJ972484 Eucalyptus sp. 

1 

CBS: CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; CPC: Pedro Crous working collection housed at CBS; DISTEF: Dipartimento di Scienze e Tecnologie Fitosanitarie, 

Catania, Italy. 

2 

GenBank accession numbers. 

3 

Isolates used for the pathogenicity trials. 

4 

Ex-type cultures; Isolates in bold obtained during survey. 

in a growth chamber at 25 ± 1 °C for 14 d. Five plants for each 

isolate and host were used and the same number of control 

plants were treated using sterile water. Pathogenicity tests were 

evaluated 5, 10 and 25 d after inoculation. 

Results 

Disease survey and isolates 

During the survey, a total of 46 Calonectria isolates were collected 

from ornamental hosts sampled. Majority of the isolates 

(41) were associated with leaf spots or leaf blight of Callistemon 

spp. (18), D. viscosa (1), Metrosideros spp. (17) and Myrtus 

communis (5), and the remaining (5) with crown and root rot 

of M. communis. Leaves showed minute brown spots, which 

often enlarged, forming a necrotic centre surrounded by a dark 

purple halo (Fig. 1). Young, non-lignified terminal shoots often 

exhibited dieback or lesions similar to those on the leaves. Severe 

defoliation was observed on M. communis and M. excelsa 

cv. Aurea. Several seedlings of M. communis had crown and 

root rot, and fungal sporulation occurred on the lower part of 

the crown. Initial symptoms were brown lesions that expanded 

rapidly to girdle the stem at the seedling crown, above and 

below the soil line, resulting in plant death. 

DNA sequence comparisons 

Amplicons of approximately 450 bases for HIS3 and 500 bases 

each for BT and TEF-1α were generated. The 70 % reciprocal 

bootstrap trees showed no conflict in tree topologies for the 

three gene regions and therefore they were combined in a dataset 

consisting of 1 532 characters including gaps. Of these 

characters, 1 187 were constant and parsimony uninformative. 

Analysis of the 345 parsimony informative characters yielded 

16 equally most parsimonious trees (TL = 814, CI = 0.721, 

RI = 0.923, RC = 0.666), of which the first tree is presented (Fig. 

2). A HKY+I model for BT, a GTR+I+G model for HIS3 and a 

GTR+G model for TEF-1α was selected for Bayesian analysis. 

The Bayesian consensus tree confirmed the tree topology obtained 

with maximum parsimony including bootstrap support. 

The phylogenetic tree illustrates a number of well-supported 

clades containing the Calonectria isolates obtained during the 

survey. Some of the isolates clustered in a clade representing 

C. polizzii with a bootstrap value (BP) of 97 and a Bayesian 

posterior probability (PP) value of 1.00. Several isolates also 

grouped with and close to C. mexicana in two separate wellsupported 

clades (BP = 68, PP = 0.95 and BP = 78, PP = 0.98, 

respectively), which could represent novel phylogenetic species.

76 78/1.00 DISTEF-TCV1 

C. tunisiana sp. nov Persoonia – Volume 27, 2011 

DISTEF-TCROS4 

100/1.00 

100/1.00 

Fig. 2 One of 16 most parsimonious trees obtained from a heuristic search 

with 1 000 random addition sequences of the combined sequences of β-tubulin, 

histone H3 and translation elongation factor 1α sequence alignments 

of the Calonectria isolates obtained during the survey and other closely 

related species. Scale bar shows 10 changes. Bootstrap support values (in 

bold) and Bayesian posterior probability values are shown at the nodes. 

Thickened lines indicate branches in the strict consensus tree and the consensus 

tree of the Bayesian analyses. The tree was rooted to C. chinensis 

(CBS 112744) and C. colombiensis (CBS 112220). Isolates in bold were 

10 changes 

obtained during the survey. 

72/0.99 

68/0.88 

63/0.98 

DISTEF-TME1 

CBS 130357 

C. colombiensis CBS 112220 

C. chinensis CBS 112744 

CBS 110918 

C. leucothoes CBS 109166 

73/0.69 

C. mexicana C. hawksworthii CBS 111870 

52/1.00 

CBS 130353 

CBS 130355 100/1.00 

CBS 230.51 

C. brasiliensis 

100/1.00 

99/1.00CBS 13035479/0.76 

CBS 114257 

C. pseudomexicana sp. nov 

68/0.95 

99/1.00 CBS 125248 

72/0.99 

DISTEF-TCROU4 

C. sulawesiensis 

CBS 125253 

68/0.88 CBS 130356 82/1.00 

CBS 114677 

DISTEF-TMN1 

C. variabilis 

78/1.00 

63/0.98 

100/1.00 

CBS 112691 

DISTEF-TCV1 

C. tunisiana sp. nov 

DISTEF-TCROS4 53/0.73 CBS 110666 

C. morganii 

98/1.00 

DISTEF-TME1 

CBS 130357 

CBS 119669 

CBS 114558 

C. insularis 

100/1.00 

CBS 114559 

C. leucothoes CBS 109166 

C. 100/1.00 

CBS 123693 

hawksworthii CBS 111870 C. cerciana 

52/1.00 

CBS 123695 

100/1.00 

CBS 230.51 

CPC 1675 

C. brasiliensis 

99/1.00 79/0.76 C. scopariaCBS 114257 

100/1.00 CPC 1679 

99/1.00 CBS 125248 

CBS 125257 

C. sulawesiensis 

68/0.98 

C. pseudoscoparia CBS 125253 

CBS 125256 82/1.00 

CBS 114677 

98/1.00 

C. variabilis 

100/1.00 

CBS 115127 

100/1.00 C. colombiana CBS 112691 

50/0.98 CBS 115638 

53/0.73 CBS 110666 

C. morganii 

100/1.00 CBS 112689 

98/1.00 

CBS 119669 C. spathulata 

CBS 555.92 

CBS 114558 

C. insularis 

94/1.00 CPC 971 

100/1.00 C. pauciramosa CBS 114559 

CPC 416 

100/1.00 

CBS 123693 

CBS 125268 

C. cerciana 

98/0.97 

C. zuluensis 

CBS 123695 

63/1.00 

CBS 125272 

CPC 1675 

100/1.00 

CBS 123402 C. scoparia 

CPC 1679 

CBS 125270 

CBS 125257 

68/0.98 DISTEF-TMC2 C. pseudoscoparia 

CBS 125256 

98/1.00 

CBS 130351 C. polizzii 

97/1.00 CBS 115127 

100/1.00 

DISTEF-TMEA1 C. colombiana 

50/0.98 CBS 115638 

DISTEF-TMN3 

100/1.00 CBS 112689 

10 changes 

CBS 130352 

CBS 555.92 

C. spathulata 

98/0.97 

63/1.00 

DISTEF-TCROU4 

CBS 130356 

DISTEF-TMN1 

94/1.00 CPC 971 

97/1.00 

CPC 416 

CBS 125268 

CBS 125272 

CBS 123402 

CBS 125270 

DISTEF-TMC2 

CBS 130351 

DISTEF-TMEA1 

DISTEF-TMN3 

CBS 130352 

C. pauciramosa 

C. zuluensis 

C. polizzii 

Taxonomy 

DNA sequence and morphological comparisons of the Calonectria 

isolates obtained during the survey show that these isolates 

belong to C. mexicana and C. polizzii and also constitute 

two previously undescribed taxa. Based on morphological 

comparisons, isolate CBS 130353 agrees with C. mexicana 

(Schoch et al. 1999) and isolates DISTEF-TMC2, CBS 130351, 

DISTEF-TMEA1, DISTEF-TMN3 and CBS 130352 represent 

C. polizzii (Lombard et al. 2010b). The remaining isolates are 

newly described as follows: 

Calonectria pseudomexicana L. Lombard, G. Polizzi & Crous, 

sp. nov. — MycoBank MB563138; Fig. 3 

Teleomorph unknown. 

Calonectria mexicana morphologice similes sed minus ramis conidiophorae. 

Etymology. Name reflects the fact that this species closely resembles 

C. mexicana. 

Conidiophores with a stipe bearing penicillate suites of fertile 

branches, stipe extensions, and terminal vesicles; stipe septate, 

hyaline, smooth, 38–69 × 5–9 µm; stipe extensions septate, 

straight to flexuous, 175–251 µm long, 3–6 µm wide at the apical 

septum, terminating in a fusiform to broadly ellipsoidal vesicle


77 

a 

b 

c 

d 

e 

f 

g 

Fig. 3 Calonectria pseudomexicana. a, b. Macroconidiophores; c–e. fusiform to broadly ellipsoidal vesicles with papillate apices; f, g. conidiogenous apparatus 

with conidiophore branches and doliiform to reniform phialides; h. 1-septate macroconidia. — Scale bars = 10 μm. 

h 

9–14 µm diam with papillate apex. Conidiogenous apparatus 

38–68 µm long, 32–64 µm wide; primary branches aseptate 

or 1-septate, 21–43 × 4–7 µm; secondary branches aseptate, 

13–26 × 4–7 µm; tertiary branches and additional branches 

(–4), aseptate, 10–18 × 2–6 µm, each terminal branch producing 

2–6 phialides; phialides doliiform to reniform, hyaline, 

aseptate, 6–14 × 2–6 µm; apex with minute periclinal thickening 

and inconspicuous collarette. Conidia cylindrical, rounded 

at both ends, straight, (40–)43–48(–49) × (4–)5–6 µm (av. = 

45 × 5 µm), 1-septate, lacking a visible abscission scar, held 

in parallel cylindrical clusters by colourless slime. Megaconidia 

and microconidia not seen. 

Culture characteristics — Colonies fast growing at 24 ºC on 

MEA, sienna to bay on surface, reverse sienna after 7 d; moderate 

white aerial mycelium with sparse to moderate sporulation; 

chlamydospores extensive throughout medium. 

Specimens examined. Tunisia, Carthage, Tunis, from Callistemon sp., 

Nov. 2010, G. Polizzi, (CBS H-20685, holotype of C. pseudomexicana, 

culture ex-type CBS 130354 = DISTEF-TCROU1); Carthage, Tunis, from 

Callistemon sp., Nov. 2010, G. Polizzi, culture CBS 130355 = DISTEF- 

TCROU3; Carthage, Tunis, from Callistemon sp., Nov. 2010, G. Polizzi, 

culture DISTEF-TCROU4. 

Notes — Calonectria pseudomexicana is morphologically 

similar to C. mexicana. Calonectria pseudomexicana has four 

or less conidiophore branches while C. mexicana has five as 

reported by Schoch et al. (1999). 

Calonectria tunisiana L. Lombard, G. Polizzi & Crous, sp. 

nov. — MycoBank MB563139; Fig. 4 

Teleomorph unknown. 

Calonectria mexicana morphologice similes sed minus conidiophorae ramis 

et fructibus breviores sunt stipe augue. 

Etymology. Name refers to the country Tunisia, where the fungus was 

collected. 

Conidiophores with a stipe bearing penicillate suites of fertile 

branches, stipe extensions, and terminal vesicles; stipe septate, 

hyaline, smooth, 42–95 × 7–11 µm; stipe extensions septate, 

straight to flexuous, 147–199 µm long, 4–5 µm wide at the 

apical septum, terminating in a fusiform to broadly ellipsoidal 

vesicle 8–14 µm diam with papillate apex. Conidiogenous apparatus 

40–68 µm long, 30–66 µm wide; primary branches 

aseptate or 1-septate, 17–41 × 5–7 µm; secondary branches 

aseptate, 10–22 × 4–7 µm; tertiary branches aseptate, 9–18 

× 4–5 µm, each terminal branch producing 2–6 phialides; 

phialides doliiform to reniform, hyaline, aseptate, 8–13 × 3–5 

µm; apex with minute periclinal thickening and inconspicuous 

collarette. Conidia cylindrical, rounded at both ends, straight,

78 Persoonia – Volume 27, 2011 

a 

b 

c 

d 

e 

f 

g 

h 

Fig. 4 Calonectria tunisiana. a, b. Macroconidiophores; c–e. fusiform to broadly ellipsoidal vesicles with papillate apices; f, g. conidiogenous apparatus with 

conidiophore branches and doliiform to reniform phialides; h. 1-septate macroconidia. — Scale bars = 10 μm. 

(43–)47–51(–53) × 4–6 µm (av. = 49 × 5 µm), 1-septate, lacking 

a visible abscission scar, held in parallel cylindrical clusters by 

colourless slime. Megaconidia and microconidia not seen. 

Culture characteristics — Colonies fast growing at 24 °C on 

MEA, sienna to bay on surface, and reverse sienna after 7 d; 

sparse white aerial mycelium with sparse sporulation; chlamydospores 

extensive throughout the medium. 

Specimens examined. Tunisia, Carthage, Tunis, from Callistemon laevis, 

Nov. 2010, G. Polizzi, (CBS H-20684, holotype of C. tunisiana, culture ex-type 

CBS 130357 = DISTEF-TCL1); Carthage, Tunis, from Callistemon sp., Nov. 

2010, G. Polizzi, culture CBS 130356 = DISTEF-TCROU2; Carthage, Tunis, 

from Metrosideros excelsus, Nov. 2010, G. Polizzi, culture DISTEF-TME1. 

Notes — Morphologically, C. tunisiana is similar to C. mexicana 

and C. pseudomexicana, but can be distinguished from 

both taxa by its shorter stipe extensions. The conidiophores of 

C. tunisiana (–3) also form fewer fertile branches than C. mexicana 

(–5) and C. pseudomexicana (–4) (Schoch et al. 1999). 

Pathogenicity 

All plants inoculated with the Calonectria spp. in this study developed 

leaf spot, leaf blight or crown and root rot symptoms. 

The first symptoms of leaf spot and leaf blight were observed 5 d 

after inoculation on all test plants inoculated with the Calonectria 

spp., resembling the symptoms observed during the survey. Isolates 

of C. pseudomexicana (CBS 130354, 130355), C. tunisiana 

(CBS 130356, 130357) as well as the single isolate of C. mexicana 

(CBS 130353) produced the most severe symptoms. Isolates 

of C. polizzii (CBS 130351, 130352) also caused leaf spot 

and leaf blight on all inoculated plants, but less severe than the 

other three Calonectria spp. tested. Ten days after inoculation, 

severe or moderate defoliation of M. communis and M. excelsa 

cv. Aurea plants was observed. 

All inoculated plants of M. communis developed crown rot, basal 

stem rot and root rot 25 d after inoculation with the isolate 

representing C. polizzii (CBS 130351). All un-inoculated control 

plants remained healthy and re-isolations from the test plants 

consistently yielded the test fungi. 

DISCUSSION 

During a survey of diseased plants at an ornamental nursery in 

Tunis, Tunisia, a number of Calonectria spp. were isolated from 

plants exhibiting crown, root rot and leaf spots. DNA sequence 

and morphological comparisons allowed the identification of 

two of these isolates as C. mexicana and C. polizzii as well 

as the description of two new species, C. pseudomexicana 

and C. tunisiana, both in the C. scoparia complex (Schoch et 

al. 1999).


79 

Calonectria mexicana resides in the C. scoparia complex 

(Schoch et al. 1999) and can be distinguished from the other 

seven Calonectria spp. in the complex based on their unique 

papillate vesicles (Schoch et al. 1999, Lombard et al. 2010b, 

c, Chen et al. 2011). Until now, C. mexicana has only been 

reported from soil samples collected in Mexico, and its pathogenicity 

was unknown (Schoch et al. 1999, Crous 2002). This 

study represents the first report of this fungus outside Mexico, 

and also demonstrates its pathogenicity on some plant hosts. 

Calonectria polizzii has previously been reported from ornamental 

plants collected at a nursery in Sicily, Italy (Schoch et 

al. 2001, Lombard et al. 2010b), although its pathogenicity was 

not confirmed. This study represents the first confirmation of the 

pathogenicity of C. polizzii and widens its distribution to Tunisia. 

Calonectria polizzii is a member of the C. scoparia complex and 

can be distinguished from the other members by its smaller 

macroconidial dimensions (Lombard et al. 2010b). 

The description of C. pseudomexicana and C. tunisiana adds 

two more species to the C. scoparia complex. This complex is 

characterised by 1-septate macroconidia and the formation of 

ellipsoidal to obpyriform terminal vesicles on the stipe extensions 

(Schoch et al. 1999, Crous 2002, Lombard et al. 2010b). 

Based on phylogenetic inference, both these newly described 

species are closely related to C. mexicana, which they also resemble 

in morphology. They can be distinguished from C. mexicana 

and each other by the number of fertile branches produced 

on the conidiophores. Calonectria tunisiana (av. = 49 × 5 µm) 

has slightly larger macroconidia than both C. mexicana (av. = 

45 × 4 µm; Schoch et al. 1999) and C. pseudomexicana (av. 

= 45 × 5 µm). 

The pathogenicity tests with isolates of C. mexicana, C. polizzii, 

C. pseudomexicana and C. tunisiana clearly showed that they 

are able to cause symptoms similar to those observed during 

the survey. Calonectria polizzii was less virulent than the other 

three species, but should still be regarded as an important 

nursery pathogen. This supports the view that most Calonectria 

spp. can induce leaf spots if the environmental conditions are 

favourable (Crous 2002). All four species caused similar disease 

symptoms on the nine inoculated plant species, suggesting 

that little is known about the host specificity and mechanisms 

of infection of this group of plant pathogens. 

This study stresses the importance of Calonectria spp. as 

nursery pathogens. Their soil-borne nature has contributed to 

their ease of movement globally and little is known about their 

origins. Furthermore, it is not known if these fungal pathogens 

originated from Tunisia or were introduced, and more isolates 

are needed for a study of their population dynamics. 

References 

Chen SF, Lombard L, Roux J, Xie YJ, Wingfield MJ, Zhou XD. 2011. Novel 

species of Calonectria associated with Eucalyptus leaf blight in Southeast 

China. Persoonia 26: 1–12. 

Crous PW. 2002. Taxonomy and pathology of Cylindrocladium (Calonectria) 

and allied genera. APS Press, St. Paul, Minnesota, USA. 

Crous PW, Alfenas AC, Wingfield MJ. 1993. Calonectria scoparia and Calonectria 

morganii sp. nov., and variation among isolates of their Cylindrocladium 

anamorphs. Mycological Research 97: 701–708. 

Crous PW, Gams W, Stalpers JA, Robert V, Stegehuis G. 2004. MycoBank: 

an online initiative to launch mycology into the 21st century. Studies in 

Mycology 50: 19–22. 

Crous PW, Phillips AJL, Wingfield MJ. 1991. The genera Cylindrocladium and 

Cylindrocladiella in South Africa, with special reference to forest nurseries. 

South African Forestry Journal 157: 69–89. 

Fisher NL, Burgess LW, Toussoun TA, Nelson PE. 1982. Carnation leaves 

as a substrate and for preserving cultures of Fusarium species. Phytopathology 

72: 151–153. 

Gueidan C, Roux C, Lutzoni F. 2007. Using multigene phylogeny analysis 

to assess generic delineation and character evolution in Verrucariaceae 

(Verrucariales, Ascomycota). Mycological Research 111: 1145–1168. 

Henricot B, Culham A. 2002. Cylindrocladium buxicola, a new species affecting 

Buxus spp., and its phylogenetic status. Mycologia 94: 980–997. 

Hillis DM, Bull JJ. 1993. An empirical test of bootstrapping as a method for 

assessing confidence in phylogenetic analysis. Systematic Biology 42: 

182–192. 

Katoh K, Toh H. 2010. Parallelization of the MAFFT multiple sequence alignment 

program. Bioinformatics 26: 1899–1900. 

Lombard L, Crous PW, Wingfield BD, Wingfield MJ. 2010a. Species concepts 

in Calonectria (Cylindrocladium). Studies in Mycology 66: 1–14. 

Lombard L, Crous PW, Wingfield BD, Wingfield MJ. 2010b. Multigene phylogeny 

and mating tests reveal three cryptic species related to Calonectria 

pauciramosa. Studies in Mycology 66: 15–30. 

Lombard L, Crous PW, Wingfield BD, Wingfield MJ. 2010c. Phylogeny and 

systematics of the genus Calonectria. Studies in Mycology 66: 31–69. 

Lombard L, Rodas CA, Crous PW, Wingfield BD, Wingfield MJ. 2009. Calonectria 

(Cylindrocladium) species associated with dying Pinus cuttings. 

Persoonia 23: 41–47. 

Lombard L, Zhou XD, Crous PW, Wingfield BD, Wingfield MJ. 2010d. Calonectria 

species associated with cutting rot of Eucalyptus. Persoonia 24: 

1–11. 

Nirenburg HI. 1981. A simplified method for identifying Fusarium spp. occurring 

on wheat. Canadian Journal of Botany 59: 1599–1609. 

Nylander JAA. 2004. MrModeltest v. 2. Programme distributed by the author. 

Evolutionary Biology Centre, Uppsala University. 

Overmeyer C, Lünneman S, Wallburnn C von, Meinhardt F. 1996. Genetic 

variability among isolates and sexual offspring of the plant pathogenic 

fungus Calonectria morganii on the basis of random amplification of 

polymorphic DNA (RAPD) and restriction fragment length polymorphism 

(RFLP). Current Microbiology 33: 249–255. 

Polizzi G. 2000. Prime esperienze di lotta chimica nei confronti del marciume 

del colletto e delle radici di Polygala myrtifolia causato da Cylindrocladium 

pauciramosum. Informatore Fitopatologico 11: 39–47. 

Polizzi G, Catara V. 2001. First report of leaf spot caused by Cylindrocladium 

pauciramosum on Acacia retinodes, Arbutus unedo, Feijoa sellowiana and 

Dodonaea viscosa in southern Italy. Plant Disease 85: 803. 

Polizzi G, Crous PW. 1999. Root and collar of milkwort caused by Cylindrocladium 

pauciromosum, a new record for Europe. European Journal of 

Plant Pathology 105: 407–411. 

Polizzi G, Grasso FM, Vitale A, Aiello D. 2007a. First occurrence of Calonectria 

leaf spot on mexican blue palm in Italy. Plant Disease 91: 1057. 

Polizzi G, Vitale A, Aiello D, Castello I, Guarnaccia V, Parlavecchio G. 2009. 

First record of crown and root rot caused by Cylindrocladium pauciramosum 

on brush cherry in Italy. Plant Disease 93: 547. 

Polizzi G, Vitale A, Aiello D, Dimartino MA, Parlavecchio G. 2007b. First report 

of damping-off and leaf spot caused by Cylindrocladium scoparium on different 

accessions of bottlebrush cuttings in Italy. Plant Disease 91: 769. 

Polizzi G, Vitale A, Aiello D, Parlavecchio G. 2006a. First record of crown 

and root rot caused by Cylindrocladium pauciramosum on California lilac 

in Italy. Plant Disease 90: 1459. 

Polizzi G, Vitale A, Castello I, Groenewald JZ, Crous PW. 2006b. Cylindrocladium 

leaf spot, blight and crown rot, new diseases of mastic tree seedlings 

caused by Cylindrocladium scoparium. Plant Disease 90: 1110. 

Posada D, Crandall KA. 1998. Modeltest: testing the model of DNA substitution. 

Bioinformatics 14: 817–818. 

Rayner RW. 1970. A mycological colour chart. Commonwealth Mycological 

Institute, Kew, Surry. British Mycological Society. 

Ronquist F, Heulsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference 

under mixed models. Bioinformatics 19: 1572–1574. 

Schoch CL, Crous PW, Cronwright G, Witthuhn RC, El-Gholl NE, Wingfield 

BD. 2000. Recombination in Calonectria morganii and phylogeny with other 

heterothallic small-spored Calonectria species. Mycologia 92: 665–673. 

Schoch CL, Crous PW, Polizzi G, Koike ST. 2001. Female fertility and single 

nucleotide polymorphism comparisons in Cylindrocladium pauciramosum. 

Plant Disease 85: 941–946. 

Schoch CL, Crous PW, Wingfield BD, Wingfield MJ. 1999. The Cylindrocladium 

candelabrum species complex includes four distinct mating populations. 

Mycologia 91: 286–298. 

Swofford DL. 2002. PAUP*. Phylogenetic analysis using parsimony (* and 

other methods), 4.0b10. Computer programme. Sinauer Associates, Sunderland, 

Massachusetts, USA. 

Vitale A, Polizzi G. 2008. First record of leaf spots and stem lesions on Pistacia 

lentiscus caused by Cylindrocladium pauciramosum and C. scoparium in 

Italy. Plant Pathology 57: 384.

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