First report of Pestalotiopsis biciliata causing dieback on Quercus coccifera and Pistacia lentiscus in Tunisia

Abstract

Species of Pestalotiopsis occur commonly as plant pathogens. Accordingly, the aim of this study was to identify the causal agent of a shrub disease in northeastern and northern Tunisian forests. Field surveys showed a progressive dieback of branches, twig blight and trunk cankers on Pistacia lentiscus and Quercus coccifera. Pestalotiopsis spp. were the main fungi consistently isolated from these shrub species. Morphological identification and phylogenetic analysis of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA and partial sequence of the translation elongation factor 1-alpha gene (tef1-α) identified the isolated fungi as Pestalotiopsis biciliata. This identification was confirmed by PCR analysis and Sanger sequencing, and demonstrated further using Koch’s postulates. To the best of our knowledge, this is the first report of P. biciliata associated with branch dieback and stem canker on Quercus coccifera and Pistacia lentiscus in Tunisia.

Résumé

Les espèces de Pestalotiopsis sont souvent des agents phytopathogènes. En conséquence, le but de cette étude était d’identifier l’agent causal d’une maladie des arbrisseaux des forêts du nord-est et du nord de la Tunisie. Des examens sur le terrain ont indiqué le dépérissement graduel des branches, la brûlure des rameaux et la formation de chancres sur les troncs de Pistacia lentiscus et de Quercus coccifera. Pestalotiopsis spp. étaient les principaux champignons systématiquement isolés de ces espèces d’arbrisseaux. L’identification morphologique et l’analyse phylogénétique de la région de l’espaceur transcrit interne (ITS) de l’ADN ribosomique nucléaire et la séquence partielle du gène 1-alpha (tef1-α) du facteur d’élongation de la transcription ont permis d’identifier les champignons isolés en tant que Pestalotiopsis biciliata. Cette identification a été confirmée par PCR ainsi que par séquençage de Sanger et d’autre part démontrée en y appliquant les postulats de Koch. Pour autant qu’on le sache, il s’agit du premier rapport de P. biciliata associé au dépérissement des branches et au chancre des tiges chez Quercus coccifera et Pistacia lentiscus en Tunisie.

Keywords:

• disease
• morphological identification
• Pestalotiopsis
• phylogenetic analysis

Mots clés:

• Maladie
• identification
• morphologique
• Pestalotiopsis
• analyse phylogénétique

Introduction

The species-rich genus Pestalotiopsis Steyaert includes a large number of plant pathogens as well as endophytes (Maharachchikumbura et al. 2011; Satini et al. 2013; Zhang et al. 2013). To date, 299 names of species of Pestalotiopsis are listed in the 2016 (status March 2016), the global fungal nomenclature database. The genus Pestalotiopsis Steyaer is the anamorph of Pestalosphaeria belonging to the family Amphisphaeriaceae (Sutton 1980). Barr (1975) studied the teleomorph-anamorph connections in Pestalotiopsis and described the genus Pestalosphaeria, as the teleomorph of Pestalotiopsis. Furthermore, Pestalosphaeria (teleomorph) has been observed in only a few species. This is mainly because it is difficult to develop and induce even under laboratory conditions (Judith-Hertz 2016). The Pestalotiopsis genus is a heterogeneous group of coelomycetous fungi that are differentiated primarily by their conidial characteristics such as size, septation, pigmentation, and the presence or absence of appendages (CABI Bioscience database 2001). It is characterized by fusiform conidia having mostly four-septate and pigmented median cells, with two to four apical appendages arising as tubular extensions from the apical cell and a centric basal appendage formed within compact acervuli (Jeewon et al. 2002). Hu et al. (2007) found that conidial characters such as conidial length, median cell length, conidial width and colour of median cells were stable characters within Pestalotiopsis, but that the length of the apical and basal appendages was variable. Pestalotiopsis species are ubiquitous in distribution, occurring on a wide range of plants, and may cause a serious decline phenomenon (Das et al. 2010). They often occur as endophytes (Liu et al. 2006; Watanabe et al. 2010), or as pathogens causing economic losses (Maharachchikumbura et al. 2011). Indeed, numerous Pestalotiopsis species are regarded primarily as opportunistic pathogens that affect stressed plants. They have been consistently associated with a variety of plant diseases, including canker lesions, shoot dieback, stem blights, leaf spots and leaf blotches (Tagne and Mathur 2001; Espinoza et al. 2008; Zhang et al. 2012; Maharachchikumbura et al. 2013a, 2013b; Morales-Rodríguez et al. 2019). Pestalotiopsis spp. have been reported on hazelnut in Turkey, Iran and Serbia (Karakaya 2001; Arzanlou et al. 2012; Tanja et al. 2017). Furthermore, P. chamaeropis has been shown to be associated with shoot blight and stem necrosis of Erica arborea in Tunisia (Hlaiem et al. 2018).

The genus Pestalotiopsis has gained considerable attention from the scientific community in recent years, not only because of its role as a plant pathogen, but also as a commonly isolated endophyte which has been shown to produce a wide range of secondary diverse metabolites (Li and Strobel 2001; Yasuda et al. 2003; Xu et al. 2010).

During surveys of the Rimel and Henchir Kort forests of Tunisia, a widespread withering affecting Pistacia lentiscus L. and Quercus coccifera L., respectively, was noted. In fact, the presence of shoot blight, branch cankers and light brown necrosis on twigs, as well as black acervuli on infected tissues, was observed on many individuals. These symptoms, along with the morphological characteristics of the fungi isolated from diseased tissues, indicated that Pestalotiopsis species are the causal agents of P. lentiscus and Q. coccifera diseases.

Until now, data regarding the identity of Pestalotiopsis species in North Africa and in Tunisia have been limited. Thus, the objectives of this work were to (i) characterize and cluster isolates of Pestalotiopsis associated with twig dieback symptoms on shrub species and (ii) confirm their aggressiveness.

Material and methods

Sample collection

Samples were obtained from symptomatic branches of declining Q. coccifera and P. lentiscus in Northeastern (Henchir Kort forest, Cap-Bon) and Northern (Rimel forest, Bizerte) Tunisia, respectively. From each shrub species, 10 shoots showing necrosis were collected and carried to the laboratory for fungal isolation.

Fungal isolation and morphological characterization

Fragments (3 × 3 mm) were taken from the margins between the necrotic and healthy tissues of the branches. Samples were then surface-disinfected in 70% ethanol for 2 min and rinsed three times in sterilized water before being placed in Petri dishes containing potato dextrose agar (PDA) containing streptomycin sulphate (0.05 g/l), and incubated in darkness at 25°C for 3 days (Franceschini et al. 2005). Cultures were purified by the hyphal tip technique and incubated under the same conditions described above. Pestalotiopsis species were identified based on morphological traits of 10-day-old cultures and conidial morphology, shape and size according to the key of Maharachchikumbura et al. (2014).

DNA extraction, PCR amplification and sequencing

Based on morphological characterization, two representative isolates of Pestalotiopsis spp. were selected for molecular identification. Total DNA from fresh mycelium grown on PDA was extracted using an innuPREP Plant DNA Kit (Analytik Jena AG, Germany) according to the manufacturer’s instructions. The ITS rDNA region and fragments of a nuclear protein coding gene (tef1-α) were amplified using the universal primer pairs ITS1/ITS4 (White et al. 1990) and EF1-728F/ EF2 (Carbone and Kohn 1999). Amplification conditions for the ITS rDNA region and tef1-α were followed Crous et al. (2013). Amplified products were sent for Sanger sequencing (Laboratory of the Interdepartmental Center for Biotechnology Services of Agricultural, Chemical and Industrial Interest-CIBIACI, Italy). New sequences were edited with FinchTV v1.4.0 (Geospiza, Inc., Seattle, Washington, USA; (http://www.geospiza.com/finchtv) and compared with those deposited in GenBank through BLAST searches (http://www. ncbi.nih.gov/) (Table 1). The DNA sequence alignment was performed using MUSCLE 3.8 (https://www.ebi.ac.uk/Tools/msa/muscle/). ITS and tef1-α sequences were deposited in the National Center for Biotechnology Information (NCBI) as GenBank accession (https://www.ncbi.nlm.nih.gov/) under the accession numbers indicated in Table 2.

Table 1. Pestalotiopsis isolates included in the phylogenetic analysis (*).

Strain	Species	Host	Country	GenBank accession numbers
				ITS	TEF
CBS 186.71	Pestalotiopsis chamaeropis	Chamaerops humilis	Italy	KM199326	KM199473
CBS 113604	P. chamaeropis	-	-	KM199323	KM19947
CBS 790.68	P. biciliata	Taxus baccata	Netherlands	KM199305	KM199507
CBS 124463	P. biciliata	Platanus hispanica	Slovakia	KM199308	KM199505
CBS 236.38	P. biciliata	Paeonia sp.	Italy	KM199309	KM199506
TN.20	P. biciliata	Quercus coccifera	Tunisia	MN841932	MN942034
TN.109	P. biciliata	Pistacia lentiscus	Tunisia	MN846272	MT241507
CBS 442.67	P. kenyana	Coffea sp.	Kenya	KM199302	KM199502
CBS 911.96	P. kenyana	-	-	KM199303	KM199503
IFRDCC 2403	P. trachicarpicola	Podocarpus macrophyllus	China	KC537809	KC53781
IFRDCC 2440	P. trachicarpicola	Podocarpus macrophyllus	China	JQ845947	JQ845946
CBS 122616	Seiridium cupressi	Cupressus sp.	Greece	LT853082	LT853179

*Isolates generated in this study are indicated in italic.

Table 2. Identity of the isolates, GenBank accession numbers of the sequences, spores size and necrosis length.

Sites	Hosts*	Isolates	GenBank accession numbers		Spores size (µm)		Necrosis length** (cm)
			ITS	tef1-α	Length	Width
Henchir kort	Qc	TN.20 Control	MN841932 -	MN942034 -	21.84 ± 0.38 -	4.94 ± 0.19 -	5 ± 0.00 0.9 ± 0.3
Rimel	Pl	TN.109 Control	MN846272-	MT241507-	22.62 ± 0.55-	5.77 ± 0.34-	4.66 ± 0.19 1.1 ± 0.0

*Qc = Quercus coccifera, Pl = Pistacia lentiscus.

**Mean values correspond to the sum of both upward and downward extent of vascular necrosis (5 repetitions per isolate) measured from the point of inoculation ± SE.

Phylogenetic analysis

The ITS and tef1-α sequences obtained in this study were combined and then supplemented with further sequences of Pestalotiopsis spp. retrieved from GenBank (Table 1) based on BLAST searches and the literature (Maharachchikumbura et al. 2014). Sequences of all isolates were aligned with ClustalX v. 1.83 (Thompson et al. 1997). Phylogenetic trees were generated using the Neighbour-Joining (NJ) method (Saitou and Nei 1987; Tamura et al. 2004) with MEGA v. 6 (Tamura et al. 2013).

Pathogenicity assay

Pathogenicity assay was conducted according to the methodology of González et al. (2012) by inoculating two Pestalotiopsis isolates on healthy excised branches (n = 10) collected from their host species and measuring about 30 cm [Q. coccifera (n = 5; isolate TN.20) and P. lentsicus (n = 5; isolate TN.109)]. Briefly, after removal of the leaves, branches were surface-disinfected with ethanol (70%) and wounded with a sterilized scalpel. For each isolate, a mycelial plug (6 mm⁻diam.) was taken from the margin of an actively growing, 7-day-old colony and placed on the wounds of the branches. The inoculation point was covered with cotton wool soaked in sterile water and wrapped in Parafilm. The inoculated branches were then placed in a beaker containing 200 mL of sterile water, whereas the bottom and the top ends of each cane were sealed with a synthetic grafting resin to prevent drying and contamination and enclosed in a transparent plastic bag. Ten control branches for each species [Q. coccifera (n = 5) and P. lentsicus (n = 5)] were inoculated with sterile PDA plugs. All inoculated branches were kept at room temperature (20–26°C) in the laboratory and monitored for necrosis development over 30 days. The pathogen was re-isolated at the end of the experiment.

Data analysis

Average lesion lengths caused by the two Pestalotiopsis species were reported as the mean ± standard error of mean (MSE). The results were evaluated statistically by analysis of variance (ANOVA) with SPSS v.20.

Results

Morphological characterization

A collection of 38 isolates was obtained from symptomatic branches of Q. coccifera in Henchir Kort and P. lentiscus in Rimel (Fig. 1). Colony colour was whitish with moderately dense mycelium on PDA and reaching 80 mm in diameter after 7 days at 25°C. Black globulose acervular conidiomata on the surface of the mycelium were produced after about 10 days (Fig. 2a). Conidiophores indistinct reduced to conidiogenous cells discrete, filiform, clavate or subcylindrical and hyaline with immersed to semi-immersed conidiomata. Conidia were fusoid, ellipsoid, straight to slightly curved, 4-septate. There were three median cells doliiform dark brown concolourous and septa darker than the rest of the cell. Both apical and basal cells were hyaline, the apical cell with 2 or 3 tubular appendages (mostly 3) and the basal cell with two tubular appendages with a truncate base measured 21 to 29.5 × 5 to 7.5 μm in size (Fig. 2b). These morphological characteristics were congruous with the description of Pestalotiopsis species (Espinoza et al. 2008; Maharachchikumbura et al. 2014).

Fig. 1 Dieback symptoms caused by Pestalotiopsis: necrotic lesion with numerous black acervuli on infected shoots of Pistacia lentiscus (a) and Quercus coccifera (b).

Fig. 2 Morphological characteristics of Pestalotiopsos biciliata; colony with conidiomata on PDA after 10 d growth at 25°C (a) and conidia with concolorous median cells (b). Scale bars: 10 μm.

Molecular identification and phylogenetic analysis

The two representative Pestalotiopsis isolates (TN.20) and (TN.109) collected from Q. coccifera and P. lentiscus, respectively, showed 100% identity at all sequenced sites with Pestalotiopsis biciliata (CBS 124463). These isolates were clustered unambiguously with P. biciliata strains CBS 124463, CBS 236.38 and CBS 790.68 used in the phylogenetic analysis. The combined ITS and tef1-α dataset of Pestalotiopsis spp. included 12 taxa (11 in-group and 1 out-group) and contained 788 characters (including gaps). Seiridium cupressi CBS 122616 was included as the out-group (Fig. 3).

Fig. 3 Phylogenetic tree obtained from combined ITS and tef1-α sequence data of Pestalotopsis biciliata. Bootstrap support values (%) from 1000 replications are shown at the nodes. The scale bar indicates 0.005 substitutions per site. The tree is rooted to Seiridium cupressi CBS122616.

Pathogenicity assay

Thirty days after inoculation, necrosis developed extending from the inoculation points, confirming the virulence of the two Pestalotiopsis isolates (TN20 and TN.109) on the shrub species (Q. coccifera and P. lentiscus) (Fig. 4 a2,b2). Stem lesions measured respectively 5.00 ±0.00 cm and 4.66 ± 0.19 cm (Table 2). Statistical analysis of variance (ANOVA) showed no significant difference in pathogenicity between the two isolates (F (1,8) = 1.854, P = 0.577). Disease symptoms were absent on control twigs and without fungi development (Fig. 4 a1,b1). Both pathogenic isolates were successfully re-isolated from the margins of the necrotic lesions of each inoculated excised branch. Their identity was confirmed based on morphological and cultural characters.

Fig. 4 Pathogenicity assay: asymptomatic control (a1, b1) and vascular necrosis caused by Pestalotiopsis biciliata on excised branches of Pistacia lentiscus (a2) and Quercus coccifera (b2) six weeks after inoculation.

Discussion

During surveys in Tunisia, disease symptoms, including shoot dieback, branch cankers and vascular discoloration of the wood were observed on P. lentiscus and Q. coccifera. Similar symptoms were reported previously on grapevines and found to be associated with Pestalotiopsis spp. (Maharachchikumbura et al. 2011). McQuilken and Hopkins (2004) reported that Pestalotiopsis isolates were not host-specific and induced typical symptoms and signs, including browning of the foliage, stems and roots, and the presence of black acervuli, on various species of ericaceous plants, herbaceous and woody hosts such as Eucalyptus spp. (Morales-Rodríguez et al. 2019) and grapevine (Úrbez-Torres et al. 2009). Moreover, Pestalotiopsis spp. are responsible for a number of plant diseases (Keith et al. 2006; Espinoza et al. 2008; Chen et al. 2013).

Considering both the morphological and molecular data, we identified Pestalotiopsis biciliata as the causal agent of brown vascular necrosis on Q. coccifera in Kenchir Kort and P. lentiscus in Rimel. Furthermore, P. biciliata was isolated from dry needles of Taxus baccata L. in the Netherlands and in the UK (Liu et al. 2019), from Paeonia sp. in Italy and from Platanus hispanica in Slovakia (Maharachchikumbura et al. 2014). The fungus was referred as the causal agent of fruit rot on withered grapes and as a foliar pathogen of Eucalyptus spp. in Italy (Lorenzini and Zapparoli 2018; Morales-Rodríguez et al. 2019). It also was reported as a foliar pathogen of Ceratonia siliqua in Algeria (Louanchi et al. 2021). P. biciliata was also associated with grapevine trunk diseases in France (Maharachchikumbura et al. 2017) and as pathogen of stone pine orchard in Portugal (Silva et al. 2020). Recently, P. biciliata has been recorded to cause dieback and branch necrosis on Pinus pinea in Tunisia (Hlaiem et al. 2021).

The pathogenicity assay confirmed the virulence of P. biciliata on wounded branches of Q. coccifera and P. lentiscus in this study, with the fungus-causing symptoms similar to those observed in the field. In contrast, control branches remained asymptomatic. A previous report by Hopkins and McQuilken (2000) indicated that wounding prior to inoculation was necessary for disease development. Similarly, studies conducted with other Pestalotiopsis species have also found that nonwounded samples remained symptomless (Rivera and Wright 2000).

Our paper provides valuable information for further study of P. biciliata, reported here as a new causal agent of shoot dieback and stem blight of Q. coccifera and P. lentiscus in the world and specifically in Tunisia. Given the high value of these evergreen shrubs, with high economic and medicinal interests, considerable attention should be given to the serious decline phenomena that have occurred in these regions. Pestalotiopsis species may represent a threat to the health status of forests in the Mediterranean basin and in Tunisia.

Acknowledgements

The authors thank Dr. Mohamed Rabeh HAJLAOUI (Laboratory of Applied Biotechnology in Agriculture, National Agricultural Research Institute of Tunisia) and Dr. Hanen ZAIER (Laboratory of the Improvement and Protection of the Genetic Resources of the olive tree, Olive tree institute) for their help during the course of the molecular work.

Disclosure statement

No potential conflict of interest was reported by the author(s).