Paraconiothyrium fuckelii, Diaporthe eres and Neocosmospora parceramosa causing cane blight of red raspberry in Northern Italy

Red raspberry (Rubus idaeus L.) represents a relevant crop which production has largely increased worldwide during the last decade. Cane blight is one of the most common diseases of red raspberry and it can produce considerable losses. During 2019–2021, surveys were conducted in red raspberry orchards in Cuneo province, Northern Italy, to investigate the etiology and pathogen diversity in association with cane blight. Eleven isolates were collected from symptomatic plants of the cultivars ‘Diamond Jubilee’ and ‘Grandeur’, and were identified through multi-locus phylogenies and morphological features. Three fungal species were identified: Paraconiothyrium fuckelii, Diaporthe eres and Neocosmospora parceramosa. Four different genomic regions were included in the molecular analyses: ITS, tef1, tub2 and rpb2. The species pathogenicity was confirmed and P. fuckelii was revealed as the most aggressive. This study provides an insight into raspberry cane blight in Italy and the genomic loci sequencing lay the basis to develop specific diagnostic protocols to monitor the presence of pathogens and to prevent disease spread, adopting effective integrated control strategies.


Introduction
Red raspberry (Rubus idaeus L., Rosaceae) is a deciduous shrub with a biennial cane habit. Raspberry cultivars can be primocane and floricane based on their fruiting habit (Martin et al. 2017). Primocane raspberries produce fruit during the fall of the first year and during the summer of the second year after plantation. Whilst, floricane fruiting raspberries produce berries only on the second year of growth. Primocane cultivars offer the convenience to extend the cropping season, with reduction of costs and higher fruit quality. Thus, different breeding programmes were conducted worldwide to promote the use of these cultivars and to increase yield, fruit quality and biotic and abiotic stress tolerance (Graham and Brennan 2018).
Red raspberry is considered a crop of global significance for its production and economic value and, due to the provided health benefits, its cultivation has increased worldwide (Graham and Brennan 2018; Retamales and Hancock 2012;Zhang et al. 2018). Europe is a leading region for raspberry production with more than 103,000 ha cultivated mainly in the northern and eastern regions (FAOSTAT 2020). In Italy, during the last decade, an increasing interest was observed in berry fruit cultivation and raspberry production achieved 2,280 tonnes with 360 ha cultivated in 2020 (FAOSTAT 2020). The rising importance of this crop led to more intensive cultivation systems (Graham and Brennan 2018). All berry fruit crops represent an expanding sector within Italian agriculture; they are included among the major fresh fruit exported during the last decade (FAOSTAT 2020). Orchards are usually established under plastic nets and fibre cover or in plastic tunnels to increase the temperature and to extend the growing season of the crop (Graham and Brennan 2018; Retamales and Hancock 2012). However, the intensive production system, the introduction of plant material from foreign countries as well as new cultivars, can promote the introduction of emerging or new pathogens (Dolan et al. 2018). Cane blight represents one of the most relevant diseases caused by fungal pathogens on raspberry plants (Martin et al. 2017). The disease affects canes that were wounded during their vegetative year causing xylem infections, bud failure, lateral shoot wilt and, in severe cases, cane death. Lesions are usually represented by dark red to purple areas around wounds with irregular dark purple borders (Graham and Brennan 2018). The fungal species Paraconiothyrium fuckelii is reported as the causal agent of raspberry cane blight (Martin et al. 2017). Synonyms of this species are Leptosphaeria coniothyrium, Coniothyrium fuckelii, Microsphaeropsis fuckelii and Kalmusia coniothyrium (Damm et al. 2008;De Gruyter et al. 2013;Mikulic-Petkovsek et al. 2014;Verkley et al. 2004). Verkley et al. (2014) renamed it after a reassessment within the Montagnulaceae family. This pathogen is also known to cause stem canker of Rosa sp. (Horst and Cloyd 2007;Jamali 2020) and it was reported in association with other woody hosts species in the genera Malus, Prunus, Ribes and Vaccinium (Bérubé and Nicolas 2015;Chen et al. 2017;Farr and Rossman 2021). Paraconiothyrium fuckelii usually overwinters on dead floricanes as pseudothecia that produce ascospores or as pycnidia that produce conidia. Thus, old floricanes represent the primary source of inoculum in Rubus orchards (Martin et al. 2017). Little information is known about the infectivity of ascospores, whilst conidia could be dispersed by splashing rains from early spring to late autumn and could enter through cane wounds causing infection and considerable losses in raspberry production (Dolan et al. 2018). Girichev et al. (2018) reported raspberry cane disease as caused by a complex of different phytopathogenic fungi. Moreover, several fungal species can survive as saprophytes on canes with a role in disease development (Girichev et al. 2018). Thus, it is necessary to clearly understand the etiology of raspberry cane blight to identify the fungal species associated with symptomatic or asymptomatic plant materials and their role in the disease development. Previous studies reported the influence of the raspberry genotype on the resistance to cane diseases caused by Botrytis cinerea, Didymella applanata and Leptosphaeria coniothyrium (White et al. 1990). Therefore, etiological studies on pathogens related to different raspberry cultivars could set the basis for new breeding programs.
Several berry fruit orchards were monitored over a three years period (2019-2021) in Piedmont, Northern Italy.
Etiological and pathogenic studies were conducted to (i) assess the fungal species diversity associated with raspberry cane blight using molecular and phylogenetic tools, (ii) to characterize the species phenotypes, and (iii) to test the pathogenicity of the species found on different cultivars, fulfilling Koch's postulates.

Field sampling and fungal isolation
During the period May 2019 to April 2021, surveys were conducted in three raspberry plantations in Falicetto, Lagnasco and Manta (Cuneo, Piedmont, Italy) ( Table 1). Samples from whole symptomatic plants of the primocane cultivars 'Grandeur' and 'Diamond Jubilee' were collected from the part showing stripe lesions on canes, and transferred in the laboratory. Disease incidence (DI) was assessed on 100 observed plants in each of the three orchards, and the average percentage of symptomatic plants was determined. Twenty symptomatic plants were collected from each orchard. The wood samples (5-10 mm) were surface sterilised in 1% sodium hypochlorite for 1 min, rinsed in sterile distilled water (SDW) for 1 min and dried on sterile absorbent paper. Small fragments (2-3 mm) were cut from the margin between healthy and necrotic tissues and plated on potato dextrose agar (PDA, Oxoid) amended with 25 ppm of streptomycin sulphate (PDA-S, Sigma-Aldrich). The plates were incubated at 25 ± 1 °C under a 12 h photoperiod. Following 48 to 72 h of incubation, mycelium plugs from the margins of resulting colonies were placed on new PDA plates. After 5 d, pure cultures were established from single hyphal tip transfers. A total of 11 isolates were obtained and 11 of these were selected based on colony aspect and used for molecular characterization (Table 2). Stock cultures of these isolates are kept at -80 °C in the AGROINNOVA Centre of Competence (University of Torino) culture collection, Torino, Italy.

DNA extraction, polymerase chain reaction (PCR) amplification, and sequencing
Genomic DNA was extracted from 0.1 g of mycelium grown on PDA, using the E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-Tek), following the manufacturer's instructions. Species Lagnasco (Cuneo, Piedmont) 44°37′20.9"N 7°33′05.6"E 'Diamond jubilee' 70% 3 Verzuolo (Cuneo, Piedmont) 44°35′51.8"N 7°30′02.4"E 'Grandeur' 80%  identification was achieved through DNA amplification and sequencing of a combined dataset of genes: the nuclear ribosomal internal transcribed spacer (ITS) region, partial regions of the β-tubulin (tub2), translation elongation factor-1α (tef1) and RNA polymerase second largest subunit (rpb2) genes. The primers used for each locus found are reported in Table 3. The PCR amplification mixtures and cycling conditions adopted for the analyses of ITS, tub2, tef1 conducted on Paraconiothyrium isolates were followed as described in Aveskamp et al. (2009) and Zhang et al. (2009). The ITS, tub2, tef1 regions analysed for Diaporthe isolates were amplified according to PCR protocols reported by . For the isolates identified as Neocosmospora isolates, the protocols were adopted according to Guarnaccia et al. (2021) for the ITS, tef1 and first part of rpb2 loci. For the part of rpb2 region amplified with the primers RPB2-7cf and RPB2-11ar, the PCR protocol by Yilmaz et al. (2020)

Phylogenetic analyses
New sequences obtained in this study were BLASTed against the NCBI's GenBank nucleotide database to determine the closest relatives for a taxonomic framework of the studied isolates. Alignments of different gene regions, including sequences obtained from this study and sequences downloaded from GenBank, were initially   (Katoh and Standley 2013), and then manually adjusted in MEGA v. 7 (Kumar et al. 2016). After an initial analysis of the ITS locus (data not shown), a subset of representative isolates was selected to clearly distinguish the isolates at species level. Phylogeny was processed through different analyses conducted individually for each locus (data not shown) and as multilocus sequence analyses, as described above. The phylogenies were based on Bayesian Inference (BI) and Maximum Parsimony (MP) for the multi-locus analyses. For BI, the best evolutionary model for each partition was determined using MrModeltest v. 2.3 (Nylander 2004) and incorporated into the analyses. MrBayes v. 3.2.5 (Ronquist et al. 2012) 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 set at 0.2 and trees were sampled every 1,000 generations. Analyses stopped when the average standard deviation of split frequencies was below 0.01. The MP analyses were performed using Phylogenetic Analysis Using Parsimony (PAUP) v. 4.0b10 (Swofford and Sullivan 2003). Phylogenetic relationships were estimated by heuristic searches with 100 random addition sequences. Tree bisectionreconnection was used, with the branch swapping option set on 'best trees', with all characters weighted equally and alignment gaps treated as fifth state. Tree length, consistency index, retention index and rescaled consistence index were calculated for parsimony, and the bootstrap analyses (Hillis and Bull 1993) were based on 1,000 replications. Sequences generated in this study were deposited in Gen-Bank (Table 2).

Phenotypic characterization
Agar plugs (5 mm diam.) of representative strains were taken from the edges of actively growing cultures on PDA-S and transferred onto the centre of 9 cm diam. Petri dishes containing PDA or malt extract agar (MEA; Oxoid). Diaporthe eres representative strain was also transferred on 1% water agar supplemented with sterile pine needles (PNA; Smith et al. 1996). All the plates were then incubated at 20-21 °C under a 12 h photoperiod to induce sporulation. Colony characters were observed after 10 d, and culture colours were determined (Rayner 1970). Cultures were examined periodically for the development of conidiomata. Conidia characteristics were examined by mounting fungal structures in SDW, and the lengths and widths of 30 conidia were measured for each isolate using a light microscope at × 40 magnification. The average and standard deviations of conidia dimensions were calculated.

Pathogenicity tests
Pathogenicity tests with representative isolates of the isolated species (P. fuckelii, D. eres and N. parceramosa) were performed to fulfil Koch's postulates. Representative isolates of each species (Table 2) were selected and inoculated on healthy raspberry plants (cv 'Grandeur' and 'Diamond Jubilee'). Each of the three fungal pathogens was inoculated onto 8 raspberry plants of each cultivar, with a total of 24 plants inoculated with pathogens. A sterile scalpel was used to remove the outer bark of the cane at 3-4 cm from the crown. Mycelium plugs (4 mm diam.) were taken from 10-day-old cultures on PDA and placed with the mycelium in contact with the internal plant tissues. The inoculation points were wrapped with Parafilm R . A further 8 plants of each cultivar were treated with sterile PDA plugs as inoculation controls. All the plants were placed in a growth chamber at 24 °C for 6 weeks. After this period, the lengths of lesions developed on the internal cane tissues were measured after peeling off the bark. Fifteen days after inoculation, the symptom severity (SS) associated with each inoculated species was evaluated as the length of vascular discolouration which were measured upward and downward from the inoculation points. Small portions (0.3 cm) of symptomatic tissue from the edge of cane lesions were placed onto PDA-S to re-isolate the fungal species, and these were identified based on their colony characteristics and ITS sequencing. This trial was conducted twice and each trial was considered a replicate. Because no normal distribution was observed in the lesion dimension data, the Kruskal-Wallis non-parametric test (at P = 0.05) was performed to determine significant differences among the strains. The data analysis was conducted using SPSS software 26 (IBM Corporate).

Field sampling and fungal isolation
Symptoms of cane blight were observed in all raspberry orchards surveyed. All the investigated crops were covered by anti-hail nets and presented relative humidity condition up to 78%. Brown stripe lesions in correspondence of wounds were detected in the early stage of the disease. Later, the lesions progressively extended longitudinally trough the canes, showing cracking and causing the death of buds and lateral shoots. In several cases, lesions affected the vascular tissues with consequent cane decay and blight. Considerable vascular damage and cane death occurred in all the orchards and on both the cultivars investigated (Fig. 1). DI was assessed as reported in Table 1. Based on colony morphology, phylogenetic analyses, and conidia characteristics (see below), D. eres was isolated from plant of the cultivar 'Diamond Jubilee', whilst P. fuckelii and N. parceramosa were isolated from both 'Diamond Jubilee' and 'Grandeur' plants.

Phylogenetic analyses
The combined-locus phylogeny of Paraconiothyrium consisted of 21 sequences, Diaporthe consisted of 23 sequences and Neocosmospora of 40, including outgroups. A total of 1,457 characters (ITS: 1-569, tub2: 576-990 and rpb2: 997-1457) were included in the Paraconiothyrium phylogenetic analyses. A total of 1,400 characters (ITS: 1-554, tub2: 561-955 and tef1: 962-1400) were included in the Diaporthe phylogenetic analyses. The analyses for the Neocosmospora group consisted of 2,641 nucleotides (ITS: 1-467, tef1: 474-1131 and rpb2: 1138-2641). A maximum of 1,000 equally most parsimonious trees were saved, and characteristics of the combined gene partitions used for each phylogenetic analysis are reported in Table 4. Bootstrap support values from the parsimony analysis were plotted on the Bayesian phylogenies presented in Figs. 2, 3 and 4. Unique site patterns for each partition and all the parameters of the Bayesian analyses are reported in Table 4. For both of the Bayesian analyses, MrModeltest recommended the models reported in Table 5. In the Paraconiothyrium species analysis four isolates from symptomatic raspberry plants grouped with four reference strains of P. fuckelii (Fig. 2), whilst two isolates of Diaporthe clustered with four reference strains of D. eres (Fig. 3). The final tree generated for Neocosmospora species showed that five isolates clustered with the type specimen and two reference strains of N. parceramosa (Fig. 4).

Phenotypic characterization
Morphological observations, supported by phylogenetic inference, were described for three known species (Fig. 5).

Pathogenicity tests
After 60 days, all the tested isolates caused lesions and internal discolouration on the inoculated plants. In some cases, a severe dieback was observed and plant death occurred. Symptoms were similar to those found in the field-grown plants (Fig. 6). Re-isolation of pathogens was obtained with a frequency of grown colonies from small portions of affected tissues placed onto PDA-S between 90 and 95%. The identification of the re-isolated species was confirmed using culture and molecular features, fulfilling Koch's postulates. Paraconiothyrium fuckelii has revealed to be the most aggressive species on both the inoculated cultivars causing vascular lesion of average length 5.1 and 4.0 cm on 'Grandeur' and 'Diamond Jubilee', respectively. Moreover, P. fuckelii caused the death of 2 of the inoculated 'Grandeur' and 1 of 'Diamond Jubilee' plants. Diaporthe eres and N. parceramosa caused less severe symptoms with mean vascular lesions length of 3.0 and 2.7 cm on 'Grandeur' and 'Diamond Jubilee', respectively, and 1.3 cm on both tested cultivars. The pairwise comparison obtained from the Kruskal-Wallis test, showed significant differences (P < 0.05) among the three isolates (Fig. 7).

Discussion
The increasing interest in berry fruit production promoted the development of new and improved commercial cultivars through several breeding programmes (Foster et al. 2019). Moreover, an intensification of the production systems through agronomic practices, use of irrigation systems and use of fertilizers, occurred to supply the consumers demand (Graham and Brennan 2018;Mezzetti 2014). However, the consequent consistent movement of plant materials and fruit favoured the spread of emerging diseases in different production areas (Dolan et al. 2018;Guarnaccia et al. 2020). The present study was conducted in the most important fruit production area of Piedmont where a strong interest for raspberry cultivation is present. The study provides an investigation of the fungal pathogens associated with raspberry cane blight observed during the conducted surveys. Paraconiothyrium fuckelii, generally considered the causal agent of cane blight (Martin et al. 2017), was consistently isolated from symptomatic raspberry canes. Moreover, D. eres and N. parceramosa were found in association with cane blight disease and produced symptoms on artificially inoculated raspberry plants. The occurrence of different fungal species demonstrated the presence of a complex of fungi involved in the development of raspberry cane diseases as described by Girichev et al. (2018). Thus, considering the difficulty to distinguish different pathogens from symptoms or based on morphological traits, it becomes necessary the use of molecular tools to provide a correct identification (Girichev et al. 2018). The multi-locus analyses conducted in this work identified the three fungal pathogens. Paraconiothyrium fuckelii is known worldwide as the only causal agent of raspberry cane blight disease. It has been reported, with its previous taxonomic synonyms, on R. idaeus in Australia, Bulgaria, Canada, New Zealand, Poland, Russia and USA (Farr and Rossman 2021). In Italy, it has been found in association with withered grapes (Lorenzini et al. 2016).
Diaporthe spp. are important plant pathogens reported in association with stem bight and cankers of different fruit trees (Guarnaccia et al. 2020;Hilário et al. 2020;Yang et al. 2018). Their identification, mainly based on morphological traits and plant hosts, has resulted in a high number of described species. Thus, during the last years, multilocus phylogenies were adopted to reassess species within Diaporthe genus (Santos et al. 2017). The genomic loci sequencing provided in this study demonstrated the identification of  (Guarnaccia et al. , 2019Sandoval-Denis et al. 2018), however, N. parceramosa (synonym: Fusarium parceramosum) has been mainly isolated as presumed human pathogen and as present in plumbing systems, whilst the type specimen (CBS 115,695) was obtained from a soil sample collected in South Africa The correct identification of this species was achieved by adding the second part of the rpb2 region to the multi-locus sequence alignments analysed. Thus, demonstrating the importance of molecular diagnostic tools as reliable, objective, and necessary for correct species analysis. To our knowledge, this work represents the first report of N. parceramosa on R. idaeus in Italy and worldwide, and the first report of P. fuckelii and D. eres on raspberry in Italy.
The species collected during the surveys were confirmed as pathogenic. However, a different susceptibility among the tested cultivars was registered. Plants of the cv 'Grandeur' were consistently affected by the fungal pathogens and cane death occurred abundantly, whilst lower lesion length was observed on 'Diamond Jubilee' plants cv. Varietal resistance is a fundamental point to consider before orchard establishment, along with the use of certified plant materials. Favourable temperatures and high levels of relative humidity can also play a major role, thus, the interaction between found pathogens and environment temperature should be assayed. Moreover, good agronomic practices should be adopted to minimize the occurrence of wounds that can facilitate the pathogens entry within the vascular tissues. Providing correct distances among plants and regular cane thinning, reducing wound formation during berry harvesting, removing pruning debris and infected canes could be useful methods to reduce fungal pathogen spread, thus should be investigated.
Furthermore, it should be considered that weather conditions could promote pathogen and disease development.
Studies conducted on D. eres in Greece showed that this pathogen has an optimal growth at 25 °C, however, considerable mycelial growth was assessed within the range 20-30 °C (Thomidis and Michailides 2009). Other analyses on water availability and relative humidity confirmed the influence of these abiotic factors on D. eres mycelial growth and conidia germination (Arciuolo et al. 2021). Wet and humid conditions have been reported as favourable also for Paraconiothyrium fuckelii development (Martin et al. 2017). Thus, periods with high temperature and high relative humidity could contribute to the spread of this pathogen. Moreover, cross infections within different plant hosts might occur, concurring to the diffusion of the disease among different orchards . Further studies are needed to understand the epidemiology of raspberry cane blight with aim of monitoring disease occurrence and to develop prediction models for the adoption of effective management strategies.
In conclusion, the present work is the first insight into raspberry cane blight in Italy. The provided phylogenetic analyses and genomic loci sequencing set the basis for the development of species-specific diagnostic protocols to detect pathogen presence early in nursery and orchard environment. Further analyses are necessary to establish the role of abiotic factors and the relationships within the fungal species found in disease development and spread. The investigation of these aspects could permit the adoption of more sustainable management strategies and contribute to the constant reduction of chemical fungicides. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. The data that support the findings of this study are available from.
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Fig. 7
Box plot showing the results of the pathogenicity tests. Boxes represent the interquartile range, while the horizontal line within each box indicates the average value. The Kruskal-Wallis test was carried out to compare the mean lesion lengths (cm) from inoculation with three representative isolates. Significant difference was accepted for P < 0.05. ° Outliers. *: Extreme values