New records of Penicillium and Aspergillus from withered grapes in Italy, and description of Penicillium fructuariae-cellae sp. nov.

Abstract

Penicillium and Aspergillus are common pathogenic fungi of grapes, that occur frequently on withered berries used in the Italian passito wine production. Members of these genera isolated from withered grapes were identified using molecular and morphological approaches. The isolates were examined by amplification of internal transcribed spacer region, β-tubulin, calmodulin and RNA polymerase II second largest subunit. Penicillium bilaiae, Aspergillus pallidofulvus and A. puulaauensis are reported for the first time from Vitis vinifera. Two Penicillium isolates showed a distinct phylogenetic position and different morphological characteristics from P. bissettii and P vasconiae, the two most closely related species. These isolates are assigned to the new species Penicillium fructuariae-cellae, that is here described. An in vitro pathogenicity assay was carried out to evaluate the infectivity to grape berries by Penicillium and Aspergillus isolates recovered in this study. All examined isolates colonized the berries when artificially inoculated, but to a lesser extent than Botrytis cinerea. This suggests that these fungi may contribute, with other pathogenic species, to the onset of post-harvest diseases of grapes.

Headnote

Summary. Penicillium and Aspergillus are common pathogenic fungi of grapes, that occur frequently on withered berries used in the Italian passito wine production. Members of these genera isolated from withered grapes were identified using molecular and morphological approaches. The isolates were examined by amplification of internal transcribed spacer region, ß-tubulin, calmodulin and RNA polymerase II second largest subunit. Penicillium bilaiae, Aspergillus pallidofulvus and A. puulaauensis are reported for the first time from Vitis vinifera. Two Penicillium isolates showed a distinct phylogenetic position and different morphological characteristics from P. bissettii and P vasconiae, the two most closely related species. These isolates are assigned to the new species Penicillium fructuariae-cellae, that is here described. An in vitro pathogenicity assay was carried out to evaluate the infectivity to grape berries by Penicillium and Aspergillus isolates recovered in this study. All examined isolates colonized the berries when artificially inoculated, but to a lesser extent than Botrytis cinerea. This suggests that these fungi may contribute, with other pathogenic species, to the onset of post-harvest diseases of grapes.

Keywords. Saprophytic/pathogenic fungi, grapes, phytogeny, taxonomy, post-harvest diseases.

INTRODUCTION

Fungal contamination of grapes causes severe and economically important losses for world food and beverage industries. Table, wine and raisin grapes can be infected by several species of fungi, on grapevines and/or during postharvest processes. Grapes for Italian passito wine production are particularly vulnerable to fungal infections, during withering carried out in fruit drying rooms (fruttaio) (off-vine withering) (Mencarelli and Tonutti, 2013).

Penicillium and Aspergillus spp. are among the most frequent saprophytic fungal pathogens on withered grapes (Törelli et ai, 2006; Lorenzini et al., 2016; Stefanini et al., 2017). Their presence is very important since they are causal agents of bunch rot and can be mycotoxin producers (Törelli et al., 2006; Somma et al., 2012). In addition, grape contamination by these fungi can lower the quality of the resulting wines. The detrimental effects of withered grapes infected by P. expansum and P. crustosum on the quality of Amarone wine, a dry red passito wine, has recently been documented (Zapparoli et al., 2018).

During previous surveys on fungi associated with withered grapes (Lorenzini et al., 2016, 2018), eight species of Penicillium (P. adametzoides, P. expansum, P. crocicola, P. crustosum, P. glabrum, P. griseofulvum, P. oxalicum and P. ubiquetum) and five species of Aspergillus (A. įlavus, A. sydowii, A. tubingensis, A. uvarum and A. welwitschiae) were identified by phylogenetic analyses. The placements of two isolates (Penicillium sp. P3 and Aspergillus sp. AS100) were not clear enough for reliable species delimitations.

In the present study, isolates P3 and AS100 were phylogenetically and morphologically analyzed to clarify their taxonomic positions. Three isolates of Penicillium and Aspergillus, recovered from withered grapes during the current survey, were also identified. These fungi belonged to species that are reported for the first time from Vitis vinifera. Two isolates were assigned to a new species of Penicillium. The pathogenicity of fungi isolated from grape berries was also assayed.

MATERIALS AND METHODS

On the basis of our previous studies (Lorenzini et al., 2016; 2018) carried out on grape berries of the Garganega and Corvina varieties, collected from fruit-drying rooms located in two Northern Italian winemaking areas (Soave and Valpolicella), five representative strains of Aspergillus and Penicillium were isolated and identified. Three isolates recovered from withered grapes in this study (designated Pdbl, Pls8 and ASİSİ3) were isolated according to Lorenzini et al. (2016). The other two isolates (P3 and AS100) were obtained during a previous sampling (Lorenzini et al., 2016). These isolates are deposited at the Westerdijk Fungal Biodiversity Institute (CBS, Utrecht, the Netherlands) and ITEM Agro-Food Microbial Culture Collection of the Institute of Science and Food Production (CNR-ISPA, Bari, Italy) (Table 1).

DNA was extracted from pure culture of each isolate as previously described (Lorenzini and Zapparoli, 2014). Each DNA extract was used to amplify the internal transcribed spacer (ITS) region, using primers ITS1/ITS4 (White et al., 1990), partial ß-tubulin gene (benA), using primers Bt2a/Bt2b (Glass and Donaldson, 1995), partial calmodulin gene (CaM), using primers cmd5/cmd6 (Hong et al., 2006), and parts of the second largest subunit of RNA polymerase II (rpb2), using primers fRPB25F2/fRPB2-7C (Liu et al, 1999). The amplified products were purified using the NucleoSpin Gel and PCR Cleanup Kit (Macherey-Nagel), and were sequenced in both directions using the same primers applied for amplification (Eurofins Genomics, Edersberg, Germany). Die generated sequences were deposited at GenBank (Table 2).

Combined and individual analyses were conducted using the partial DNA sequences of five isolates recovered from withered grapes, and other reference taxa belonging to the genus Penicillium (sections Lanatadivaricata and Sclerotiora) and Aspergillus (A. versicolor clade and section Circumdati), retrieved from GenBank (Table 2). Maximum Likelihood (ML) analysis of the combined data sets was performed using MEGA7 v. 7.0.25 software. The combined data sets were analysed as three or four distinct partitions. For each individual data set, the most optimal substitution model was calculated in MEGA7 (Kumar et al, 2016) using the Akaiké Information Criterion (AIC). Maximum Likelihood analyses of the individual data sets were also conducted using MEGA7, and robustness of the trees was evaluated by 1,000 bootstrap (BS) replicates. A second measure for statistical support was performed using Bayesian Evolutionary Analysis Sampling Trees (BEAST) Version vl.10.1, 2002-2018 (Drummond and Rambaut, 2007), and the previously obtained most optimal substitution model was used in the analyses. The Markov Chain Monte Carlo (MCMC) analysis used four chains and started from a random tree topology. Burn-in was set to 25% and Tracer v. 1.5.0 (Rambaut and Drummond, 2009) was used to confirm the convergence of chains. The phylograms obtained through the ML analyses were used for presenting the data. Phylograms were redrawn from the tree files using FigTree vl.4 2006-2012. Bootstrap values less than 70% and posterior probability (pp) values less than 0.95 were removed from the phylograms. All ambiguous positions were removed for each sequence pair. Evolutionary analyses were conducted in MEGA7. All isolates of Aspergillus and Penicillium (Pdbl, P3, Pls8, ASİSİ3 and AS100) were inoculated onto Czapek yeast extract agar (CYA), yeast extract sucrose agar (YES), creatine sucrose agar (CREA) (Visagie et al. 2014) and malt extract agar (MEA, 2% w v1 malt extract, 0.1% w w1 peptone, 2% w w1 dextrose, 1.5% w w1 agar) at 25°C in the darkness. The colony diameters (mm) were measured daily for 7 d (three replicate plates for each isolate) and the experiments were performed twice. The colony diameters were also measured on CYA at 15, 30 and 37°C. The cultural characteristics and micro-morphology of each strain was examined on CYA, YES and MEA after 7, 14 or 30 d at 25°C. Colony morphology was also examined on CREA after 7 d incubation.

Growth tests for the fungal isolates were carried out under acidic, neutral and alkaline conditions, as reported by Diao et al. (2018).

Preparations for microscopy were made from colonies grown on CYA, YES and MEA after 7, 14 or 30 d. Lactic acid (60% v v1) was used as mounting fluid and excess conidia were washed away with ethanol (70% v v1), ethanol. Characters were recorded and analyzed using stereomicroscopy (Leica EZ4D, Leica Microsystems). Measurements of fungal components were carried out using light microscopy (Leica DM750) equipped with camera module (Leica ICC50W). Lengths and widths were determined for 20 conidiophores, metulae and phialides, 50 conidia, 20 cleistothecia and 30 ascospores (when present) from each isolate.

The five isolates were tested for their ability to cause disease on grape berries (white fresh table and red withered wine grapes). The berries were surface sterilized by immersion for 5 min in 0.5% NaOCl solution, then rinsed twice with sterile distilled water and placed in compartmentalized square culture dishes. Suspensions of conidia were prepared, adjusted to 104 conidia ml/1 and then inoculated by berry piercing, as reported by Lorenzini and Zapparoli (2014). Mock inoculation (controls) consisted of berries wounded and inoculated with sterile water, and a positive control consisted of berries wounded and inoculated with Botrytis cinerea ITEM 17200. Hie experiment was performed twice, each with three replicates, which each consisted of 25 berries. After 7 and 14 d at 22°C, the disease index (DI) was assessed on a scale of 0 to 4, as previously described (Lorenzini and Zapparoli, 2014). Variance analysis (ANOVA) was used for the DI data to evaluate isolate differences in pathogenicity. Tukey's multiple comparison test (Tukey, HSD) was applied to determine statistically significant differences.

RESULTS

Phylogenetic analysis

Using the BLASTn tool in GenBank, the benA, CaM, ITS and rpb2 gene sequences of Pdbl (ITEM 18277) showed 99% similarity to Penicillium sp. P3 (ITEM 182761) (Lorenzini et al. 2016) for benA, greater than 89% similarity to P. bissettii and P. annulatum for CaM, greater than 98% similarity to different Penicillium species (e.g. P. janthinellum, P. reticulisporum, P. ochrochloron, P. bissettii and P. javanicum) for ITS, and 95% similarity to Penicillium sp. for rpb2. The alignment results of the benA, CaM, ITS and rpb2 gene sequences of P3 were similar to those of Pdbl. Based on these data, the phylogenetic position of both isolates (Pdbl and P3) was evaluated using members of Penicillium section Lanatadivaricata, according to Diao et al. (2018). The ML combined phylogenetic tree (benA+CaM+lTS+rpb2) with the greatest log likelihood (-21693.88) is shown in Figure la. The Pdbl and P3 isolates were grouped together (BS/pp = 100/1), and were distantly related to P. bissettii DOAMC 167011 and P. vasconiae CBS 339.79. Data from phylogenetic analyses using benA, CaM, ITS and rpb2 individually (data not shown) were in concordance with those based on the combine dataset. However, analyses of the combined dataset provided greater support than the individual datasets. Die molecular identification of all isolates recovered in this study is based, therefore, on phylogeny from the combined dataset of gene sequences.

The comparative analysis by GenBank database of benA, CaM and ITS gene sequences of isolate Pls8 (ITEM 18278) showed 99% similarity to different strains of Penicillium bilaiae (section Sclerotiord). The phylogenetic position of Pls8 was therefore evaluated using members of Penicillium section Sclerotiora, reported by Wang et al. (2017). The combined phylogenetic tree (ЬепА+СаМ+nS) with the greatest log likelihood (-11066.6111) confirmed the identification (Figure lb), as the Pls8 (ITEM 18278) was grouped with P. bilaiae NRRL3391 (BS/pp = 100/1).

Comparative analysis using GenBank of benA, CaM and ITS gene sequences of ASİSİ3 (ITEM 18279) showed 99% similarity to A. melleus, A. pallidofulvus, A. sulphureus and A. petrakii for benA, 99% similarity to A. pallidofulvus and different strains of Aspergillus sp. for CaM, and 99% similarity to different Aspergillus species (e.g. A. ochraceus, A. melleus and A. pallidofulvus) for ITS. Based on these results, the phylogenetic analysis was performed using the Aspergillus taxa of section Circumdati, according to Siqueira et al. (2017). The ML combined phylogenetic tree (ЬепА+СаМ+nS) with the greatest log likelihood (-3612.040) showed that ASİSİ3 strongly belongs to A. pallidofulvus, as it grouped with the relevant strain, NRRL4789 (BS/pp = 100/1) (Figure 2a).

The GenBank comparative analysis of benA, CaM and ITS sequences of AS100 (ITEM 18280) showed 99% similarity to A. puulaauensis, A. versicolor and A. jensenii for benA, and 99% similarity to different species of Aspergillus (e.g. A. puulaauensis, A. cvjetkovicii, A. tennesseensis, A. versicolor, A. jensenii and A. creber) for CaM and ITS. In the ML combined tree (ЬепА+СаМ+nS) with the greatest log likelihood (-3938.2941), the clustering of AS100 with A. puulauensis NRRL35641 was highly supported (BS/pp = 78/1), as shown in Figure 2b. Phylogenetic analysis based on the ITS dataset placed AS100 in a group containing most of the Aspergillus taxa of the A. versicolor clade (data not shown).

Culture and morphological characteristics

Penicillium isolates Pdbl (ITEM 18277) and P3 (ITEM 182761) had similar colony morphology on the different media (Figure 3). On CYA, colonies were compact, velvety, with entire margins, and were white; initially yellowish (for Pdbl) or cream (for P3) then becoming gray-green due to abundant sporulation. Spherical or suboval cleistothecia were observed, often covered with a network of hyphae. 'Die cleistothecia matured after three or more weeks, containing evanescent asci and hyaline ascospores, which were smooth-walled and globose to subglobose (Table 3). The colonies were surrounded by diffused soluble pigment in the agar developing as a red-brown colony halos. Hyaline exudates were also observed. Reverse sides of the colonies were light brown and pale cream shades (Figures 3a, b and c).

The growth test revealed little variability among the isolates. The colony diameters at 25°C were 36 to 41 mm for isolate Pdbl and 43 to 48 mm for P3, at 15°C were 16 to 20 mm for Pdbl and 17 to 20 mm for P3 mm, and at 30°C were 46 to 49 mm for Pdbl and 47 to 49 mm for P3. The colony diameters at 37°C were 8 to 10 mm for Pdbl and 10 to 13 mm for P3. On YES, the colonies were moderately deep and radially sulcate, with regular margins. The mycelium was white and pale yellow in the centre for Pdbl, or white and greenish to grayish in the centre for P3. Sporulation was moderate and pale yellow for Pdbl and pale gray-green for P3. The cleistothecia were spherical or suboval. The colonies were surrounded by diffused soluble pigment in the agar, as a faint yellow zones surrounded by faint purpuric-brown halos. The colony textures were pubescent and exudates were not observed. The reverse sides of the colonies were orange to brown in colour (Figures 3b, e and f). The colony diameters were 43 to 50 mm for isolate Pdbl and 42 to 46 mm for P3. On MEA, the colonies were compact, velvety, sometimes radially wrinkled, with entire and plane margins. The mycelia were whitish and non-sporulating mycelium. The cleistothecia were spherical or suboval, and hyaline exudates sometimes were observed. The reverse sides of colonies were yellow and white for Pdbl and white to pale cream for P3 (Figures 3g, h and i). The colony diameters were 38 to 40 mm for isolate Pdbl and 43 to 45 mm for P3. The colony diameters at 25°C after 7 d on PDA pH 4 were 37 to 40 mm for Pdbl and 39 to 41 for P3, on ⅛ strength PDA pH 7 were 35 to 38 mm for Pdbl and 38 to 42 mm for P3, and on Horikoshi agar pH 10 were 29 to 30 mm for Pdbl and 32 to 33 mm for P3. On CREA, the isolates grew well and produced acid (Figures 3j, k, 1). On CYA and YES, conidiophores were monoverticillate or biverticillate, and a minor proportion were divaricate. Metulae were oblong and divergent, phialides were ampuliform, and conidia were smooth walled and globose to subglobose (Figure 4; Table 3). On MEA, conidiophores were rarely observed.

On CYA, colonies of P. bilaiae Pls8 were convex, with concentric folds, conidia were abundantly produced, and aerial mycelium was lanose and floccose and grey-green to white. The colony margins were margin entire and white and sporulation was heavy. Exudates were dark, superficial or embedded, and the colonies were surrounded by diffused soluble pigment into agar as faint purpuric-brown halos. The colony reverse sides were orange-yellow. On YES, colonies were heavily wrinkled, white and pubescent at the margins, green-gray in colour, and felty in the centres. The colonies sporulated heavily. The margins entire and exudates were not observed. The colonies were surrounded by soluble pigment diffused into the agar as faint yellow zones surrounded by faint purpuric-brown halos. The colony reverse sides were orange to brown. On MEA, the colonies were velutinous, floccose and raised in the centre, with villose white aerial mycelium and entire margins which were entire, low, plane and white. The colonies were; sporulating heavily, without exudates, and were surrounded by diffused soluble pigment into the agar as faint yellow zones. The reverse sides of the colonies were yellow to pale orange. Colony diameters were 22 to 26 mm on CYA, 20 to 22 mm on YES and 16 to 20 mm on MEA. On CREA, the fungal growth was weak with good acid production. On CYA, YES, and MEA the conidiophores were monoverticillate, the stipes were smooth walled and mostly globose vesiculate or subglobose to ellipsoidal. The phialides were ampulliforms and wide at the bases. Conidia were globose to subglobose (Table 3).

On CYA, colonies of A. pallidofulvus ASİSİ3 was velutinous, the mycelium was white, and sporulation was pale yellow to cream, without exudates. Reverse sides of colonies were light brown to brown. On YES, the colonies were moderately powdery to velutinous, the mycelium was white without exudates, and sporulation was light yellow Colony reverse sides were pale brown to yellow. On MEA, the colonies were velutinous, felty and floccose and the mycelium was white with light yellow sporulation, without exudates. Colony reverse sides were cream to pale brown. Colony diameters were 56 to 60 mm on CYA, 70 to 75 mm on YES and 55 to 61 mm on MEA. On CREA, growth was weak with no acid production. On CYA, YES and MEA, the conidiophores were biseriate, stipes were hyaline to pale brown, vesicles were globose, metulae were oblong covering entire vesicles, and phialides were ampulliform. Conidia were globose, subglobose to ovoid and smooth (Table 3).

On CYA, colonies of A. puulaauensis AS100 was sulcate and raised in the centre. Sporulation green to gray. Colony margins were regular, plane and white without exudates. Colony reverse sides were yellow to light orange. On YES, colonies were moderately powdery to felty, sulcate, raised in the centre, and white with green to grey and light pink shadows. Sporulation was green to gray. Colony margins were regular and no exudates were produced. Colony reverse sides were yellow to light brown. On MEA, colonies were sulcate, and raised in the centre. Mycelium was white and sporulation was light yellow. Colony margins were regular, plane and white, without exudates. Colony reverse sides were yellow to light orange. The colony diameters were 18 to 22 mm on CYA, 24 to 25 mm on YES and 18 to 20 mm on MEA. On CREA, growth was moderate growth without acid production. On CYA, YES and MEA, the conidiophores were biseriate, and stipes were smooth, and hyaline to light yellow. Vesicles were spatulate or subspherical, and metulae were oblong covering entire vesicles. Phialides were oblong from which globose or subglobose conidia developed (Table 3).

Pathogenicity assay

All Penicillium and Aspergillus isolates obtained in this study displayed ability to infect grape berries, although infection was much less than for B. cinerea (Table 4). Wounded inoculated berries initially showed mycelium around the inoculation sites and subsequently necrotic areas became visible, particularly in white fresh table berries. Red withered berries inoculated by Penicillium isolates Pdbl, P3 and Pls8 caused typical symptoms of Penicillium infection characterized by tufts of white and green mycelium erupting from the berry skins (Figure 5). Abundant sporulation was observed on berries infected by A. pallidofulvus isolate ASİSİ3. The pathogens were re-isolated from inoculated berries, fulfilling Koch's postulates.

Taxonomy

The phylogenetic analysis based on four different loci and the morphological analysis showed that two isolates of Penicillium (Pdbl and P3) recovered from withered grapes were distinct from any known species within the Penicillium section Lanata-divaricata. Therefore, these isolates are here described as members of a new Penicillium species.

Penicillium fructuariae-cellae Lorenzini, Zapparoli & Perrone sp. nov.

MycoBank: MB 831228 - Figures 3 and 4

In: subgenus Aspergilloides, section Lanata-divaricata.

ITS barcode: MK039434. Alternative markers: benA =KU554679; CaM = MK045337; rpb2 = MK520927.

Etymology. Latin, fructuariae-cellae, meaning fruitdrying room for grape withering, the place where two representative strains were isolated.

Type specimen. ITALY, Verona, Maraño di Valpolicella, on Corvina withered grapes stored in fruit-drying room, Dec. 2013, coll. M. Lorenzini and G. Zapparoli, isol. M. Lorenzini and G. Zapparoli, P3 (holotype CBS 145110'; ex-type strain ITEM 18276'1').

Colony morphology. Colony diameters (mm), 7 d: CYA 43-48; YES 42-46; MEA 43-45; CREA 35-37; CYA 15°C 17-20; CYA 30°C 47-49; CYA 37°C 10-13.

Colonies on CYA after 7 d at 25°C were compact, velvety; margins entire and white; sporulation abundant, conidia en masse pale gray-green; cleistothecia spherical or suboval covered with networks of hyphae; asci evanescent, ascospores hyaline, smooth-walled, globose to subglobose; soluble red-brown pigments and hyaline exudates produced; colony reverse sides pale brown and pale cream. Colonies on YES after 7 d at 25°C were moderately deep, radially sulcate, with regular margins; mycelia white and green to gray; sporulation moderate, conidia en masse pale gray-green; cleistothecia observed; soluble yellow and faint purpuric-brown pigments produced from colonies; exudates absent; colony reverse sides orange to brown. Colonies on MEA after 7 d at 25°C were compact, sometimes radially wrinkled, with entire and plain margins with velvety texture; mycelia white; sporulation poor; cleistothecia observed; hyaline exudates sometimes observed; colony reverse sides white yellow-pale cream. Colonies grew well on CREA after 7 d at 25°C, with good acid production.

Conidiophores (on CYA) monoverticillate, biverticillate, with a minor proportion divaricate; stipes smooth, 55-313 x 2-3.5 pm, metulae divergent, 2-4 per stipe or branch, 9-47 x 2-3.5 pm; phialides ampulliform, 2-10 per metula, 3.5-10 x 2-3 pm; conidia smooth walled, globose to subglobose, 2-3.5 pm (mean = 2.5 pm ± 0.4 pm n = 50); cleistothecia covered with a network of hyphae, 74-221 x 67-194 pm (n = 20); asci evanescent; ascospores hyaline, smooth-walled, globose to subglobose 2.5-4.5 pm (mean = 3.0 pm ± 0.4 pm, n = 30).

Other strains examined. ITALY, Verona, Montecchia di Crosara, Garganega withered grapes stored in a fruit-drying room, Nov. 2017, coll. M. Lorenzini and G. Zapparoli, isol. M. Lorenzini and G. Zapparoli, Pdbl, ITEM 18277 = CBS 145111, ITS barcode: MK039435. Alternative markers: benA = MK045333; CaM = MK045338; rpb2 = MK520928.

Notes. Penicillium fructuariae-cellae is classified in section Lanata-divaricata, and is distantly related to other Penicillium species. The multi-locus phylogeny placed it closed to P. bissettii KAS1951 and P. vasconiae CBS 339.79 (Figure la). Penicillium fructuariae-cellae produces red-brown pigments in CYA compared with P. bissettii and P. vasconiae that do not produce pigments. Penicillium fructuariae-cellae mainly differs from P. vasconiae and P. bissettii in conidiophore structure and size as it has longer conidiophores than P. vasconiae, and shorter conidiophores than P. hissetti. Penicillium fructuariae-cellae also differs from P. bissettii by having smooth stipes. Penicillium fructuariae-cellae differs from P. vasconiae in phialide shape (P. vasconiae has long tapped neck phialides) and having smooth and small conidia.

DISCUSSION

Phylogenetic analysis and morphological observations of the isolates recovered in this study from withered grapes compile the first report of P. bilaiae, A. pallidofulvus and A. puulaauensis from Vitis vinifera.

The species identification of Pls8 (ITEM 18278) as P. bilaiae was taxonomically clear due to its congruence with phylogenetic and morphological data from the P. bilaiae holotype NRRL 3391. However, isolate Pls8 showed slower growth in agar media and some micro-morphological differences (i.e. longer stipes, wider and more numerous phialides) than holotype PB-50 described by Pitt (1979) and Savard et al. (1994). Prior to the present study, P. bilaiae was detected in Portuguese grapes through morphological observations (Serra et al., 2005), a method that does not provide sufficient data for reliable identification at the species level. Penicillium bilaiae is morphologically similar to both P. alexiae and P. adametzioides (Visagie et al., 2013). Hence, the present study provides the first taxonomic evidence of the occurrence of P. bilaiae on Vitis vinifera.

The assignment of isolate ASİSİ3 (ITEM 18279) to A. pallidofulvus was confirmed by its genealogy and macromorphology, according to the description of the holotype A. pallidofulvus NRRL 4749 (Visagie et al., 2014). However, micro-morphological observations of ASİSİ3 showed differences in the size of its stipes and phialides (respectively smaller and shorter for ASİSİ3 than the holotype), and with no production of sclerotia by ASİSİ3, in contrast with the A. pallidofulvus holotype. This spe- cies, recently introduced into section Circumdati, has also been isolated from green coffee beans in India and clinical samples (Visagie et al. 2014; Masih et ai, 2016). The recovery of this species from grapes and the results of pathogenicity assays show that A. pallidofulvus may exhibit pathogenic behaviour in grapevine. Nevertheless, further investigation is required to determine occurrence for this fungus on withered grapes and its infectivity under post-harvest environmental conditions.

The use of multi-locus phylogenetic analysis (CaM, benA and ITS) resolved the taxonomic position of isolate AS100 (ITEM 18280), identifying it as A. puulaauensis. Assignment of the isolate to this species has previously proved impossible using only the CaM gene sequence (Lorenzini et ai, 2016). Moreover, AS100 and the holotype A. puulaauensis NRRL 35641 (Jurjevic et ai, 2012) both showed identical colony macro- and micro-morphological characters. The recovery of A. puulaauensis from grape berries further supports its worldwide and cosmopolitan distribution, since this species has previously been reported from disparate environments including Hawaiian plants, Atlantic sponges, Italian cheese, air samples in North America and clinical samples (Jurjevic et ai, 2012; Siqueira et ai, 2017; Bovio et ai, 2018; Decontardi et ai, 2018).

Based on multi-locus phylogenetic analyses, isolates Pdbl (ITEM 18277) and P3 (ITEM 18276T) represented a distinct species, herein named P. fructuariae-cellae. These two isolates form a phylogenetic cluster based on benA+CaM+nS+rpb2 combined gene genealogies, distinct from any currently described species in section Lanata-divaricata, which was recently updated with 13 new Penicillium species collected from Chinese acidic soils (Diao et ai, 2018). Penicillium fructuariae-cellae is acid-preferential, like most of the new species described by Diao et ai (2018). This is a physiological characteristic congruent with the acidic habitat (grapes) from which it was isolated. Isolates of P3 and Pdbl are distantly related to P. vasconiae CBS 339.79 (Ramirez and Martinez, 1980) and the recently described species P. bissettii (Visagie et ai, 2016). Moreover, P. fructuariaecellae displays some differences in macro-morphological characteristics (e.g. colony colour, texture and pigment production) compared with related species. The pigment production by these two isolates distinguishes them from P. vasconiae and P. bissettii that produce no pigments. Penicillium fructuariae-cellae produces monoverticillate, sometimes biverticillate conidiophores, whereas P. vasconiae is strictly monoverticillate and P. bissettii is biverticillate/terverticillate. The conidiophores of P. fructuariae-cellae are longer than those of P. vasconiae (< 50 pm; Ramírez and Martinez, 1980) and shorter than conidiophores of P. bissettii (190-670 pm; Visagie et ai, 2016). Its stipes are smooth, while in P. bissettii they are rough. Its phialides are also shorter than those of P. vasconiae (9-13 pm; Ramírez and Martinez, 1980). As well, of P. fructuariae-cellae produced smaller conidia than P. vasconiae (4-4.5 pm; Ramírez and Martinez, 1980). The conidial surfaces of P. fructuariae-cellae were smooth, while those of P. vasconiae are conspicuously echinulate (Ramírez and Martinez, 1980). These morphological differences together with phylogenetic information, support the uniqueness of P. fructuariae-cellae.

Based on the results of pathogenicity assays, the detrimental effects of these fungi on withered grapes was quite significant. Although these Penicillium and Aspergillus isolates were less pathogenic than B. cinerea, they could make major contributions to berry rotting. Decay of berry surfaces, like that observed on infected berries caused by each isolate due to mycelial growth and necrosis, is important for susceptibility to subsequent fungal infections by the same and other pathogens (Padgett and Morrison, 1990). Incidence and symptoms of grape diseases caused by these fungi under fruit-drying room conditions require further investigation.

In conclusion, this study describes isolates belonging to species of Penicillium and Aspergillus from withered grape berries that have not been previously reported from V. vinifera. A new species of Penicillium from this host, P. fructuariae-cellae, is herein described. The recovery of these species highlights the complexity of fungal species affecting withered grapes (Lorenzini et ai, 2016; 2018). According to pathogenicity assays, P. fructuariae-cellae, P. bilaiae, A. pallidofulvus and A. puulaauensis are able to infect grapes but with much lower infectivity than B. cinerea, which is the most important pathogen occurring on withered grapes. It is likely that the species identified in this study are less pathogenic than other Penicillium and Aspergillus species frequently reported on withered grapes (e.g. P. expansum, P. crustosum, A. tubingensis and A. uvarum) (Lorenzini et ai, 2016). Further investigations are necessary to ascertain the pathogenic role of P. fructuariae-cellae, P. bilaiae, A. pallidofulvus and A. puulaauensis under withering conditions, as well as their interaction with other causal fungal agents causing rots of grape berries.

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Citation: Lorenzini M., Cappello M.S., Perrone G., Logrieco A., Zapparoli G. (2019) New records of Penicillium and Aspergillus from withered grapes in Italy, and description of Penicillium fructuariae-cellae sp. nov.. Phytopathologia Mediterranea 58(2): 323-340. doi: 10.14601 /PhytopathoLMediter-10619

Accepted: June 115, 2019

Published: September 14, 2019

Copyright: © 2019 Lorenzini M., Cappello M S., Perrone G., Logrieco A., Zapparoli G. 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: Dimitrios I. Tsitsigiannis, Agricultural University of Athens, Greece.

References

LITERATURE CITED

Bovio E., Garzoli L., Poli A., Prigione V, Firsova D., ... Varese G.C., 2018. Die culturable mycobiota associated with three Atlantic sponges, including two new species: Thelebolus balausformis and T. spongiae. Fungal Systematics and Evolution 1: 141-167.

Decontardi S., Soares C.( Lima N.. Battilani R, 2018. Polyphasic identification of Penicillia and Aspergilli isolated from Italian grana cheese. Food Microbiology 73: 137-149.

Diao Y-Z.( Chen Q., Jiang X.-Z., Houbraken J., Barbosa R.N.Wu W.-R, 2018. Penicillium section Lanatadivaricata from acidic soil. Cladistics, 0: 1-36.

Drummond A.J., Rambaut A., 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology, 7: 214.

Glass N.L., Donaldson G.C., 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology, 61: 1323-1330.

Hong S.B., Cho H.S., Shin H.D., Frisvad J.C., Samson R.A., 2006. Novel Neosartorya species isolated from soil in Korea. International Journal of Systematics and Evolutionary Microbiology 56: 477-486.

Jurjevic Z., Peterson S.W., Horn B.W., 2012. Aspergillus section Versicolores: nine new species and multilocus DNA sequence based phytogeny. IMA Fungus 3: 59-79.

Kumar S., Stecher G.( Tamura K., 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870-1874.

Liu Y., Whelen S., Hall B.D., 1999. Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. Molecular Biology and Evolution 16: 1799-1808.

Lorenzini M., Zapparoli G., 2014. Characterization and pathogenicity of Alternaría spp. strains associated with grape bunch rot during post-harvest withering. International Journal of Food Microbiology 186: 1-5.

Lorenzini M., Cappello M.S., Logrieco A., Zapparoli G., 2016. Polymorphism and phylogenetic species delimitation in filamentous fungi from predominant mycobiota in withered grapes. International Journal of Food Microbiology 238: 56-62.

Lorenzini M., Simonato B., Favati F, Bernardi R, Sbarbati A., Zapparoli G., 2018. Filamentous fungi associated with natural infection of noble rot on withered grapes. International Journal of Food Microbiology 272: 83-86.

Masih A, Singh P.K., Kathuria S., Agarwal K., Meis J.F., Chowdhary A., 2016. Identification by molecular methods and matrix-assisted laser desorption ionization-time of flight mass spectrometry and antifungal susceptibility profiles of clinically significant rare Aspergillus species in a referral chest hospital in Delhi, India. Journal of Clinical Microbiology 54: 2354-2364.

Mencarelli E, Tonutti R, 2013. Sweet, Reinforced and Fortified Wines: Grape Biochemistry, Technology and Vinification. John Wiley & Sons, Ltd, Chichester, UK.

Padgett M., Morrison J.C., 1990. Changes in grape berry exudates during fruit development and their effect on mycelial growth of Botrytis cinerea. Journal of the American Society for Horticultural Science 115: 269-273.

Pitt J.I., 1979. The genus Penicillium and its teleomorphic states Eupenicillium and Talalvmyces. London, Academic Press, UK.

Rambaut A., Drummond A.J., 2009. Tracer v. 1.5. Available from: http://tree.bio.ed.ac.uk/software/tracer/.

Ramirez C., Martinez A.T., 1980. Some new species of Penicillium recovered from the atmosphere in Madrid and from other substrata. Mycopathologia 72: 181-191.

Savard M.E., Miller J.D., Blais L.A., Seifert K.A., Samson R.A., 1994. Secondary metabolites of Penicillium bilaii strain PB-50. Mycopathologia 127: 19-27.

Serra R., Braga A., Venancio A., 2005. Mycotoxin-producing and other fungi isolated from grapes for wine production, with particular emphasis on ochratoxin A. Research in Microbiology 156: 515-521.

Siqueira J.P.Z., Sutton D.A., Genė J., García D., Wiederhold, N., ... Guarro J., 2017. Multilocus phylogeny and antifungal susceptibility of Aspergillus section Circumdati from clinical samples and description of A. pseudosclerotiorum sp. nov. Journal of Clinical Microbiology 55: 947-958.

Somma S., Perrone G., Logrieco A., 2012. Diversity of black Aspergilli and mycotoxin risks in grape, wine and dried vine fruits. Phytopathologia Mediterranea 51: 131-147.

Stefanini L, Carlin S„ Tocci N„ Albanese D„ Donati C„ ... Cavalieri D., 2017. Core microbiota and metabolome of Vitis vinifera L. cv. Corvina grapes and musts. Frontiers in Microbiology 8: 457.

Törelli E., Firrao G., Locci R., Gobbi E., 2006. Ochratoxin А-producing strains of Penicillium spp. isolated from grapes used for the production of "passito" wines. International Journal of Food Microbiology 106: 307-312.

Visagie C.M., Houbraken J., Rodriques C., Silva Pereira C., Dijksterhuis J., ... Samson R.A., 2013. Five new Penicillium species in section Sclerotiora: a tribute to the Dutch Royal family. Persoonia 31, 42-62.

Visagie C.M., Varga J., Houbraken J., Meijer M., Kocsube S.Samson S.A., 2014. Ochratoxin production and taxonomy of the yellow aspergilli (Aspergillus section Circumdati). Studies in Mycology 78: 1-61.

Visagie C.M., Renaud J.B., Burgess K.M.N., Mailoch D.W., Clark D., ... Seifert K.A., 2016. Fifteen new species of Penicillium. Persoonia 36: 247-280.

Wang X.C., Chen K., Zeng Z.Q., Zhuang W.Y., 2017. Phylogeny and morphological analyses of Penicillium section Sclerotiora (Fungi) lead to the discovery of five new species. Scientific Reports 7: 8233.

White T.J., Bruns T., Lee S., Taylor J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: a Guide to Methods and Applications (M.A. Innis, D.H. Gelfand, J.J. Sninský and T.J. White eds.), Academic Press San Diego, CA, USA, 315-322.

Zapparoli G., Lorenzini M., Tosi E., Azzolini M., Slaghenaufi D., ... Simonato B., 2018. Changes in chemical and sensory properties of Amarone wine produced by Penicillium infected grapes. Food Chemistry 263: 42-50.