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Article

Identification and Characterization of Diaporthe spp. Associated with Twig Cankers and Shoot Blight of Almonds in Spain

by
Maela León
1,
Mónica Berbegal
1,
José M. Rodríguez-Reina
2,
Georgina Elena
1,
Paloma Abad-Campos
1,
Antonio Ramón-Albalat
1,
Diego Olmo
3,
Antonio Vicent
4,
Jordi Luque
5,
Xavier Miarnau
6,
Carlos Agustí-Brisach
7,
Antonio Trapero
7,
Nieves Capote
8,
Francisco T. Arroyo
8,
Manuel Avilés
9,
David Gramaje
10,
Marcos Andrés-Sodupe
10 and
Josep Armengol
1,*
1
Instituto Agroforestal Mediterráneo, Universitat Politècnica de València, Camino de Vera S/N, 46022 Valencia, Spain
2
Departamento de Ecosistemas Agroforestales, Universitat Politècnica de València, Camino de Vera S/N, 46022 Valencia, Spain
3
Laboratori de Sanitat Vegetal, Serveis de Millora Agrària, Conselleria d’Agricultura, Medi Ambient i Territori, Govern Balear, 07009 Palma de Mallorca, Spain
4
Centre de Protecció Vegetal i Biotecnologia, Institut Valencià d’Investigacions Agràries (IVIA) Moncada, 46113 Valencia, Spain
5
Plant Pathology, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Carretera de Cabrils km 2, 08348 Cabrils, Spain
6
Fruit Production, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Fruitcentre Building, PCiTAL, Park of Gardeny, 25003 Lleida, Spain
7
Departamento de Agronomía, ETSIAM, Universidad de Córdoba, Campus de Rabanales, Edif. C4, 14071 Córdoba, Spain
8
IFAPA Centro Las Torres, Ctra. Sevilla-Cazalla km 12,2, 41200 Alcalá del Río, Sevilla, Spain
9
Departamento de Ciencias Agroforestales, Escuela Técnica Superior de Ingeniería Agronómica, Universidad de Sevilla, Ctra. Utrera km 1, 41013 Sevilla, Spain
10
Instituto de Ciencias de la Vid y del Vino (ICVV), Consejo Superior de Investigaciones Científicas–Universidad de la Rioja–Gobierno de La Rioja, Ctra. de Burgos Km. 6, 26007 Logroño, Spain
*
Author to whom correspondence should be addressed.
Agronomy 2020, 10(8), 1062; https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy10081062
Submission received: 4 July 2020 / Revised: 21 July 2020 / Accepted: 22 July 2020 / Published: 23 July 2020
(This article belongs to the Special Issue New Trends in Disease and Pest Management: Challenges and Opportunities)
Browse Figures
Versions Notes

Abstract

:
Two hundred and twenty-five Diaporthe isolates were collected from 2005 to 2019 in almond orchards showing twig cankers and shoot blight symptoms in five different regions across Spain. Multilocus DNA sequence analysis with five loci (ITS, tub, tef-1α, cal and his), allowed the identification of four known Diaporthe species, namely: D. amygdali, D. eres, D. foeniculina and D. phaseolorum. Moreover, a novel phylogenetic species, D. mediterranea, was described. Diaporthe amygdali was the most prevalent species, due to the largest number of isolates (85.3%) obtained from all sampled regions. The second most frequent species was D. foeniculina (10.2%), followed by D. mediterranea (3.6%), D. eres and D. phaseolorum, each with only one isolate. Pathogenicity tests were performed using one-year-old almond twigs cv. Vayro and representative isolates of the different species. Except for D. foeniculina and D. phaseolorum, all Diaporthe species were able to cause lesions significantly different from those developed on the uninoculated controls. Diaporthe mediterranea caused the most severe symptoms. These results confirm D. amygdali as a key pathogen of almonds in Spain. Moreover, the new species, D. mediterranea, should also be considered as a potential important causal agent of twig cankers and shoot blight on this crop.

1. Introduction

The worldwide cultivated area for almond (Prunus dulcis (Mill.) D.A. Webb) is over 2,000,000 ha. Spain, with 657,768 ha, is the country with the largest area for almond production in the world, followed by the United States, with 441,107 ha [1]. Almond is the second largest tree crop in Spain, after olive, and it is widely distributed in all regions of the country [2]. Nevertheless, Spain only contributes approximately 10% to world almond production, because the trees have been traditionally grown under rain-fed conditions and planted in marginal areas with poor soils, low rainfall and a high incidence of frost [3], thus presenting low average yields (5154 kg ha−1) [1].
In recent years, almond production in Spain has been experiencing a highly favorable period, in which crop intensification, with the introduction of drip irrigation and the use of new highly productive cultivars, has increased the yield in new plantations [4]. However, the incidence of almond-associated fungal diseases, such as twig cankers and shoot blight caused by Diaporthe spp., is increasing and compromises crop productivity, especially in coastal areas with higher humidity and milder temperatures [5,6].
Diaporthe amygdali (Delacr.) Udayanga, Crous and K.D. Hyde is considered the causal agent of twig canker and shoot blight of almond and peach (Prunus persica (L.) Batsch) [7,8]. Symptoms of this disease are characterized by the quick desiccation of buds, flowers and leaves in late winter or early spring. Brown lesions (1 to 5 cm diameter), initially formed around buds on green shoots, further develop into annual sunken cankers, sometimes with a gummy exudate, as well as withering of twigs. As a result, leaves wilt and, when the disease is severe, defoliation can occur. In summer, pycnidia develop just under the dry canker epidermis [7,9,10].
The species D. amygdali was first described as Fusicoccum amygdali Delacr., associated with almond cankers in France [11]. Tuset and Portilla [9] re-examined the type specimen of F. amygdali and, based on morphology and symptomatology, they re-classified this fungus into Phomopsis as P. amygdali (Delacr.) J.J Tuset and M.T. Portilla. Additionally, they also considered P. amygdalina Canonaco to be a synonym of P. amygdali. Diogo et al. [8] used morphological, molecular and pathogenicity data to clarify the identity of a collection of Phomopsis isolates obtained from almond in Portugal. In this research, as no cultures of P. amygdali were linked unequivocally to any existing type, the authors proposed the fungus in voucher CBS-H 20420 (from Portugal) as the epitype for this species (isolate CBS 126679). Udayanga et al. [12] re-evaluated the phylogenetic species recognition in the genus Diaporthe using a multi-locus phylogeny based on the internal transcribed spacer (ITS) region of the nuclear rDNA, and partial sequences from translation elongation factor 1-α (tef-1α), β-tubulin (tub) and calmodulin (cal) genes. In this study, P. amygdali was transferred into Diaporthe as D. amygdali based on multi-locus DNA sequence data.
In recent years, the taxonomy of the genus Diaporthe has been deeply revised. The generic names Diaporthe and Phomopsis are no longer used to distinguish different morphs of this genus, as Rossman et al. [13] proposed that the genus name Diaporthe should be retained over Phomopsis because: (i) it was introduced before Phomopsis and (ii) Diaporthe represents the majority of species described, and therefore it has priority over Phomopsis. Diaporthe was historically considered as monophyletic based on its typical sexual morph and Phomopsis’s asexual morph [14]. However, Gao et al. [15] revealed its paraphyletic nature. Recent studies have demonstrated that morphological characters are inadequate to define species in this genus [16], due to their variability under changing environmental conditions [14]. Therefore, genealogical concordance methods based on multi-gene DNA sequence data provide a better approach to resolving the taxonomy for Diaporthe [17].
Literature about recent characterization studies of collections of Diaporthe isolates, obtained exclusively from almonds or including them together with isolates from other fruit or nut crops, is very scarce. Diogo et al. [8] examined Diaporthe isolates from almond and other Prunus species in Portugal through combining morphology, pathogenicity data and a phylogenetic study based only on ITS sequences. These authors concluded that D. amygdali was the main species on almond, reported D. neotheicola for the first time on this host and a third species represented by a single isolate, which could not be unequivocally identified. Later, Lawrence et al. [18] characterized morphologically different Diaporthe isolates associated with wood cankers of fruit and nut crops in northern California, including three almond isolates, which were assigned to the species D. australafricana and D. novem, based on multi-gene, ITS, tef-1α and cal sequence analyses.
In Spain, the studies of Tuset and Portilla [9] and Tuset et al. [10] described almond diseases and their associated pathogens, including D. amygdali. These studies were based solely on the morphological characterization of the isolates. Additional studies using molecular tools to ascertain the identity of representative sets of Diaporthe isolates from almond in this country are lacking. Gramaje et al. [19] reported only one isolate of D. amygdali, which was collected from a survey of wood-associated fungal trunk pathogens of almond trees on the island of Mallorca. Thus, the objectives of the present study were: (i) to characterize a wide collection of Diaporthe isolates collected from almond trees in Spain by means of phenotypical characterization (fungal morphology and temperature growth) and DNA sequence analyses and (ii) to evaluate the pathogenicity of these Diaporthe isolates to almond twigs. The final goal was to obtain updated and more complete information about the Diaporthe species causing twig cankers and shoot blight of almonds in Spain.

2. Materials and Methods

2.1. Sampling and Isolation

A total of 225 Diaporthe isolates were collected from 2005 to 2019 in almond orchards showing twig cankers and shoot blight symptoms (Figure 1) in five different regions across Spain (Andalucía (n = 56), Islas Baleares (n = 39), Cataluña (n = 43), Comunidad Valenciana (n = 76) and La Rioja (n = 11)). For isolation, wood segments with cankers were cut from the affected branches, washed under running tap water, surface disinfected for 1 min in a 1.5% sodium hypochlorite solution and rinsed twice in sterile distilled water. Small pieces of affected tissues taken from the margin of the lesions were plated on potato dextrose agar (PDA; Biokar-Diagnostics, Zac de Ther, France) supplemented with 0.5 g/L of streptomycin sulphate (Sigma-Aldrich, St. Louis, MO, USA) (PDAS). Plates were incubated at 25 °C in the dark for 7 to 10 d, and all colonies were transferred to PDA. All isolates were hyphal-tipped and maintained in 15% glycerol solution at −80 °C in 1.5 mL cryovials in the fungal collection of the Instituto Agroforestal Mediterráneo–Universitat Politècnica de València (IAM-UPV) (Spain) (Table 1).

2.2. DNA Extraction, PCR Amplification and Sequencing

Mycelium was scraped from 10-day-old fungal cultures grown on PDA medium. Total fungal DNA was extracted using the E.Z.N.A. Plant DNA Kit (Omega Bio-tek, Norcross, GA, USA), following the manufacturer’s short protocol instructions.
The ITS region and fragments of tub and tef-1α genes were amplified and sequenced. Based on these preliminary results, representative isolates were selected for amplifying and sequencing cal and histone H3 (his) genes. Amplification by polymerase chain reaction (PCR) was performed in a total volume of 25 μL using HotBegan™ Taq DNA Polymerase (Canvax Biotech SL, Córdoba, Spain), according to the manufacturer’s instructions on a Peltier Thermal Cycler-200 (MJ Research). One reaction was composed of 2.5 μL of 10× PCR Buffer B, 2.5 μL of MgCl2 (25 mM), 2.5 μL of dNTPs (8 mM), 1 μL of each primer (10 μM), 0.2 μL of HotBegan Taq DNA Polymerase (5 U/μL), 1 μL of purified template DNA and 14.3 μL of nuclease-free water. The thermal cycle consisted of an initial step of 3 min at 94 °C, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing for 30 s and elongation at 72 °C for 45 s. A final extension was performed at 72 °C for 5 min. The primers pairs and the annealing temperatures (Ta) for each locus were as follows: ITS1-F and ITS4 for ITS (Ta = 55 °C) [20,21], EF1-688F and EF1-1251R for tef-1α (Ta = 55 °C) [22], BtCadF and BtCadR or T1 and BT2b for tub (Ta = 55 °C for both pairs) [23,24,25], CYLH3F and H3-1b for his (Ta = 58 °C) [25,26], CL1C and CL2C or CAL-563F and CL2C for cal (Ta = 58 °C for both pairs) [27,28]. PCR products were analyzed by 1% agarose gel electrophoresis, purified and sequenced by Macrogen Inc. (Madrid, Spain) using both PCR primers. Each consensus sequence was assembled using Sequencher software 5.0 (Gene Codes Corp., Ann Arbor, Michigan).

2.3. Phylogenetic Analyses

Sequences generated in this study were compared with reference sequences in the GenBank nucleotide database to determine the closest relatives for the phylogenetic studies. For each of the five loci (ITS, tub, tef-1α, cal and his), the DNA sequences obtained in this study (Table 1), together with those retrieved from GenBank (Table 2), were aligned using the ClustalW algorithm included in the MEGAX software package [29,30]. The alignments were analyzed and adjusted manually when necessary. Ambiguous sequences at either end of the alignments were excluded prior to analyses. Concatenated datasets were built in Sequence Matrix v.1.8 [31].
Phylogenetic analyses were based on Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP). Bayesian analyses were performed using MrBayes v 3.2 on the CIPRES Science Gateway v 3.3 [32,33]. The best-fitting model of nucleotide evolution for each partition was determined by MrModeltest 2.3 using the Akaike information criterion (AIC) [34]. Four simultaneous analyses were run for 100 million generations, sampling every 10,000, with four Markov chain Monte Carlo (MCMC) chains. The first 25% of saved trees were discarded and posterior probabilities were determined from the remaining trees. The ML analyses were done with the tool Randomized Axelerated Maximum Likelihood RAxML-HPC2 on XSEDE implemented on CIPRES Science Gateway v 3.3 [35]. ML tree searches were performed under the generalized time-reversible with gamma correction (GTR + Γ) nucleotide substitution model using 1000 pseudoreplicates. The other parameters were used as default settings. MP analyses were performed in MEGA X with the tree Bisection and reconnection (TBR) algorithm, where gaps were treated as missing data. The robustness of the topology was evaluated by 1000 bootstrap replications [36]. Measures for the maximum parsimony as tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency index (RC) were also calculated.
New sequences obtained in this study were deposited in GenBank (Table 1) and the multilocus alignment in was deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S26453).

2.4. Taxonomy

Agar plugs (6-mm diameter) were taken from the edge of actively growing cultures on PDA and transferred onto the center of 9-cm diameter Petri dishes containing one of the following culture media: malt extract agar (MEA; Sigma-Aldrich Laboratories), PDA, 2% tap water agar supplemented with sterile pine needles (PNA) or and oatmeal agar (OA; 60 g oatmeal, 12.5 g agar, Difco, Le Pont de Claix, France). Plates were then incubated at 21–22 °C under a 12 h/12 h near-ultraviolet light/darkness cycle to induce sporulation as described by Guarnaccia et al. (2018). Cultures were examined periodically for the development of ascomata and conidiomata. Colony colors were rated only on PDA after 15 days of incubation according to Rayner [37]. Morphological characteristics were examined using an Axio Scope A.1 microscope (Zeiss, manufacturer data) after mounting single pycnidia in lactic acid. Fungal structures were measured (30 measurements per type of structure) using the Zeiss AxioVision LE imaging device. Photos were captured using a Zeiss AxioCam MRm digital camera from images recorded with the 40× objective. Descriptions, nomenclature and illustrations of taxonomic novelties were deposited in MycoBank (MB 836048).
The effect of temperature on the mycelial growth of selected isolates of the species D. mediterranea (DAL24, DAL34 and DAL174) was measured on PDA. For this purpose, agar plugs (6-mm diameter) obtained from the growing edge of colonies were transferred to the center of PDA plates, which were incubated at 5, 10, 15, 20, 25, 30, 35 or 40 °C in darkness. Four replicates for each isolate and temperature combination were used. Growth was determined after 7 days in two orthogonal directions, and the mean growth rate was calculated in mm/day using a simplified version of the non-linear equation proposed by Duthie et al. [38]. Regression curves were fitted to the data using the R function “nls” included in the “stats” package [39,40].

2.5. Pathogenicity Tests

Pathogenicity tests were conducted as described by Diogo et al. [8]. One-year-old twigs of almond cv. Vayro, about 30 cm long, were inoculated with a set of 14 representative isolates of the five Diaporthe species found associated with P. dulcis in this study: D. amydgali (isolates DAL-3, DAL-4, DAL-45, DAL-70, DAL-105, DAL-140 and DAL-159), D. eres (DAL-102), D. foeniculina (DAL-27 and DAL-61), D. phaseolorum (DAL-222) and D. mediterranea (DAL-24, DAL-34 and DAL-174). These isolates were selected to represent diverse geographical origins. The twigs were surface sterilized by immersion in 70% ethanol for 30 s, 1.5% sodium hypochlorite solution for 1 min and ethanol for 30 s. Then, they were air dried in a laminar flow cabinet.
Wounds were made in the center of each twig with a 6-mm cork borer. Colonized agar plugs with mycelium of about the same size, which were obtained from active 10-day-old colonies growing on PDA, were inserted underneath the epidermis and the wounds were sealed with Parafilm. Inoculated twigs were kept in an upright position with their lower ends immersed in 1 L jars with 500 mL of sterile water in a growth chamber at 23 °C with 12 h of light per day. The twigs were covered with a plastic bag during the first 4 days to keep a moist environment. Six twigs per isolate were used and a negative control was prepared using uncolonized PDA plugs. Jars were arranged in a completely randomized design and the water was changed every 3 days. The experiment was repeated once.
Lesion lengths were measured 15 days after inoculation. Immediately after lesion measurements, two representative shoots per inoculated isolate and replicate were surface sterilized as described above. Small internal fragments were cut from the margin of the healthy and necrotic tissue and placed onto PDA. Plates were incubated at 25 °C in the dark for 7 to 10 d, and all fungal growths resembling Diaporthe were transferred to PDA. A representative subsample, one culture from each of the 14 isolates and replicates, were subjected to DNA extraction and molecular identification as described above to satisfy Koch’s postulates.
Significance levels for mean values of lesion length (cm), corresponding to different Diaporthe spp. isolates inoculated and control detached twigs, were determined. The analyses were performed considering individual isolates and groups of isolates from each Diaporthe spp. ANOVA assumptions were verified using Shapiro–Wilk and Levene’s tests. The datasets did not meet ANOVA assumptions, thus the analysis was performed using the Kruskal–Wallis test. Control twigs were compared with the inoculated ones considering individual isolates, and different species were compared with D. amygdali using the Wilcoxon rank sum test (p < 0.01). The analyses were performed in R using the agricolae and stats packages [39,40].

3. Results

3.1. Phylogenetic Analyses

Three loci (ITS region and fragments of tub and tef-1α genes) were sequenced in all Diaporthe isolates (n = 225) obtained in this study and compared with those in GenBank. The BLAST search showed high identity with D. amygdali, D. eres, D. foeniculina and D. phaseolorum accessions. For phylogenetic analyses, two representative isolates of closely related species, i.e., the ex-type together with one additional isolate when possible, were selected as references, and their corresponding sequences were retrieved from GenBank (Table 2). These sequences (n = 38), including Diaporthella corylina strain CBS 121124 which was used as outgroup, were added to those of the Spanish isolates (n = 225). The MP three-locus phylogeny showed that Spanish isolates of Diaporthe grouped into five distinct clades, four of them with known Diaporthe species (data not shown). The most abundant group, with 192 isolates, clustered with the ex-type isolate of D. amygdali (CBS 126679), the second (n = 23) with the ex-type of D. foeniculina (CBS 111553) and two single isolates each grouped with the ex-type of D. eres (CBS 109767), and with D. phaseolorum (CBS 116019). The remaining isolates (n = 8) clustered together, closely related to, but separated from, D. sterilis (CBS136969), suggesting that they could belong to a new species.
For accurate resolution of the species limits of our isolates, fragments of his and cal genes were sequenced in a set of 70 and 39 representative D. amygdali isolates, respectively, and for all isolates of the other groups (Table 1). The selection of the D. amygdali isolates was based on the province/region of origin and year of isolation. In addition, all GenBank sequences (ITS and tub) of two undescribed Diaporthe isolates (PMM 1657 and PMM 1660), which shared 100% identity with these loci of the potential new species, were included in the analyses (Table 2). Then, MP, ML and BI phylogenetic trees were constructed for the five-locus combined dataset, which included all taxa (n = 265) regardless of the level of completeness. A total of 2826 characters, including gaps (ITS: 1–564, tub: 565–1384, tef-1α: 1385–1814, his: 1815–2300 and cal: 2301–2826), were used in phylogenetic analyses, of which 1494 were constant and 817 were parsimony informative. The MP analysis yielded a single most parsimonious tree (TL = 2387; CI = 0.647; RI = 0.955; RC = 0.618). The ML analysis resulted in a single best tree with the final ML optimization likelihood = −15029.27737. In the BI analysis, the ITS/tub/tef-1α/his/cal partitions had 158/341/310/210/308 unique site patterns, respectively, and the analysis read a total of 40,004 trees, sampling 30,004 of them. The topologies and branching order of the inferred trees were compared visually, and they were fully congruent among themselves and with the previous ITS/tub/tef-1α multilocus phylogeny. The ITS/tub/tef-1α/his/cal ML tree is presented with the support of all phylogenetic methods at the branches (Figure 2).
Diaporthe amygdali represented 85.3% of the studied isolates and they were obtained from all sampled regions. The second most frequent species was D. foeniculina, with 23 isolates (10.2% of total), and it was recovered in all sampled regions except in La Rioja. Diaporthe eres and D. phaseolorum, each with only one isolate, were recovered from the Andalucía region. The remaining isolates (n = 8, 3.6% of total) were grouped together with 92% and 98% bootstrap support for MP and ML, respectively, and with 1 of BI posterior probability, but not with any known Diaporthe species. Therefore, they were putatively identified as belonging to a novel species described here and named D. mediterranea. This new species was obtained from the Islas Baleares and Comunidad Valenciana regions.

3.2. Taxonomy

Based on both the results of the phylogenetic inference and morphological characters, one new species of Diaporthe is described below (Figure 3).
Diaporthe mediterranea M. León, J. M. Rodríguez-Reina and J. Armengol, sp. nov.—MycoBank MB 836048; Figure 3.
Typification: Alcalalí, Alicante province (Comunidad Valenciana), Spain. From Prunus dulcis twig canker, 2017, J. Armengol, DAL-34 (holotype; CBS H-24368—ex-type culture CBS 146754).
Etymology. Named after the Mediterranean Sea, because this species was found on almond trees from orchards located in the Alicante province (Comunidad Valenciana) and Mallorca (Islas Baleares) in Mediterranean coastal areas of Spain.
Known distribution: Spain.
Description: Conidiomata pycnidial, globose or irregular, solitary on PNA but also aggregated on MEA, PDA and OA, erumpent, dark brown to black, (mean diameter ± SD = 527 ± 104.8 μm, n = 30), whitish translucent to creamy conidial drops exuded from the ostioles. Conidiophores densely aggregated lining the inner cavity, smooth and hyaline, cylindrical, straight, reduced to conidiogenous cells (mean ± SD =15.5 ± 2.7 × 2.2 ± 0.4 μm, n = 30). Paraphyses not observed. Alpha conidia produced in all the tested media, aseptate, fusiform, hyaline, multi-guttulate and acute at both ends, (mean ± SD = 6.6 ± 0.5 × 2.4 ± 0.2 μm, n = 30). Beta and gamma conidia not observed.
Culture characteristics: Colonies covering the medium within 7 d at 25 °C, with moderate aerial mycelium. Colonies on MEA, PDA and OA white at first, becoming light cream, mycelium flat on MEA and OA, denser and more felted on PDA. Reverse pale brown with light to dark grayish dots with age, with visible solitary and aggregate conidiomata at maturity on MEA, PDA and OA. Optimum growth temperature on PDA was 25.4 °C. Growth rates of colonies on PDA at 5, 10, 15, 20, 25, 30 and 35 °C were 0.02, 0.11, 0.36, 0.44, 0.67, 0.57 and 0.01 mm per day, respectively. No growth was observed at 40 °C.
Additional materials examined: DAL24 Sant Llorenç d’Escardassar, Mallorca, Islas Baleares, Spain, 2014 and DAL174 Altea la Vella, Alicante, Comunidad Valenciana, Spain, 2018.
Notes: Diaporthe mediterranea was collected from P. dulcis in Spain. The BLASTn search showed 100% identity with the available sequences (ITS and tub) of two isolates named Phomopsis sp. 5 (PMM 1657 and PMM 1660), collected from Vitis vinifera in South Africa [41,42], which were not described as new species by any of the authors. Nevertheless, other loci are needed to better resolve the identity of these isolates. Phylogenetic analysis combining five gene loci showed that all the isolates of D. mediterranea clustered together in a highly supported clade (92/98/1) and displayed a close relationship but they were clearly differentiated from D. sterilis. Based on alignments of the separate loci, D. mediterranea differs from D. sterilis [43] in seven positions (6 nt and one indel of 1 nt) of 426 bp in tub (p-distance = 1.4%), 20 positions (4 nt and one indel of 16 nt) of 342 bp in tef1-α (p-distance = 1.5%), 21 nt of 434 bp in his (p-distance = 4.8%), and 3 nt of 469 bp in cal (p-distance = 0.6%). The ITS sequences of both species showed 100% identity. Morphologically, D. mediterranea mainly differs from D. sterilis in its capacity to produce alpha conidia, because all isolates representing D. sterilis could not be induced to sporulate on any of the culture media used by Lombard et al. [43], when this new Diaporthe species collected from Vaccinium corymbosum was described.

3.3. Pathogenicity Tests

All Diaporthe isolates inoculated on one-year-old twigs of almond cv. Vayro caused necrotic lesions of variable length (Figure 4). There was no effect of the experiment on the lesion length (p = 0.5032). Mean lesion length in canes inoculated with different Diaporthe isolates (n = 12 per inoculated isolate) ranged from 1.4 to 13.7 cm and control twigs treated with uncolonized PDA plugs showed a mean lesion length of 0.6 cm (Figure 5). Statistical analysis revealed significant differences in lesion length between the control and twigs inoculated with all isolates, except those of D. foeniculina, namely DAL-27 and DAL-61 (p = 0.7224 and p = 0.0117, respectively) and D. phaseolorum DAL-222 (p = 0.0239).
When isolates of the different Diaporthe species were grouped, significant differences in mean lesion length (cm) were also observed (p < 0.01). Twigs inoculated with D. mediterranea showed significantly longer mean lesions (11.3 cm) compared with D. amygdali. (Figure 6). There were no statistical differences among mean lesion length values caused by D. amygdali (7.7 cm), D. eres (8.4 cm) or D. phaseolorum (6.2 cm). However, twigs inoculated with D. foeniculina showed significantly shorter lesions (2.6 cm) compared with the other Diaporthe spp., except for D. phaseolorum.

4. Discussion

The survey conducted on almond orchards showing twig cankers and shoot blight symptoms in five different regions of Spain from 2005 to 2019 resulted in a collection of 225 Diaporthe isolates, which were used to elucidate the diversity of Diaporthe species associated with this host using both phenotypical data and DNA sequence analyses.
This is the first study in which a collection of Diaporthe isolates from almond has been characterized using multilocus DNA sequence analysis with five loci (ITS, tub, tef-1α, cal and his), which has been recommended in previous phylogenetic studies of the genus Diaporthe for species identification and separation [14,17,44]. This analysis allowed the identification of four known Diaporthe species, namely: D. amygdali, D. eres, D. foeniculina and D. phaseolorum. Moreover, it also confirmed that eight isolates represented a novel phylogenetic species, newly described here as D. mediterranea.
Diaporthe amygdali was the most prevalent species, due to the largest number of isolates collected from widely separated almond growing regions in Spain. This fungus has been described on this crop in other Mediterranean countries, such as France [11], Greece [45], Hungary [46], Italy [47], Portugal [8,48] and Tunisia [49], where it is considered the main pathogen associated with twig cankers and shoot blight symptoms. In Mediterranean areas, D. amygdali has also been reported as a damaging agent in other fruit and nut crops, such as apricot [50], peach [9,51] and English walnut [52]. Diaporthe amygdali is also present in other continents, affecting diverse hosts: on almond and peach in the USA [53,54]; grapevine in South Africa [55]; peach in Japan [56]; peach and nectarine in Uruguay [57,58]; and peach, pear and walnut in China [59,60,61].
Regarding the other Diaporthe species found in our study: D. eres was previously reported on P. dulcis in Portugal [8], and D. foeniculina is present on almond in Italy, with one isolate (CBS 171.78) deposited at the Westerdijk Fungal Biodiversity Institute (Utrecht, the Netherlands) [62]. To our knowledge, our study represents the first report of D. phaseolorum on almond.
The isolates described in our work as belonging to the new taxon, D. mediterranea, were found only in two almond-growing regions in Spain: coastal areas of Alicante province (Comunidad Valenciana) and Mallorca (Islas Baleares). It is interesting to note that the ITS and tub sequences of two Diaporthe isolates, namely Phomopsis sp. 5 (PMM 1657 and PMM 1660), which were collected from V. vinifera in South Africa [41,42], showed 100% identity with the ITS and tub sequences of D. mediterranea. Further studies including other loci would be needed to resolve the identity of the South African isolates (PMM 1657 and PMM 1660).
Pathogenicity tests were performed using one-year-old almond twigs, as described by [8], who determined the capacity of Diaporthe spp. isolates from Portugal to cause lesions on this crop. Except for D. foeniculina and D. phaseolorum, all Diaporthe species inoculated to almond twigs cv. Vayro were able to cause lesions significantly different from those developed on the uninoculated controls. The most severe symptoms were detected on almond twigs inoculated with D. mediterranea. Therefore, this study provides novel information about the ability of this species to cause disease on P. dulcis, being more aggressive than the well-known pathogen D. amygdali. Diaporthe eres was also pathogenic to almond, but the incidence of this species and D. phaseolorum in the survey conducted in this study was extremely low, with only one isolate found in each species.
The present study is the first comprehensive attempt to characterize Diaporthe species associated with P. dulcis in Spain, combining morphology and multilocus DNA sequence analysis. Our results confirm D. amygdali as a key pathogen of almonds in Spain. Moreover, the new species D. mediterranea should also be considered as a potentially important causal agent of twig cankers and shoot blight on this crop, according to the high virulence shown in the pathogenicity tests. In Spain, the lack of information regarding the identity of Diaporthe species on almond and their pathogenicity hinders the development of efficient control strategies and the development of resistant varieties. These aspects have been addressed for the first time in this work and will contribute to the development of improved integrated disease management programs against twig canker and shoot blight disease.

Author Contributions

Conceptualization, M.L., M.B., G.E. and J.A.; Methodology, M.L., M.B., J.M.R.-R., G.E., P.A.-C., A.R.-A. and J.A.; Software, M.L. and M.B.; Validation, M.L., M.B., J.M.R.-R. and J.A.; Formal Analysis, M.L., M.B., J.M.R.-R. and J.A.; Investigation, M.L., M.B., J.M.R.-R., G.E., P.A.-C., A.R.-A. and J.A.; Resources, D.O., A.V., J.L., X.M., C.A.-B., A.T., N.C., F.T.A., M.A., D.G. and M.A.-S.; Data Curation, M.L., J.M.R.-R. and J.A.; Writing—Original Draft Preparation, M.L., M.B. and J.A.; Writing—Review and Editing, M.L., M.B., J.M.R.-R., G.E., P.A.-C., A.R.-A., D.O., A.V., J.L., X.M., C.A.-B., A.T., N.C., F.T.A., M.A., D.G., M.A.-S. and J.A.; Visualization, M.L., M.B. and J.A. All authors have read and agreed to the published version of the manuscript.

Funding

Research funded by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), grants RTA2017-00009-C04-01, -02, -03 and -04 and with matching funds from the European Regional Development Fund (ERDF). G. Elena and C. Agustí-Brisach were supported by the Spanish post-doctoral grants “Juan de la Cierva-Formación” and “Juan de la Cierva-Incorporación”, respectively. J. Luque and X. Miarnau were partially supported by the CERCA program, Generalitat de Catalunya. D. Gramaje was supported by the Ramón y Cajal program, Spanish Government (RYC-2017-23098).

Acknowledgments

We thank P. Yécora for her technical support.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Twig canker and shoot blight symptoms caused by Diaporthe spp. on almond.
Figure 1. Twig canker and shoot blight symptoms caused by Diaporthe spp. on almond.
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Figure 2. Randomized Axelerated Maximum Likelihood (RAxML) tree based on analysis of a combined dataset of ITS, tub, tef-1α, his and cal sequences. Bootstrap support values for Maximum Parsimony (MP) and ML higher than 70% and Bayesian posterior probabilities (PP) higher than 0.90 are shown at the branches (MP/ML/PP). Clades highlighted contain the isolates identified in the current study and the novel taxa is shown in red. Ex-type strains are indicated in bold. The tree is rooted using Diaporthella corylina (CBS121124). The scale bar represents the expected number of nucleotide substitutions per site.
Figure 2. Randomized Axelerated Maximum Likelihood (RAxML) tree based on analysis of a combined dataset of ITS, tub, tef-1α, his and cal sequences. Bootstrap support values for Maximum Parsimony (MP) and ML higher than 70% and Bayesian posterior probabilities (PP) higher than 0.90 are shown at the branches (MP/ML/PP). Clades highlighted contain the isolates identified in the current study and the novel taxa is shown in red. Ex-type strains are indicated in bold. The tree is rooted using Diaporthella corylina (CBS121124). The scale bar represents the expected number of nucleotide substitutions per site.
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Figure 3. Diaporthe mediterranea (DAL-34). (A–C) Colonies on Malt Extract Agar (MEA), Oat Agar (OA) and Potato Dextrose Agar (PDA), respectively; (D) conidiomata sporulating on PDA; (E) alpha conidia—scale bar = 20 μm.
Figure 3. Diaporthe mediterranea (DAL-34). (A–C) Colonies on Malt Extract Agar (MEA), Oat Agar (OA) and Potato Dextrose Agar (PDA), respectively; (D) conidiomata sporulating on PDA; (E) alpha conidia—scale bar = 20 μm.
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Figure 4. Necrotic lesions induced by the Diaporthe spp. inoculated on almond detached canes. (A) Uninoculated control; (B) D. amygdali (DAL-4); (C) D. eres (DAL-102); (D) D. foeniculina (DAL-61); (E) D. mediterranea (DAL-34) and (F) D. phaseolorum (DAL-222).
Figure 4. Necrotic lesions induced by the Diaporthe spp. inoculated on almond detached canes. (A) Uninoculated control; (B) D. amygdali (DAL-4); (C) D. eres (DAL-102); (D) D. foeniculina (DAL-61); (E) D. mediterranea (DAL-34) and (F) D. phaseolorum (DAL-222).
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Figure 5. Box plot of lesion length (cm) caused by isolates of Diaporthe spp. on almond detached twigs (n = 12 per isolate) at 15 days after inoculation. Black lines in the boxes show medians. Isolate labels: Da: D. amygdali, De: D. eres, Df: D. foeniculina, Dp: D. phaseolorum, Dm: D. mediterranea. Asterisks (*) indicate that values are significantly different from the control according to the Wilcoxon rank sum test (p < 0.01).
Figure 5. Box plot of lesion length (cm) caused by isolates of Diaporthe spp. on almond detached twigs (n = 12 per isolate) at 15 days after inoculation. Black lines in the boxes show medians. Isolate labels: Da: D. amygdali, De: D. eres, Df: D. foeniculina, Dp: D. phaseolorum, Dm: D. mediterranea. Asterisks (*) indicate that values are significantly different from the control according to the Wilcoxon rank sum test (p < 0.01).
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Figure 6. Box plot of lesion length (cm) caused by Diaporthe spp. on almond detached twigs inoculated (n = 12 per isolate) with isolates of D. amygdali (seven isolates), D. eres (one isolate), D. foeniculina (two isolates), D. phaseolorum (one isolate) and D. mediterranea (three isolates). Black lines in the boxes show medians. Asterisks (*) indicate that values are significantly different than D. amygdali according to the Wilcoxon rank sum test (p < 0.01).
Figure 6. Box plot of lesion length (cm) caused by Diaporthe spp. on almond detached twigs inoculated (n = 12 per isolate) with isolates of D. amygdali (seven isolates), D. eres (one isolate), D. foeniculina (two isolates), D. phaseolorum (one isolate) and D. mediterranea (three isolates). Black lines in the boxes show medians. Asterisks (*) indicate that values are significantly different than D. amygdali according to the Wilcoxon rank sum test (p < 0.01).
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Table
Table 1. Collection details and GenBank accession numbers of isolates included in this study.
Table 1. Collection details and GenBank accession numbers of isolates included in this study.
SpeciesStrain NumberYearLocationProvince/RegionGenBank Accession Numbers
ITStef-1αtubhiscal
D. amygdaliDAL-12014Sant JoanMallorca/Islas BalearesMT007292MT006769MT006466--
DAL-22014Sant JoanMallorca/Islas BalearesMT007293MT006770MT006467--
DAL-32014Santa MargalidaMallorca/Islas BalearesMT007294MT006771MT006468MT006997MT006694
DAL-42014Santa MargalidaMallorca/Islas BalearesMT007295MT006772MT006469MT006998MT006695
DAL-52014CalviàMallorca/Islas BalearesMT007296MT006773MT006470--
DAL-72014CalviàMallorca/Islas BalearesMT007297MT006774MT006471MT006999-
DAL-92014CalviàMallorca/Islas BalearesMT007298MT006775MT006472MT007000MT006696
DAL-122014BinissalemMallorca/Islas BalearesMT007299MT006776MT006473MT007001-
DAL-132014LlucmajorMallorca/Islas BalearesMT007300MT006777MT006474--
DAL-142014LlucmajorMallorca/Islas BalearesMT007301MT006778MT006475--
DAL-152014MarratxíMallorca/Islas BalearesMT007302MT006779MT006476MT007002-
DAL-162014Sa PoblaMallorca/Islas BalearesMT007303MT006780MT006477MT007003MT006697
DAL-172014Sa PoblaMallorca/Islas BalearesMT007304MT006781MT006478--
DAL-182014IncaMallorca/Islas BalearesMT007305MT006782MT006479MT007004-
DAL-192014BinissalemMallorca/Islas BalearesMT007306MT006783MT006480MT007005-
DAL-202014PalmaMallorca/Islas BalearesMT007307MT006784MT006481--
DAL-212014BinissalemMallorca/Islas BalearesMT007308MT006785MT006482--
DAL-222014LlucmajorMallorca/Islas BalearesMT007309MT006786MT006483MT007006-
DAL-232014IncaMallorca/Islas BalearesMT007310MT006787MT006484--
DAL-322017AlcalalíAlicante/Comunidad ValencianaMT007313MT006790MT006487--
DAL-332017AlcalalíAlicante/Comunidad ValencianaMT007314MT006791MT006488--
DAL-352017AlcalalíAlicante/Comunidad ValencianaMT007315MT006792MT006489--
DAL-362017AlcalalíAlicante/Comunidad ValencianaMT007316MT006793MT006490--
DAL-372017AlcalalíAlicante/Comunidad ValencianaMT007317MT006794MT006491--
DAL-382017AlcalalíAlicante/Comunidad ValencianaMT007318MT006795MT006492--
DAL-392017AlcalalíAlicante/Comunidad ValencianaMT007319MT006796MT006493--
DAL-402017AlcalalíAlicante/Comunidad ValencianaMT007320MT006797MT006494--
DAL-412017AlcalalíAlicante/Comunidad ValencianaMT007321MT006798MT006495--
DAL-422017AlcalalíAlicante/Comunidad ValencianaMT007322MT006799MT006496MT007008MT006699
DAL-432017BunyolaMallorca/Islas BalearesMT007323MT006800MT006497MT007009MT006700
DAL-442017BunyolaMallorca/Islas BalearesMT007324MT006801MT006498--
DAL-452017BunyolaMallorca/Islas BalearesMT007325MT006802MT006499MT007010MT006701
DAL-462017BunyolaMallorca/Islas BalearesMT007326MT006803MT006500--
DAL-472017BunyolaMallorca/Islas BalearesMT007327MT006804MT006501--
DAL-482017BunyolaMallorca/Islas BalearesMT007328MT006805MT006502MT007011MT006702
D. amygdali (cont.)DAL-492017BunyolaMallorca/Islas BalearesMT007329MT006806MT006503--
DAL-502017BunyolaMallorca/Islas BalearesMT007330MT006807MT006504MT007012-
DAL-512017BunyolaMallorca/Islas BalearesMT007331MT006808MT006505--
DAL-522017PalmaMallorca/Islas BalearesMT007332MT006809MT006506--
DAL-532017PalmaMallorca/Islas BalearesMT007333MT006810MT006507--
DAL-542017PalmaMallorca/Islas BalearesMT007334MT006811MT006508--
DAL-552017PalmaMallorca/Islas BalearesMT007335MT006812MT006509--
DAL-562017PalmaMallorca/Islas BalearesMT007336MT006813MT006510--
DAL-572017PalmaMallorca/Islas BalearesMT007337MT006814MT006511MT007013-
DAL-652017La RinconadaSevilla/AndalucíaMT007338MT006815MT006512MT007014-
DAL-702018GodelletaValencia/Comunidad ValencianaMT007339MT006816MT006513MT007015MT006703
DAL-712018GodelletaValencia/Comunidad ValencianaMT007340MT006817MT006514--
DAL-722018GodelletaValencia/Comunidad ValencianaMT007341MT006818MT006515--
DAL-732018GodelletaValencia/Comunidad ValencianaMT007342MT006819MT006516--
DAL-742018GodelletaValencia/Comunidad ValencianaMT007343MT006820MT006517--
DAL-752018GodelletaValencia/Comunidad ValencianaMT007344MT006821MT006518--
DAL-762018MontserratValencia/Comunidad ValencianaMT007345MT006822MT006519MT007016MT006704
DAL-772018MontserratValencia/Comunidad ValencianaMT007346MT006823MT006520--
DAL-782018MontserratValencia/Comunidad ValencianaMT007347MT006824MT006521--
DAL-792018MontserratValencia/Comunidad ValencianaMT007348MT006825MT006522--
DAL-802018MontserratValencia/Comunidad ValencianaMT007349MT006826MT006523--
DAL-812018MontserratValencia/Comunidad ValencianaMT007350MT006827MT006524--
DAL-822018ViverCastellón/Comunidad ValencianaMT007351MT006828MT006525MT007017MT006705
DAL-832018ViverCastellón/Comunidad ValencianaMT007352MT006829MT006526--
DAL-842018ViverCastellón/Comunidad ValencianaMT007353MT006830MT006527--
DAL-852018ViverCastellón/Comunidad ValencianaMT007354MT006831MT006528MT007018MT006706
DAL-862018ViverCastellón/Comunidad ValencianaMT007355MT006832MT006529--
DAL-872018ViverCastellón/Comunidad ValencianaMT007356MT006833MT006530--
DAL-882018ViverCastellón/Comunidad ValencianaMT007357MT006834MT006531--
DAL-892018ViverCastellón/Comunidad ValencianaMT007358MT006835MT006532--
DAL-902018ViverCastellón/Comunidad ValencianaMT007359MT006836MT006533--
DAL-912018ViverCastellón/Comunidad ValencianaMT007360MT006837MT006534--
DAL-922018ViverCastellón/Comunidad ValencianaMT007361MT006838MT006535--
DAL-932018ViverCastellón/Comunidad ValencianaMT007362MT006839MT006536--
DAL-942018ViverCastellón/Comunidad ValencianaMT007363MT006840MT006537MT007019-
D. amygdali (cont.)DAL-952018ViverCastellón/Comunidad ValencianaMT007364MT006841MT006538MT007020-
DAL-962018ViverCastellón/Comunidad ValencianaMT007365MT006842MT006539--
DAL-972018Fuente la HigueraValencia/Comunidad ValencianaMT007366MT006843MT006540--
DAL-982018Fuente la HigueraValencia/Comunidad ValencianaMT007367MT006844MT006541--
DAL-1032017GibraleónHuelva/AndalucíaMT007368MT006845MT006542MT007021MT006707
DAL-1042016El ContadorAlmería/AndalucíaMT007369MT006848MT006543MT007022MT006708
DAL-1052017Alcalá del RíoSevilla/AndalucíaMT007370MT006846MT006544MT007023MT006709
DAL-1082018BiarAlicante/Comunidad ValencianaMT007371MT006847MT006545MT007024MT006710
DAL-1092018BiarAlicante/Comunidad ValencianaMT007372MT006849MT006546--
DAL-1102018Fuente la HigueraValencia/Comunidad ValencianaMT007373MT006850MT006547--
DAL-1112018Fuente la HigueraValencia/Comunidad ValencianaMT007374MT006851MT006548--
DAL-1122018Fuente la HigueraValencia/Comunidad ValencianaMT007375MT006852MT006549--
DAL-1132018Fontanars dels AlforinsValencia/Comunidad ValencianaMT007376MT006853MT006550MT007025MT006711
DAL-1142018Fontanars dels AlforinsValencia/Comunidad ValencianaMT007377MT006854MT006551MT007026MT006712
DAL-1162018AlcublasValencia/Comunidad ValencianaMT007378MT006855MT006552MT007027-
DAL-1172018AlcublasValencia/Comunidad ValencianaMT007379MT006856MT006553--
DAL-1182018CasinosValencia/Comunidad ValencianaMT007380MT006857MT006554--
DAL-1192018CasinosValencia/Comunidad ValencianaMT007381MT006858MT006555--
DAL-1202018CasinosValencia/Comunidad ValencianaMT007382MT006859MT006556--
DAL-1212018Vall d’AlbaCastellón/Comunidad ValencianaMT007383MT006860MT006557MT007028-
DAL-1222018Vall d’AlbaCastellón/Comunidad ValencianaMT007384MT006861MT006558--
DAL-1252018Vall d’AlbaCastellón/Comunidad ValencianaMT007385MT006862MT006559--
DAL-1262018Vall d’AlbaCastellón/Comunidad ValencianaMT007386MT006863MT006560--
DAL-1282018GodelletaValencia/Comunidad ValencianaMT007387MT006864MT006561--
DAL-1292018GodelletaValencia/Comunidad ValencianaMT007388MT006865MT006562--
DAL-1302018TorremendoAlicante/Comunidad ValencianaMT007389MT006866MT006563MT007029-
DAL-1312018TorremendoAlicante/Comunidad ValencianMT007390MT006867MT006564--
DAL-1322018RequenaValencia/Comunidad ValencianaMT007391MT006868MT006565MT007030MT006713
DAL-1332018RequenaValencia/Comunidad ValencianaMT007392MT006869MT006566MT007031-
DAL-1342018RequenaValencia/Comunidad ValencianaMT007393MT006870MT006567MT007032-
DAL-1352018L’ElianaValencia/Comunidad ValencianaMT007394MT006871MT006568MT007033-
DAL-1362018L’ElianaValencia/Comunidad ValencianaMT007395MT006872MT006569--
DAL-1382005ConstantíTarragona/CataluñaMT007396MT006873MT006570--
DAL-1392005ConstantíTarragona/CataluñaMT007397MT006874MT006571MT007034-
DAL-1402012UlldeconaTarragona/CataluñaMT007398MT006875MT006572MT007035MT006714
D. amygdali (cont.)DAL-1412016GandesaTarragona/CataluñaMT007399MT006876MT006573--
DAL-1432018GandesaTarragona/CataluñaMT007400MT006877MT006574--
DAL-1442018GandesaTarragona/CataluñaMT007401MT006878MT006575--
DAL-1452018GandesaTarragona/CataluñaMT007402MT006879MT006576--
DAL-1462018GandesaTarragona/CataluñaMT007403MT006880MT006577MT007036-
DAL-1472018ConstantíTarragona/CataluñaMT007404MT006881MT006578MT007037-
DAL-1482018ConstantíTarragona/CataluñaMT007405MT006882MT006579MT007038-
DAL-1492018ConstantíTarragona/CataluñaMT007406MT006883MT006580MT007039MT006715
DAL-1512018ConstantíTarragona/CataluñaMT007407MT006884MT006581--
DAL-1522018ConstantíTarragona/CataluñaMT007408MT006885MT006582MT007040MT006716
DAL-1532018ConstantíTarragona/CataluñaMT007409MT006886MT006583--
DAL-1542018La Selva del CampTarragona/CataluñaMT007410MT006887MT006584MT007041MT006717
DAL-1552018La Selva del CampTarragona/CataluñaMT007411MT006888MT006585MT007042MT006718
DAL-1562018La Selva del CampTarragona/CataluñaMT007412MT006889MT006586--
DAL-1582018La Selva del CampTarragona/CataluñaMT007413MT006890MT006587--
DAL-1592018La Selva del CampTarragona/CataluñaMT007414MT006891MT006588--
DAL-1602018La Selva del CampTarragona/CataluñaMT007415MT006892MT006589--
DAL-1612018ConstantíTarragona/CataluñaMT007416MT006893MT006590--
DAL-1622018ConstantíTarragona/CataluñaMT007417MT006894MT006591--
DAL-1632018EstepaSevilla/AndalucíaMT007418MT006895MT006592--
DAL-1642018EstepaSevilla/AndalucíaMT007419MT006896MT006593MT007043MT006719
DAL-1672018Los PalaciosSevilla/AndalucíaMT007420MT006897MT006594MT007044MT006720
DAL-1682018Los PalaciosSevilla/AndalucíaMT007421MT006898MT006595--
DAL-1692018Los PalaciosSevilla/AndalucíaMT007422MT006899MT006596--
DAL-1702018Los PalaciosSevilla/AndalucíaMT007423MT006900MT006597--
DAL-1712018Los PalaciosSevilla/AndalucíaMT007424MT006901MT006598--
DAL-1722018Los PalaciosSevilla/AndalucíaMT007425MT006902MT006599MT007045-
DAL-1812018CórdobaCórdoba/AndalucíaMT007426MT006903MT006600MT007046MT006721
DAL-1822018CórdobaCórdoba/AndalucíaMT007427MT006904MT006601--
DAL-1832018CórdobaCórdoba/AndalucíaMT007428MT006905MT006602--
DAL-1842018Mairena del AlcorSevilla/AndalucíaMT007429MT006906MT006603MT007047-
DAL-1852018Mairena del AlcorSevilla/AndalucíaMT007430MT006907MT006604--
DAL-1862018Mairena del AlcorSevilla/AndalucíaMT007431MT006908MT006605--
DAL-1872018Mairena del AlcorSevilla/AndalucíaMT007432MT006909MT006606--
D. amygdali (cont.)DAL-1882018Mairena del AlcorSevilla/AndalucíaMT007433MT006910MT006607--
DAL-1892018Mairena del AlcorSevilla/AndalucíaMT007434MT006911MT006608MT007048-
DAL-1902018Mairena del AlcorSevilla/AndalucíaMT007435MT006912MT006609MT007049MT006722
DAL-1912018Mairena del AlcorSevilla/AndalucíaMT007436MT006913MT006610--
DAL-1922018Mairena del AlcorSevilla/AndalucíaMT007437MT006914MT006611--
DAL-1932018RondaMálaga/AndalucíaMT007438MT006915MT006612MT007050MT006723
DAL-1942018RondaMálaga/AndalucíaMT007439MT006916MT006613--
DAL-1952018RondaMálaga/AndalucíaMT007440MT006917MT006614--
DAL-1962018RondaMálaga/AndalucíaMT007441MT006918MT006615--
DAL-1972018RondaMálaga/AndalucíaMT007442MT006919MT006616MT007051MT006724
DAL-1982018RondaMálaga/AndalucíaMT007443MT006920MT006617--
DAL-1992018RondaMálaga/AndalucíaMT007444MT006921MT006618--
DAL-2002018RondaMálaga/AndalucíaMT007445MT006922MT006619--
DAL-2012018RondaMálaga/AndalucíaMT007446MT006923MT006620--
DAL-2022018RondaMálaga/AndalucíaMT007447MT006924MT006621MT007052-
DAL-2032018ReusTarragona/CataluñaMT007448MT006925MT006622--
DAL-2042018ReusTarragona/CataluñaMT007449MT006926MT006623MT007053MT006725
DAL-2052018ReusTarragona/CataluñaMT007450MT006927MT006624MT007054MT006726
DAL-2062018RiudomsTarragona/CataluñaMT007451MT006928MT006625--
DAL-2072018RiudomsTarragona/CataluñaMT007452MT006929MT006626--
DAL-2082018RiudomsTarragona/CataluñaMT007453MT006930MT006627MT007055-
DAL-2092018RiudomsTarragona/CataluñaMT007454MT006931MT006628MT007056-
DAL-2102018RiudomsTarragona/CataluñaMT007455MT006932MT006629MT007057-
DAL-2112018RiudomsTarragona/CataluñaMT007456MT006933MT006630--
DAL-2122018RiudomsTarragona/CataluñaMT007457MT006934MT006631--
DAL-2132018RiudomsTarragona/CataluñaMT007458MT006935MT006632--
DAL-2142018BotarellTarragona/CataluñaMT007459MT006936MT006633--
DAL-2152018BotarellTarragona/CataluñaMT007460MT006937MT006634MT007058-
DAL-2162018BotarellTarragona/CataluñaMT007461MT006938MT006635MT007059-
DAL-2192018Les Borges BlanquesLérida/CataluñaMT007462MT006939MT006636MT007060MT006727
DAL-2202018Isona i Conca DellàLérida/CataluñaMT007463MT006940MT006637MT007061MT006728
DAL-2212018Isona i Conca DellàLérida/CataluñaMT007464MT006941MT006638MT007062-
DAL-2252019MurilloLogroño/La RiojaMT007465MT006942MT006639MT007063MT006729
DAL-2262019MurilloLogroño/La RiojaMT007466MT006943MT006640--
D. amygdali (cont.)DAL-2272019Santa Engracia de JuberaLogroño/La RiojaMT007467MT006944MT006641MT007064MT006730
DAL-2282019Santa Engracia de JuberaLogroño/La RiojaMT007468MT006945MT006642--
DAL-2292019Santa Engracia de JuberaLogroño/La RiojaMT007469MT006946MT006643--
DAL-2302019Santa Engracia de JuberaLogroño/La RiojaMT007470MT006947MT006644--
DAL-2312019Santa Engracia de JuberaLogroño/La RiojaMT007471MT006948MT006645--
DAL-2322019Santa Engracia de JuberaLogroño/La RiojaMT007472MT006949MT006646--
DAL-2332019LagunillaLogroño/La RiojaMT007473MT006950MT006647MT007065MT006731
DAL-2342019Santa Engracia de JuberaLogroño/La RiojaMT007474MT006951MT006648--
DAL-2362019Alcalá del RíoSevilla/AndalucíaMT007475MT006952MT006649MT007066-
DAL-2372019Alcalá del RíoSevilla/AndalucíaMT007476MT006953MT006650--
DAL-2382019Alcalá del RíoSevilla/AndalucíaMT007477MT006954MT006651--
DAL-2392019CórdobaCórdoba/AndalucíaMT007478MT006955MT006652--
DAL-2402019CórdobaCórdoba/AndalucíaMT007479MT006956MT006653MT007067MT006732
DAL-2412019CórdobaCórdoba/AndalucíaMT007480MT006957MT006654--
DAL-2422019Santa CruzCórdoba/AndalucíaMT007481MT006958MT006655--
DAL-2432019CórdobaCórdoba/AndalucíaMT007482MT006959MT006656--
DAL-2442019Villamanrique de la CondesaSevilla/AndalucíaMT007483MT006960MT006657MT007068MT006733
DAL-2452019Villamanrique de la CondesaSevilla/AndalucíaMT007484MT006961MT006658--
DAL-2462019Santa Engracia de JuberaLogroño/La RiojaMT007485MT006962MT006659--
D. eresDAL-1022016CórdobaCórdoba/AndalucíaMN997106MT007104MT006462MT007106MT006465
D. foeniculinaDAL-102014Santa Margalida i CalviàMallorca/Islas BalearesMT007497MT006963MT006660MT007069MT006734
DAL-112014 Mallorca/Islas BalearesMT007498MT006964MT006661MT007070MT006735
DAL-272017AlcalalíAlicante/Comunidad ValencianaMT007499MT006965MT006662MT007071MT006736
DAL-282017AlcalalíAlicante/Comunidad ValencianaMT007500MT006966MT006663MT007072MT006737
DAL-302017AlcalalíAlicante/Comunidad ValencianaMT007501MT006967MT006664MT007073MT006738
DAL-312017AlcalalíAlicante/Comunidad ValencianaMT007502MT006968MT006665MT007074MT006739
DAL-612016Alcalá del RíoSevilla/AndalucíaMT007503MT006969MT006666MT007075MT006740
DAL-622016Alcalá del RíoSevilla/AndalucíaMT007504MT006970MT006667MT007076MT006741
DAL-632016Alcalá del RíoSevilla/AndalucíaMT007505MT006971MT006668MT007077MT006742
DAL-642016Alcalá del RíoSevilla/AndalucíaMT007506MT006972MT006669MT007078MT006743
DAL-662017La RinconadaSevilla/AndalucíaMT007507MT006973MT006670MT007079MT006744
DAL-672017La RinconadaSevilla/AndalucíaMT007508MT006974MT006671MT007080MT006745
DAL-682017La RinconadaSevilla/AndalucíaMT007509MT006975MT006672MT007081MT006746
DAL-692017La RinconadaSevilla/AndalucíaMT007510MT006976MT006673MT007082MT006747
DAL-992018Fuente la HigueraValencia/Comunidad ValencianaMT007511MT006977MT006674MT007083MT006748
DAL-1002018Fuente la HigueraValencia/Comunidad ValencianaMT007512MT006978MT006675MT007084MT006749
D. foeniculina (cont.)DAL-1012018Fuente la HigueraValencia/Comunidad ValencianaMT007513MT006979MT006676MT007085MT006750
DAL-1072018MarchenaSevilla/AndalucíaMT007514MT006980MT006677MT007086MT006751
DAL-1422018CabrilsBarcelona/CataluñaMT007515MT006981MT006678MT007087MT006752
DAL-1502018ConstantíTarragona/CataluñaMT007516MT006982MT006679MT007088MT006753
DAL-1572018La Selva del Camp Tarragona/CataluñaMT007517MT006983MT006680MT007089MT006754
DAL-1652018EstepaSevilla/AndalucíaMT007518MT006984MT006681MT007090MT006755
DAL-2172018Les Borges BlanquesLérida/CataluñaMT007519MT006985MT006682MT007091MT006756
D. mediterraneaDAL-62014CalviàMallorca/Islas BalearesMT007486MT006986MT006683MT007092MT006758
DAL-82014ConsellMallorca/Islas BalearesMT007487MT006987MT006684MT007093MT006759
DAL-242014Sant Llorenç d’EscardassarMallorca/Islas BalearesMT007488MT006988MT006685MT007094MT006760
DAL-342017AlcalalíAlicante/Comunidad ValencianaMT007489MT006989MT006686MT007095MT006761
DAL-1732018Altea la VellaAlicante/Comunidad ValencianaMT007493MT006993MT006691MT007099MT006765
DAL-1742018Altea la VellaAlicante/Comunidad ValencianaMT007494MT006994MT006690MT007100MT006766
DAL-1752018Altea la VellaAlicante/Comunidad ValencianaMT007495MT006995MT006692MT007101MT006767
DAL-1762018Altea la VellaAlicante/Comunidad ValencianaMT007496MT006996MT006693MT007102MT006768
D. phaseolorumDAL-2222016Alcalá del RíoSevilla/AndalucíaMN997107MT007103MT006463MT007105MT006464
Table
Table 2. Additional Diaporthe species used in the phylogenetic analyses.
Table 2. Additional Diaporthe species used in the phylogenetic analyses.
SpeciesStrainHostCountryGenBank Accession Numbers
ITStef-1αtubhiscal
D. acaciigenaCBS 129521Acacia retinodesAustraliaKC343005KC343731KC343973KC343489KC343247
D. amygdaliCBS 126679Prunus dulcisPortugalKC343022KC343748KC343990KC343506KC343264
CBS 111811Vitis viniferaSouth AfricaKC343019KC343745KC343987KC343503KC343261
D. celastrinaCBS 139.27Celastrus scandensUSA KC343047KC343773KC344015KC343531KC343289
D. celerisCBS 143349Vitis viniferaUKMG281017MG281538MG281190MG281363MG281712
CBS 143350Vitis viniferaUKMG281018MG281539MG281191MG281364MG281713
D. chamaeropisCBS 454.81Chamaerops humilisGreeceKC343048KC343774KC344016KC343532KC343290
CBS 753.70 Spartium junceumCroatiaKC343049KC343775KC344017KC343533KC343291
D. chongqingensisPSCG 435Pyrus pyrifoliaChinaMK626916MK654866MK691321MK726257MK691209
PSCG 436Pyrus pyrifoliaChinaMK626917MK654867MK691322MK726256MK691208
D. cinerascensCBS 719.96Ficus caricaBulgariaKC343050KC343776KC344018KC343534KC343292
D. endophyticaCBS 133811Schinus terebinthifoliusBrazilKC343065KC343791KC344033KC343549KC343307
LGMF911Schinus terebinthifoliusBrazilKC343066KC343792KC344034KC343550KC343308
D. eresCBS 138594Ulmus laevisGermanyKJ210529KJ210550KJ420799KJ420850KJ434999
CBS 109767Acer campestreAustriaKC343075KC343801KC344043KC343559KC343317
D. foeniculinaCBS 111553Foeniculum vulgareSpainKC343101KC343827KC344069KC343585KC343343
CBS 187.27Camellia sinensisItalyKC343107KC343833KC344075KC343591KC343349
D. fusicolaCGMCC 3.17087Lithocarpus glabraChinaKF576281KF576256KF576305-KF576233
CGMCC 3.17088Lithocarpus glabraChinaKF576263KF576238KF576287-KF576221
D. garethjonesiiMFLUCC 12-0542AUnknown dead leafThailandKT459423KT459457KT459441-KT459470
D. helicisCBS 138596HederahelixGermanyKJ210538KJ210559KJ420828KJ420875KJ435043
D. kadsuraeCFCC 52586Kadsura longipedunculataChinaMH121521MH121563MH121600MH121479MH121439
CFCC 52587Kadsura longipedunculataChinaMH121522MH121564MH121601MH121480MH121440
D. masireviciiBRIP 54120cZea maysAustraliaKJ197278KJ197240KJ197258--
BRIP 57892aHelianthus annuusAustraliaKJ197276KJ197239KJ197257--
D. ovalisporaICMP20659Citrus limonChinaKJ490628KJ490507KJ490449KJ490570-
D. ovoicicolaCGMCC 3.17092Lithocarpus glabraChinaKF576264KF576239KF576288-KF576222
CGMCC 3.17093Citrus sp.ChinaKF576265KF576240KF576289-KF576223
D. phaseolorumCBS 113425Olearia cf. raniNew ZealandKC343174KC343900KC344142KC343658KC343416
CBS 116019Caperonia palustrisUSAKC343175KC343901KC344143KC343659KC343417
D. pullaCBS 338.89Hedera helixCroatiaKC343152KC343878KC344120KC343636KC343394
D. pustulataCBS 109742Acer pseudoplatanusAustriaKC343185KC343911KC344153KC343669KC343427
CBS 109784Prunus padusAustriaKC343187KC343913KC344155KC343671KC343429
D. sojaeCBS 100.87Glycine sojaItalyKC343196KC343922KC344164KC343680KC343438
CBS 116017Euphorbia nutansUSAKC343197KC343923KC344165KC343681KC343439
D. sterilisCBS 136969Vaccinium corymbosumItalyKJ160579KJ160611KJ160528MF418350KJ160548
CBS 136970Vaccinium corymbosumItalyKJ160580KJ160612KJ160529-KJ160549
D. subellipicolaMFLUCC 17-1197On dead woodChinaMG746632MG746633MG746634--
Diaporthella corylinaCBS 121124Corylus sp.ChinaKC343004KC343730KC343972KC343488KC343246
Phomopsis sp. 5PMM1657Vitis viniferaSouth AfricaKY511331-KY511363--
PMM1660Vitis viniferaSouth AfricaKY511333-KY511365--
Note. BRIP: Queensland Plant Pathology Herbarium, Brisbane, Queensland, Australia; CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; CFCC: China Forestry Culture Collection Center; CGMCC: China General Microbiological Culture Collection, Beijing, China; ICMP: International Collection of Microorganisms from Plants, Auckland, New Zealand; LGMF: Culture collection of the Laboratory of Genetics of Microorganisms, Federal University of Parana, Curitiba, Brazil; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; PMM: Lesuthu et al., 2019. Ex-type isolates are indicated in bold.

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MDPI and ACS Style

León, M.; Berbegal, M.; Rodríguez-Reina, J.M.; Elena, G.; Abad-Campos, P.; Ramón-Albalat, A.; Olmo, D.; Vicent, A.; Luque, J.; Miarnau, X.; et al. Identification and Characterization of Diaporthe spp. Associated with Twig Cankers and Shoot Blight of Almonds in Spain. Agronomy 2020, 10, 1062. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy10081062

AMA Style

León M, Berbegal M, Rodríguez-Reina JM, Elena G, Abad-Campos P, Ramón-Albalat A, Olmo D, Vicent A, Luque J, Miarnau X, et al. Identification and Characterization of Diaporthe spp. Associated with Twig Cankers and Shoot Blight of Almonds in Spain. Agronomy. 2020; 10(8):1062. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy10081062

Chicago/Turabian Style

León, Maela, Mónica Berbegal, José M. Rodríguez-Reina, Georgina Elena, Paloma Abad-Campos, Antonio Ramón-Albalat, Diego Olmo, Antonio Vicent, Jordi Luque, Xavier Miarnau, and et al. 2020. "Identification and Characterization of Diaporthe spp. Associated with Twig Cankers and Shoot Blight of Almonds in Spain" Agronomy 10, no. 8: 1062. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy10081062

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