Soilborne diseases caused by Fusarium and Neocosmospora spp. on ornamental plants in Italy

Abstract

During surveys conducted in 2010-2014, several disease symptoms were observed on eight ornamental plant species in different nurseries located in Sicily (Southern Italy). Two Neocosmospora and 31 Fusarium isolates were recovered from symptomatic plants. Fungus identification was based on morphological characteristics and phylogenetic analyses of fragments of the intergenic spacer (IGS), internal transcribed spacer (ITS) and large subunit (LSU) regions of the rDNA; partial fragments of the beta-tubulin (TUB), RNA polymerase largest subunit (RPB1), RNA polymerase second largest subunit (RPB2) and translation elongation factor 1-alpha (EF-1a) genes. The Fusarium species recovered from ornamental plants were F. agapanthi (from Agapanthus africanus), F anthophilum (Dasylirion longissimum), F fujikuroi (Trachycarpus princeps), F. oxysporum (Bougainvillea glabra, Cordyline australis 'Purpurea', Dasylirion longissimum, Eremophila laanii and Philoteca myoporoides) and F proliferatum (T. princeps), while N. solani was isolated from crown and root rot of Ficus carica. The pathogenicity of representative isolates collected from each host was tested on seedlings or cuttings grown in a growth chamber. All the Fusarium and Neocosmospora isolates tested were pathogenic and reproduced symptoms identical to those observed in the field, except for F. fujikuroi on T. princeps and F. oxysporum on D. longissimum that were non-pathogenic.

Headnote

Abstract. During surveys conducted in 2010-2014, several disease symptoms were observed on eight ornamental plant species in different nurseries located in Sicily (Southern Italy). Two Neocosmospora and 31 Fusarium isolates were recovered from symptomatic plants. Fungus identification was based on morphological characteristics and phylogenetic analyses of fragments of the intergenic spacer (IGS), internal transcribed spacer (ITS) and large subunit (LSU) regions of the rDNA; partial fragments of the beta-tubulin (TUB), RNA polymerase largest subunit (RPB1), RNA polymerase second largest subunit (RPB2) and translation elongation factor 1-alpha (EF-1a) genes. The Fusarium species recovered from ornamental plants were F. agapanthi (from Agapanthus africanus), F anthophilum (Dasylirion longissimum), F fujikuroi (Trachycarpus princeps), F. oxysporum (Bougainvillea glabra, Cordyline australis 'Purpurea', Dasylirion longissimum, Eremophila laanii and Philoteca myoporoides) and F proliferatum (T. princeps), while N. solani was isolated from crown and root rot of Ficus carica. The pathogenicity of representative isolates collected from each host was tested on seedlings or cuttings grown in a growth chamber. All the Fusarium and Neocosmospora isolates tested were pathogenic and reproduced symptoms identical to those observed in the field, except for F. fujikuroi on T. princeps and F. oxysporum on D. longissimum that were non-pathogenic.

Keywords. Morphology, multigene phylogeny, pathogenicity, root rot, wilt.

INTRODUCTION

During the last decade, Italy has significantly increased production of ornamental plants in nurseries, and several new species and products have been introduced for cultivation in greenhouses and open fields. Movement of ornamental plants through the peninsula led to the spread of pathogens to new areas, and introduction of new pathogens from abroad (Gullino and Garibaldi, 2006; Polizzi et al., 2012; Aiello et al., 2017, 2018).

In Sicily (Southern Italy), production of ornamentals has increased in the eastern area, where it replaced lemon orchards due to decline in demand for these fruits. Plant growth in nurseries is compromised by several foliar and root diseases, and among these diseases those caused by species of Nectriaceae are exceptionally common (Polizzi et al., 2007; Vitale et al., 2009; Aiello et al., 2014, 2015; Gullino et al, 2015).

Fusarium Link sensu lato was recently segregated into several Fusarium-like genera (i.e., Bisifusarium L. Lombard, Crous & W. Gams [Fusarium dimerum species complex (SC)], Neocosmospora E.F. Sm. [Fusarium solani SC] and Rectifusarium L. Lombard, Crous & W. Gams [Fusarium ventricosum SC]). These taxa are among the most important human, animal or plant pathogens, affecting an extensive variety of hosts (O'Donnell et al., 2008, 2010; Lombard et al., 2015). Fusarium and Fusarium-like genera are well-known as responsible for diseases on ornamental plants, including flowering crops, herbaceous ornamentals such as begonia, carnation and chrysanthemum, woody ornamentals such as Bougainvillea, Hebe, Hibiscus and Pyracantha spp. (Horst and Nelson, 1997; Sinclair and Lyon, 2005; Polizzi et al., 2010a, 2010b, 2011; Bertoldo et al., 2015; Lupien et al., 2017), and palms such as Arecastrum, Phoenix and Washingtonia spp. (Elliott et al., 2004).

Considering the importance of diseases caused by Fusarium-like fungi, the high economic losses caused by these pathogens and the relevance of these crops, surveys were conducted over a 5-year period in ornamental nurseries located in the Catania province, eastern Sicily, Italy. During the surveys conducted from 2010 to 2014, large numbers of palms, perennial herbaceous shrubs, and young cuttings were detected showing symptoms of crown and root rots, damping-off, wilt and dieback. The aims of the present study were to identify the Fusaria obtained from these affected ornamentals, using morphological characteristics and DNA sequence analyses, and to evaluate the pathogenicity of representative isolates on the hosts from which they were isolated.

MATERIALS AND METHODS

Field sampling and pathogen isolations

During 2010-2014, surveys were performed in ornamental plant-producing regions located in eastern Sicily (Table 1). The disease incidence (DI) was recorded for each host, based on the number of symptomatic plants in the total of those present in five investigated nurseries. Additionally, approx. 20 plants per species per nursery showing wilt, crown or root rot or damping-off symptoms, were randomly collected for analysis. Fragments (each 5x5 mm) of symptomatic tissues were cut from the margins of lesions, surface-sterilised in a sodium hypochlorite solution (10%) for 20 s, followed by 70% ethanol for 30 s, and rinsed three times in sterilised water. Tissue fragments were dried in sterilised filter paper, placed on 2% potato dextrose agar (PDA) amended with 100 pg mL-1 penicillin and 100 pg mL-1 streptomycin (PDA-PS), and were incubated at 25°C until characteristic Fusarium-like colonies were observed. Pure cultures were obtained by transferring single conidia to fresh PDA, with the aid of a Nikon SMZ1000 dissecting microscope.

Fungal isolates and morphological characterization

The cultural and micromorphological features of all the isolates included in this study were evaluated fol- lowing the procedures of Aoki et al. (2003), with some modification as described previously (Sandoval-Denis et al., 2018).

DNA extraction, PCR amplification and sequencing

Fungus isolates were grown on PDA for 4-7 d at room temperature, under a natural day/night photoperiod. Total genomic DNA was extracted from fresh mycelium scraped from each colony surface, using the Wizard" Genomic DNA purification Kit (Promega Corporation). Fragments of seven nuclear loci, including the translation elongation factor 1-alpha (EF-1a), the intergenic spacer region of the rDNA (IGS), the internal transcribed spacer region of the rDNA (ITS), the large subunit of the rDNA (LSU), the RNA polymerase largest subunit (RPB1), RNA polymerase second largest subunit (RPB2) and beta-tubulin (TUB), were PCR amplified as described previously (O'Donnell et al., 2009; 2010, Sandoval-Denis et al., 2018). The PCR products were sequenced using the following primer pairs: EF-1/EF-2 for EF-1a (O'Donnell et al., 2008), iNL11/iCNS1 plus the internal sequencing primer pair NLa/CNSa for IGS (O'Donnell et al., 2009), ITS4/ITS5 for ITS (White et al., 1990), LR0R/LR5 for LSU (Vilgalys and Hester, 1990; Vilgalys and Sun, 1994), Fa/G2R for RPB1 (O'Donnell et al., 2010), 5f2/7cr and 7cf/11ar for RPB2 (Liu et al., 1999; Sung et al., 2007), and 2Fd/4Rd for TUB (Woudenberg et al., 2009). Sequences generated in this study were uploaded to the GenBank and the European Nucleotide Archive (ENA) databases.

Phylogenetic analyses and molecular identification

Sequence alignments were performed individually for each locus using MAFFT on the European Bioinformatics Institute (EMBL-EBI) portal (http://www.ebi. ac.uk/Tools/msa/maffi/). BLASTn searches on GenBank, and pairwise sequence alignments on the Fusarium MLST database of the Westerdijk Fungal Biodiversity Institute (http://www.westerdijkinstitute.nl/fusarium/), were performed using EF-1a and RPB2 sequences. This was to assess the distribution of the Fusaria isolates among the different Fusarium species complexes or Neocosmospora. Following this preliminary identification, different loci combinations were selected for each of the Fusarium species complexes and Neocosmospora isolates, according to the phylogenetic informativeness for each locus as compiled in published literature. These combinations were as follows: EF-1a, ITS, RPB1, RPB2 and TUB for the F. fujikuroi species complex (FFSC) (Edwards et al. 2016); EF-1a and IGS for the F. oxysporum species complex (FOSC), and collapsed to haplotypes according to O'Donnell et al. (2009); EF-1a, ITS, LSU and RPB2 for the genus Neocosmospora (O'Donnell et al., 2008).

The different gene datasets were analysed independently and combined, using Maximum likelihood (ML) and Bayesian methods (BI) as described previously (Sandoval-Denis et al., 2018).

Pathogenicity tests

Pathogenicity tests were performed on potted healthy seedlings or cuttings of all symptomatic species recovered with a subset of 16 representative isolates (Table 2). Each experiment was conducted twice, obtaining similar results in both tests. For each experiment three replicates per isolate were used with 20 to 50 plants per replicate. All plants were inoculated by placing two colonised 1 cm2 plugs (PDA from 9-d-old mycelium cultures, grown at 25 ± 1°C in the dark) at the base of each plant stem. Uninoculated plants for all the host species served as controls. After inoculation, plants were covered with a plastic bag for 48 h and maintained at 25 ± 1°C and 95% relative humidity (RH) under a 12 h fluorescent light/dark regime until the symptoms were observed. All plants were irrigated two to three times per week, and were examined each week for disease symptoms. Disease incidence (DI) was determined for each host species. Fungi were re-isolated from symptomatic tissues and identified, to fulfil Koch's postulates.

RESULTS

Field sampling and pathogen isolations

Symptoms referable to Fusarium spp. were detected on eight ornamental species in five nurseries investigated in Eastern Sicily, Italy (Figure 1). The diseases were observed on seedlings and unrooted and rooted cuttings (1 to 12-month-old) during propagation stages in the greenhouses. Disease incidence varied from 10 to 50%, according to the host species (Table 1). The symptoms observed on ornamental plants consisted of damping-off, crown and root rot, and wilt (Table 1).

Damping-off consisted of root rot and stem decay at soil level, and occurred on young seedlings. Rotted roots were dark brown or black, and were partially or completely destroyed. Crown rot sometimes occurred in association with root rot. As consequence of crown and root rot, basal leaves turned necrotic while infected plants sometimes wilted and died. Wilted plants had conspicuous vascular brown discolourations from the crown to the canopy.

A total of 33 monosporic Fusarium-like isolates were collected (Table 2). Among these, two isolates were obtained from damping-off, 12 from root rot, and 19 were from wilted plants.

Phylogenetic analyses and species identification

Pairwise sequence alignments on the Fusarium MLST database and GenBank BLASTn searches demonstrated that 16 isolates belonged to the FOSC and 15 isolates to the FFSC, while two isolates were assigned to the genus Neocosmospora (F. solani species complex) (Table 2).

Subsequent more inclusive multilocus phylogenetic analyses identified a total of five Fusarium spp. and one Neocosmospora sp. The alignment characteristics and statistics are summarized in Table 3. The phylogenetic analyses of the 15 FFSC isolates from ornamentals revealed a total of four species (F. agapanthi O'Donnell, T. Aoki, J. Edwards & Summerell, F. anthophilum (A. Braun) Wollenw., F. fujikuroi Nirenberg and F. proliferatum) from different hosts (Figure 2). Isolates belonging to FOSC were studied based on a two-gene analysis using EF-1a and IGS sequences and incorporated in the original alignments previously published by O'Donnell et al. (2009) including representatives of 257 known FOSC haplotypes. The FOSC isolates from ornamentals belonged to 15 different haplotypes: isolates CPC 27748 and 22749, from Cordyline australis 'Purpurea' showed identical DNA sequences, and corresponded to haplotype 122 of FOSC; isolate CPC 27733 from Philoteca myoporoides belonged to haplotype 188, while each of the remaining isolates corresponded to a previously undescribed haplotype (Figure 3). The phylogeny of the genus Neocosmospora was based on EF-1a, ITS, LSU and RPB2 sequences, and showed that two isolates from Ficus carica (CPC 27736 and 27737) belonged to N. solani (Martius) L. Lombard & Crous (= F. solani) (Figure 4).

Pathogenicity tests

Fourteen Fusarium and two Neocosmospora isolates tested were pathogenic to the different inoculated original hosts, and produced symptoms similar to those observed on diseased plants in nurseries (Figure 1). Two isolates were non-pathogenic. Damping-off occurred on Agapanthus africanus, crown and root rot on F. carica and root rot with subsequent leaf chlorosis appeared on Trachycarpus princeps. The remaining host plants showed vascular discolouration and wilted. The DI (%) caused by Fusarium and Neocosmospora species on different hosts ranged from 75 to 100%, after 15 d to 3 months (Table 2).

All F. agapanthi, F. proliferatum, and N. solani isolates were pathogenic, and caused 100% DI on A. africanus, T. princeps and F. carica, whereas F. anthophilum caused disease with lower DI on Dasylirion longissimum (75%). Fusarium oxysporum isolates gave high DI (100%) on Bougainvillea glabra, C. australis 'Purpurea', Eremophila laanii and P. myoporoides, but was non-pathogenic on D. longissimum. Similarly, F. fujikuroi caused no symptoms on the original host T. princeps. The pathogens were re-isolated from the artificially inoculated plants, and were identified as previously described, fulfilling Koch's postulates. No symptoms were observed on control (uninoculated) plants.

DISCUSSION

The most important plant pathogenic Fusarium species is the soil-borne F. oxysporum Schltdl. (Gordon and Martyn, 1997; Gullino et al., 2012), currently encompassing nearly 150 formae speciales (ff. spp.) and races. The broad host plant range of this fungus includes valuable ornamental plants such as Chrysanthemum, Dianthus, Gerbera, Gladiolus, and Lilium spp. (Engelhard and Woltz, 1971; Linderman, 1981; Farr and Rossman, 2018), on which it causes symptoms ranging from vascular wilt to crown and root rot (Engelhard and Woltz, 1971; Linderman, 1981).

Fusarium proliferatum (Matsush.) Nirenberg ex Gerlach & Nirenberg is another important species, which has been described as the causal agent of blight, dieback and wilt of several palms belonging to the genera Chamaerops, Phoenix, Ravenea, Trachycarpus and Washingtonia (Polizzi and Vitale, 2003; Armengol et al., 2005).

In the present study, two Neocosmospora and 31 Fusarium isolates were recovered from eight ornamental species in Sicily over a 5-year period. Disease symptoms were observed in five ornamental nurseries, and included damping-off, crown and/or root rot, and wilt. The isolates obtained from symptomatic tissues were identified based on single and multilocus phylogenetic analyses of seven loci (EF-1a, IGS, ITS, LSU, RPB1, RPB2, and TUB), as well as morphological characters. Our study revealed considerable diversity in the composition of Fusarium-like fungal populations recovered from nurseries.

As confirmed in the pathogenicity tests, all the F. oxysporum isolates caused symptoms except on D. longissimum. However, this host showed disease symptoms when inoculated with F. anthophilum. The inoculated isolate of F. fujikuroi produced no symptoms on T. princeps, while the remaining Fusarium species investigated, F. agapanthi, F. proliferatum and N. solani, were pathogenic to the respective tested hosts from which they were isolated.

Fusarium and Neocosmospora species are widespread in nurseries in Italy (Polizzi et al., 2003; 2010a; 2010b; 2011; Bertoldo et al., 2015), where they represent a limiting factor for production of ornamental plants cultivated in Sicily. These pathogenic species have very broad host ranges worldwide (Farr and Rossman, 2018). However, there are no known reports of diseases caused by F. anthophilum on D. longissimum. Moreover, F. agapanthi was originally described as pathogenic on Agapanthus praecox in Australia and Italy (Edwards et al., 2016). However, this pathogen was isolated in the present study, causing serious seedling damping-off of A. africanus, suggesting that it may also be more prevalent on other species of Agapanthus. Previous studies have reported F. proliferatum associated with palms belonging to the genera Chamaerops, Phoenix, Trachycarpus and Washingtonia (Polizzi and Vitale, 2003; Armengol et al., 2005). Our study presents a new report for F. proliferatum as a pathogen of T. princeps.

The FOSC includes soil-borne pathogens responsible for vascular wilts, stem cankers, rots, and damping-off of a wide range of agronomical and horticulturally important crops (Baayen et al., 2000; Michielse and Rep, 2009; O'Donnell, 2009). Members of this complex collectively represent the most commonly found and economically important species complex within Fusarium. Fusarium oxysporum was the predominant species found in all the nurseries sampled, and unlike other species, it was recovered from multiple hosts. Recently, Polizzi et al. (2010a; 2010b; 2011) identified F. oxysporum associated with wilt diseases of B. glabra, E. laanii and P. myoporoides. However, no reports were previously known of diseases caused by F. oxysporum on C. australis, as reported here.

The present study is also the first report of N. solani causing crown and root rot of F. carica cuttings. This plant species is often cultivated for fruit production, and thousands of cuttings cultivated for ornamental purposes were investigated because serious losses were observed from of crown and root rot, leading to plant death. Neocosmospora is a species-rich genus containing at least 60 phylogenetically distinct species (O'Donnell, 2000; Zhang et al., 2006; O'Donnell et al., 2008; Nalim et al., 2011). These fungi generally cause crown and/or root rot of infected host plants, while symptoms on aboveground plant portions may manifest as cankers, wilting, stunting and chlorosis, or as lesions on stems and/or leaves (Coleman, 2016; Guarnaccia et al., 2018).

The high disease incidence observed in the investigated ornamental nurseries probably depends on the prevailing climatic conditions, farming practices and environmental conditions such as temperature, humidity, irrigation systems or the use of non-disinfected plant growth substrates. Potted plant production could promote infections, since plants are frequently stressed due to being containerised during the production processes. Moreover, several wounds can occur during transplanting. Thus, prevention is a major strategy to control Fusarium diseases, and an accurate diagnosis of Fusaria species occurring in a particular area is significant for the selection of effective disease management strategies.

This study provides the first overview of Fusarium and Neocosmospora diversity associated with diseased ornamental plants in Southern Italy, and includes information on the pathogenicity of these fungi. It also provides the first reports of several new pathogen/host combinations, such as N. solani associated with crown and root rot of F. carica, and F. agapanthi, F. anthophilum, F. oxysporum and F. proliferatum as pathogens, respectively, of A. africanus, D. longissimum, C. australis and T. princeps.

ACKNOWLEDGEMENTS

The authors thank Dr Pietro Formica for support with specimen collection and fungal isolations. This research was funded by Research Project 2016-2018 "Emergent Pests and Pathogens and Relative Sustainable Management Strategies", financed by University of Catania.

Sidebar

Citation: Guarnaccia V., Aiello D., Polizzi G., Crous P.W., SandovalDenis M. (2019) Soilborne diseases caused by Fusarium and Neocosmospora spp. on ornamental plants in Italy. Phytopathologia Mediterranea 58(1): 127-137. doi: 10.13128/Phytopathol_ Mediterr-23587

Accepted: November 19, 2018

Published: May 15, 2019

Copyright: © 2019 Guarnaccia V., Aiello D., Polizzi G., Crous P.W., Sandoval-Denis M.. This is an open access, peer-reviewed article published by Firenze University Press (http:// www.fupress.com/pm) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability Statement: All relevant data are within the paper and its Supporting Information files.

Competing Interests: The Author(s) declare(s) no conflict of interest.

Editor: Lizel Mostert, Faculty of AgriSciences, Stellenbosch, South Africa.

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