Paraphoma garibaldii sp. nov. causing leaf spot disease of Campanula rapunculoides in Italy.

Leaf and stem spots are among the most important diseases compromising ornamental plants worldwide. In this study, Paraphoma garibaldii sp. nov. is described from leaf lesions on Campanula rapunculoides in Piedmont, Northern Italy. The new species was characterised using a polyphasic approach including morphological characterisation and a multilocus molecular phylogenetic analysis based on partial nucleotide sequences of the translation elongation factor 1-α (tef1), the internal transcribed spacers (ITS) region and the β-tubulin (tub2) markers. Pathogenicity tests and the fulfilment of Koch's postulates confirm P. garibaldii as a novel foliar pathogen of Campanula rapunculoides. Presently, the fungal infection due to Paraphoma garibaldii is known from a single location in Italy, and further surveys are required to determine its distribution and relative importance. Citation: Guarnaccia V, Martino I, Tabone G, Crous PW, Gullino ML (2022). Paraphoma garibaldii sp. nov. causing leaf spot disease of Campanula rapunculoides in Italy. Fungal Systematics and Evolution 9: 19-26. doi: 10.3114/fuse.2022.09.03.

INTRODUCTION

The genus Phoma was introduced by Saccardo (1880), but the generic concept was significantly revised by Boerema & Bollen (1975). Boerema divided this genus in nine sections based on morphological features. The section Paraphoma was distinguished based on the presence of setose pycnidia and muriform chlamydospores. However, this classification system revealed several difficulties in understanding species boundaries and in reflecting the evolutionary relationships among species. Furthermore, molecular analyses revealed that Paraphoma is polyphyletic, and related to genera affiliated with the families Phaeosphaeriaceae (de Gruyter ), Cucurbitariaceae and Coniothyriaceae (Chen ). Paraphoma is based on P. radicina, which was isolated from roots of Prunus cerasus in Australia and from rootstocks of Malus sylvestris in the Netherlands (de Gruyter ). Currently, 14 species are included within the genus: P. chlamydocopiosa, P. chrysantemicola, P. convolvuli, P. dioscoreae, P. fimeti, P. ledniceana, P. melnickii, P. pye, P. radicina, P. rhaphiolepidis, P. salicis, P. variabilis and P. vinacea (Crous ). Species within Paraphoma are generally regarded as soil-borne pathogens. They usually cause root and crown rot disease, but they have been isolated from necrotic leaf spots on Tanacetum cinerariifolium (Moslemi , 2018). Several Paraphoma spp. have been reported in association with ornamental and herbaceous plant hosts. For instance, P. chrysanthemicola was isolated from leaf spots on Atractylodes japonica in China (Ge ). Three Paraphoma species were found in association with T. cinerariifolium in Australia: P. vinacea (Moslemi ), P. chlamydocopiosa and P. pye (Moslemi ), Paraphoma radicina was isolated from crown rot on Medicago sativa in China (Cao ), while P. convolvuli and P. melnikiae were identified in association with leaf spots of Convolvulus arvensis in Russia (Gomzhina ).

Ornamental plants represent an economically important sector of agriculture worldwide. Presently, Europe is leading in ornamental plant production, with The Netherlands ranking first, followed by Italy (DG-AGRI-G2 2020). In particular, bedding plants represent a major group in the ornamental sector with a continuous increasing commercial value and relevance. However, seeds, propagation materials and growing media could consistently influence bedding plants cultivation, as there are several diseases affecting them (Guarnaccia ). Campanula spp. are popular bedding plants, and these are planted on the borders of parks and gardens (Garibaldi ). Several fungal pathogens have been found in association with Campanula spp. in Italy including Sclerotinia sclerotium on Ca. carpatica (Garibaldi ), Coleosporium campanulae on Ca. rapunculoides and Ca. trachelium (Garibaldi , 2021), and Golovinomyces orontii on Ca. glomerata and Ca. rapunculoides (Garibaldi , 2018). Campanula spp. are also severely affected by leaf anthracnose caused by Colletotrichum lineola and C. nymphaeae (Guarnaccia ) and Alternaria leaf spot caused by Alternaria alternata which can cause severe defoliation (Garibaldi ). Moreover, different Campanula spp. were reported as susceptible to Rhizoctonia solani and as hosts of phoma-like taxa, such as Stagonosporopsis trachelii (Garibaldi , Guarnaccia ).

In this study a new Paraphoma sp. associated with leaf spots on Campanula rapunculoides was identified and characterised on the basis of morphological features and multi-locus DNA phylogeny. Pathogenicity and Koch’s postulates were tested.

MATERIALS AND METHODS

Field surveys and fungal isolation

The surveys were conducted in a garden in Piedmont, Northern Italy (45°36’43.8”N 8°03’22.7”E), a site constantly monitored as a representative area exposed to the introduction of new plant pests since the historical data known for this site and its geographical isolation.

At the end of June 2020, leaf spots and stem necrosis were observed on 6-mo-old plants of Ca. rapunculoides. The disease index was recorded as the number of symptomatic plants. Small sections (0.2–0.5 cm long) from the margin of lesions were surface disinfected with 1 % sodium hypochlorite for 1 min, rinsed once in sterile distilled water, dried on sterile filter paper and placed on 2 % potato dextrose agar (PDA) plates amended with 25 ppm streptomycin sulphate (Sigma-Aldrich, St. Louis, MO, USA). The plates were incubated at 25 ± 1 °C under a 12 h photoperiod. After 48–72 h of incubation, mycelial plugs were taken from the margin of the resulting colonies and transferred to fresh PDA plates. After 5 d, pure cultures were established from single hyphal tip transfers. Stock cultures were maintained at -80 °C in the Agroinnova (University of Torino) culture collection, Torino, Italy. Reference strains and specimens are maintained in the CBS culture collection of the Westerdijk Fungal Biodiversity Institute (WI), Utrecht, the Netherlands.

DNA extraction, PCR amplification and sequencing

Total DNA was extracted with an E.Z.N.A.® Fungal DNA Mini Kit (Omega Bio-Tek, Darmstadt, Germany), according to the manufacturer’s instructions. The nuclear ribosomal internal transcribed spacer (ITS) region was amplified using ITS1 and ITS4 primers (White ). The primers TUB2Rd and TUB4Fd (Aveskamp ) were used to amplify part of the β-tubulin (tub2) gene. The partial translation elongation factor 1-α (tef1) gene was amplified with EF1-728F (Carbone & Kohn 1999) and EF2 (O’Donnell ) primers. The amplification mixtures and cycling conditions for all three loci were followed as described in each of the cited references. Both strands of the PCR products were sequenced by Eurofins Genomics Service (Ebersberg, Germany). The sequences generated were analysed using Geneious v. 11.1.5 (Kearse , Auckland, New Zealand) and consensus sequences were processed.

Phylogenetic analyses

The newly generated sequences were analysed using BLAST search on the NCBIs GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi) database to achieve a taxonomic framework by determining the closest relatives. The MAFFT v. 7 online program (http://mafft.cbrc.jp/alignment/server/index.html) (Katoh & Standley 2013) was used to align each gene region of the sequences obtained from this study and sequences downloaded from GenBank. Alignments were then manually adjusted by MEGA v. 7 (Kumar ). The analyses were conducted individually for each locus (data not shown) and as multi-locus analysis, with the aim of identifying the isolates at species level. Reference sequences were selected based on recent studies on Paraphoma species (Moslemi , Cao , Gomzhina , Magaña-Dueñas ). The phylogeny was developed based on Maximum Parsimony (MP) approach for all individual loci, and on both MP and Bayesian Inference (BI) methods for the concatenated multilocus analyses. For BI, the best evolutionary model for each partition was selected with MrModeltest v. 2.3 (Nylander 2004) and incorporated into the analyses. MrBayes v. 3.2.5 (Ronquist ) was used to generate phylogenetic trees under optimal criteria per partition. The Markov Chain Monte Carlo (MCMC) analysis used four chains and started from a random tree topology. The heating parameter was established at 0.2 and trees were sampled every 1 000 generations. The analyses were considered done when the average standard deviation of split frequencies was less than 0.01. The MP analyses were conducted using PAUP (Swofford 2003). Phylogenetic relationships were estimated by heuristic searches with 100 random additional sequences. Tree bisection-reconnection was adopted, with the branch swapping option set at ‘best trees’ only with all characters weighted equally and alignment gaps treated as fifth state. Tree length (TL), consistency index (CI), retention index (RI) and re-scaled consistency index (RC) were calculated, and the parsimony and the bootstrap analyses (Hillis & Bull 1993) were based on 1 000 replications. Sequences generated in this study were deposited in GenBank (Table 1), and the alignments in TreeBASE (www.treebase.org; study number S29045).

Table 1.

GenBank accession numbers of Paraphoma spp. and closely related taxa included in this study.

Species	Culture No.1	GenBank accession no.2
		ITS	tub2	tef1
Juncaceicola alpina	CBS 456.84	KF251181	KF252285	KF253139
J. typharum	CBS 296.54	KF251192	KF252686	KF253148
Neosetophoma samarorum	CBS 138.96	KF251160	KF252655	KF253119
Neostagonospora caricis	CBS 135092	KF251163	KF252658	–
Paraphoma aquatica	FMR 16956T	OU612361	OU612355	–
P. chlamydocopiosa	UMPc01	KU999072	KU999084	KU999080
P. chrysanthemicola	CBS 172.70	KF251165	KF252660	KF253123
	CBS 522.66T	KF251166	KF252661	KF253124
P. convolvuli	MF 9.222	MG764055	–	–
	MF 9.265	MG764062	MG779457	–
	MF 9.301	MG764060	MG779461	–
P. dioscoreae	CBS 135100T	KF251167	KF252662	KF253125
	CPC 11355	KF251168	KF252663	KF253126
	CPC 11361	KF251169	KF252664	KF253127
P. fimeti	CBS 170.70T	KF251170	KF252665	KF253128
	CBS 368.91	KF251171	KF252666	KF253129
P. garibaldii	CBS 148459	OL435708	OL449254	OL449256
	CBS 148460	OL435709	OL449255	OL449257
P. ledniceana	CBS 146533	MT371091	MT372661	MT372654
P. melnikiae	MF 9.182	MG764058	MG779454	–
	MF 9.294T	MG764059	MG779455	–
	MF 9.88	MG764063	MG779456	–
P. pye	UMPp02	KU999073	KU999087	KU999081
P. radicina	CBS 102875T	KF251173	KF252668	KF253131
	CBS 111.79	KF251172	KF252667	KF253130
P. rhaphiolepidis	CBS 142524T	KY979758	KY979924	KY979896
P. salicis	CBS 146797	MW883437	MW890140	–
P. vinacea	UMPV002	KU176885	KU176893	KU176897
Setophoma terrestris	CBS 335.29	KF251246	KF252729	KF253196
Xenoseptoria neosaccardoi	CBS 120.43	KF251280	KF252761	KF253227

1 CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands; CPC: Culture collection of Pedro Crous housed at Westerdijk Fungal Biodiversity Institute; FMR: Faculty of Medicine and Health Sciences, Reus, Spain; MF: All-Russian Institute of Plant Protection; UMP: University of Melbourne, Ex-type and ex-epitype cultures are indicated with superscript T.

2 ITS: internal transcribed spacers 1 and 2 together with 5.8S nrDNA; tub2: beta-tubulin gene; tef1: translation elongation factor 1-α gene. Sequences newly generated in this study are indicated in bold.

Morphology

Slide preparations were mounted in lactic acid from colonies sporulating on sterilised pine needles placed on 2 % tap water agar (PNA) (Smith ). Observations were performed under a Nikon SMZ25 dissection-microscope, and a Zeiss Axio Imager 2 bright field microscope using differential interference contrast (DIC) illumination, and images recorded on a Nikon DS-Ri2 camera with associated software. Colony features and pigment production were described on 2 % malt extract agar (MEA), PDA and oatmeal agar (OA; Crous ) after 2 wk at 25 °C. Colony colours were scored using the colour charts of Rayner (1970). The taxonomic novelty was registered in MycoBank (www.MycoBank.org; Crous ).

Pathogenicity

Pathogenicity tests were performed on healthy Ca. rapunculoides plants grown in 2 L pots. The virulence of a representative isolate (CBS 148459) grown for 15 d on PDA at 25 °C, was tested. Leaves of three 5-mo-old plants of Ca. rapunculoides were sprayed with a conidial suspension (1 × 105 conidia/mL). Sterile water was sprayed on three plants used as negative control. All inoculated and non-inoculated plants were covered with a transparent plastic film to retain a high level of relative humidity (RH) and kept in a growth chamber at 23 °C with a 12 h photoperiod. The plastic film was removed after 7 d. The experiment was repeated once. All plants were irrigated 2–3 times per week and examined daily for disease symptom development. Disease incidence (DI) was recorded as described above. The inoculated fungi were re-isolated and identified by sequencing the tub2 and tef1 loci, thus fulfilling Koch’s postulates.

RESULTS

Field survey and fungal isolation

Leaf symptoms identified as those caused by Paraphoma spp. were found in the investigated site with a disease incidence value of 50 %, considered as the percentage of affected leaves. The symptoms were observed on 6-mo-old Campanula rapunculoides plants grown in open fields in a private garden. The observed symptoms consisted of grey to brown, necrotic, circular, converging lesions on leaves, chlorotic yellowing and, in some case, defoliation of the investigated host. Moreover, necrosis on stems and wilting of the apical part of the plant were observed. Several colonies resembling Paraphoma sp. appeared following isolation, and two monohyphal strains (CBS 148459, CBS 148460) were used for morphological and molecular characterisation.

Taxonomy

Guarnaccia, M.L. Gullino & Crous, MycoBank MB 842029. Fig. 1.

Fig. 1.

Paraphoma garibaldii (CBS 148459). A. Pycnidia with setae forming on PNA. B–D. Brown setae arising from outer pycnidial wall. E, F. Conidiogenous cells giving rise to conidia. G. Aseptate, guttulate conidia. Scale bars: A = 300 μm; All others = 10 μm.

Etymology: Named after Prof. Angelo Garibaldi, in recognition of his contribution to research on ornamental plant diseases.

Conidiomata pycnidial, erumpent to superficial on PNA, wall of 3-4 layers of brown, thin-walled textura angularis, globose, 200–300 μm diam, covered by brown, septate, thick-walled, subcylindrical setae, 30–70 × 3–4 μm, with obtuse ends. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lining the inner cavity, phialidic, hyaline, smooth-walled, ampulliform to doliiform, 5–8 × 4–6 μm, with prominent periclinal thickening. Conidia solitary, aseptate, hyaline, smooth-walled, guttulate, subcylindrical, obtuse at the apex and truncate at the base, (4–)5–6(–7) × (2–)2.5(–3) μm.

Culture characteristics: On MEA, PDA and OA, colonies erumpent, spreading with moderate aerial mycelium and even lobate margins, up to 70 mm diam after 2 wk, surface and reverse red.

Typus: Italy, Piedmont, Biella, on leaf spots of Campanula rapunculoides (Campanulaceae), May 2021, A. Garibaldi (holotype CBS H-24894, culture ex-type CBS 148459).

Additional material examined: Italy, Piedmont, Biella, on leaf spots of Ca. rapunculoides, May 2021, A. Garibaldi, CBS 148460.

Notes: Paraphoma garibaldii is phylogenetically distinct from all 14 species of the genus. Morphologically, its conidia are similar to those of P. variabilis (4–8 × 2–3 μm, from dung, Spain; Crous ), but distinct in that the latter has greyish colonies and shorter (7–25 × 2.5–3 μm), subhyaline setae.

Based on the results by BLAST search, all the sequences obtained in this study showed high similarity (around 96 %) with species included in the Paraphoma genus, however they were identical with no particular species. Three alignments representing single locus analyses of ITS, tub2, tef1 (data not shown), and a combined alignment of the three loci were analysed. The single phylogenetic analysis generated by each locus produced a similar tree topology. The strains of Paraphoma garibaldii formed a well-supported monophyletic clade in the ITS, tub2 and tef1 single-locus trees, with maximum bootstrap values, respectively. The multi-locus phylogeny consisted of 30 sequences, including Setophoma terrestris (CBS 335.29, Gomzhina ) as outgroup. A total of 1 172 characters (ITS: 1–502, tub2: 509–777, tef1: 784–1 172) were included in the phylogenetic analysis, 456 characters were parsimony-informative, 239 were variable and parsimony-uninformative, and 464 were constant. A maximum of 1 000 equally MP trees were saved (Tree length = 1 899, CI = 0.656, RI = 0.737 and RC = 0.483). Bootstrap support values from the MP analysis are included on the Bayesian tree in Fig. 2. For the BI, MrModeltest suggested that all partitions should be analysed with dirichlet state frequency distributions. The following models were recommended by MrModeltest and used: GTR+I+G for ITS, K80+G for tub2 and HKY+G for tef1. In the BI, the ITS partition had 219 unique site patterns, the tub2 partition had 161 unique site patterns, the tef1 partition had 245 unique site patterns and the analysis ran for 675 000 generations, resulting in 1 352 trees of which 534 trees were used to calculate the posterior probabilities.

Fig. 2.

Consensus phylogram of 1352 trees resulting from BI of the combined ITS, tub2 and tef1 datasets. Bayesian posterior probability values and bootstrap support values are indicated at the nodes. The tree was rooted with Setophoma terestris (CBS 335.29).

Isolate CBS 148459 was pathogenic for 100 % of the inoculated Ca. rapunculoides plants causing similar symptoms observed for the first time on the cultivated plants grown in the garden. Dark brown leaf spots appeared 7 d after inoculation, and leaves wilted 5 d after the appearance of large chlorotic areas and the expansion of necrotic tissues (Fig. 3). No symptoms appeared on control plants. The pathogen was consistently re-isolated from the inoculated plants and identified with molecular analysis as described above.

Fig. 3.

Symptoms caused after artificial inoculation of Paraphoma garibaldii on Campanula rapunculoides. A. Necrotic leaf area. B, C. Leaf spots. D. Necrosis surrounded by a chlorotic area.

DISCUSSION

In this study two Paraphoma isolates were recovered from Ca. rapunculoides plants showing leaf spot symptoms in Piedmont, Northern Italy during 2021, and identified based on single and multi-locus (ITS, tub2 and tef1) phylogenetic analyses, as well as morphological characters. These analyses revealed the two isolates to represent a novel species erected here as Paraphoma garibaldii.

The robust three-locus based analysis distinguished P. garibaldii from other Paraphoma species, and other genera causing foliar diseases on this crop, such as Alternaria, Coleosporium, Colletotrichum and Stagonosporopsis. In spite on the recent detection of similar leaf diseases caused by other fungal species in the same geographic area (Guarnaccia ), P. garibaldii was the only fungus associated with leaf spot disease in this survey, demonstrating it was able to cause leaf spot disease independently. Furthermore, pathogenicity tests confirmed that P. garibaldii causes the disease on Ca. rapunculoides, thereby fulfilling Koch’s postulates.

This study has revealed and characterised a novel pathogenic fungal species, P. garibaldii, associated with leaf spot on Campanula rapunculoides, which is one of the most common ornamental bedding plants in Italy. As no epidemiological data are yet available, it is not possible to suggest any control strategies to control P. garibaldii infections. Several previous studies in the same geographical area have revealed a wide diversity of soil- and air-borne fungal species (Garibaldi ), including more taxa pathogenic to Campanula spp. (Guarnaccia ). Further surveys are required to determine the distribution of P. garibaldii, as it might represent a limiting factor for future cultivation of Ca. rapunculoides.