An Assessement of the Air Quality in Apple Warehouses : New Record of Aspergillus Europaeus, Aspergillus Pulverulentus, Penicillium Allii and Penicllium Sumatraense as Decay Agents

doi:10.21203/rs.3.rs-185193/v1

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Original Paper

An Assessement of the Air Quality in Apple Warehouses : New Record of Aspergillus Europaeus, Aspergillus Pulverulentus, Penicillium Allii and Penicllium Sumatraense as Decay Agents

https://doi.org/10.21203/rs.3.rs-185193/v1

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Airborne fungi are one of the major components of aeromycobiota known to produce several fungal diseases in fruits. Their presence in indoor environment of warehouses may limit the storage period of apples. Qualitative and quantitative analysis of airborne fungal spores were conducted using gravity settling techniques to detect fungal airspora present in the atmosphere of two apple warehouses in Tunisia. In this study, 375 fungal isolates were obtained and purified. Phylogenetic analysis of Calmodulin, beta-tubulin and ITS regions coupled with phenotypic characterization helped to identify fifteen fungal species. Penicillium exhibited the highest diversity with ten species detected (Penicillium expansum, Penicillium allii, Penicillium polonicum, Penicillium solitum, Penicillium chrysogenum, Penicillium steckii, Penicillium viridicatum, Penicillium sumatraense, Penicillium italicum and Penicillium citrinum), followed by four species of Aspergillus genus (Aspergillus niger, Aspergillus pulverulentus, Aspergillus flavus and Aspergillus europaeus) and Alternaria alternata. In vivo experiments confirmed the pathogenicity of thirteen species at room temperature and under cold-storage conditions. Among them, A. europaeus, A. pulverulentus, P. allii and P. Sumatraense were described for the first time as pathogenic on apples. The present study identified the major airborne fungi associated with postharvest rot in apple storage facilities in Tunisia and may help in efficient control of post-harvest and storage fruit diseases.

General Microbiology

Applied & Industrial Microbiology

postharvest disease

cold rooms

pathogenicity

airspora

fungi

Postharvest fungal diseases are one of the significant factors that limit the shelf life of fruits (Zhang et al. 2019) by altering their organoleptic characteristics (Iqbal et al. 2019). Despite the use of improved storage technologies and the use of plant protection products, fruit losses including apples are estimated to range up to 20 -25% per year in developed countries (Nabi et al. 2017) and are more severe in developing countries (Singh et al. 2017).

In Tunisia, apples are among the most produced and consumed fruit (ONAGRI, 2018). Its production has increased from 98.300 tons in 2017 to 139.500 tons in 2018, an increase of 41% (GIFRUITS, 2018). The governorate of Kasserine is considered as the most apple producing region with 3 million apple trees followed by Siliana (30.150 tons) and greater Tunis (14.352 tons). The preservation of fruits in Tunisia depends heavily on the cold storage industry. In fact, the agricultural sector holds more than 70% of the total refrigerated storage capacities, approximately 1.100.000 per m³ are kept for the storage of fruits and vegetables. Apple storage stations in Tunisia have reported significant losses in fruit quantity (Gara et al. 2019).

These losses are due to the occurrence of postharvest fungal diseases (Bahri et al. 2019). Postharvest diseases of apple are caused by a range of fungal pathogens (Sutton 2014). The most common and destructive postharvest disease of stored apples is the blue mold caused by Penicillium expansum (da Rocha et al. 2016; Darolt et al. 2016; Spadaro 2010; Spotts et al. 1999; Yu et al. 2014). Several other species such as P. chrysogenum (Rharmitt et al. 2016), P. solitum (Sholberg et al. 2005), P. crustosum (Vico et al. 2014), Penicillium carneum (Peter et al. 2012), Penicillium griseofulvum (Spadaro et al. 2011) and Penicillium biourgeianum (Azam et al. 2016) have also been reported to be responsible for the rot.

Infected apples develop several other forms of rot such as Alternaria Rot caused by Alternaria alternata (Shtienberg 2012; Zhu et al. 2017), rot caused by Aspergillus niger and Aspergillus flavus (Hasan 2000), grey mold by Botrytis cinerea (Romanazzi and Feliziani 2014), Bitter rot by Colletotrichum acutatum (Mari et al. 2012) and Bull’s eye rot by Neofabraea vagabunda (Di Francesco et al, 2019). Wounds caused by insects and birds, as well as by physical damage occurring before or during harvest, are important entrance sites for pathogens (Wenneker and Thomma 2020). It has been reported that storage sites may represent a “reservoir” of airborne fungi (O’Gorman and Fuller 2008). These airborne fungi can cause devastating losses (Lucas et al. 1992). In addition, Fungi may produce mycotoxins such as patulin and trichothecenes in infected fruit and it may persist even if they are no longer present in food (Fernández-Cruz et al. 2010). Exposure to theses toxins pose health risk for consumers due to their properties to induce severe toxicity effects at low dose levels (Agriopoulou et al. 2020).

(Fernandez-Baldo et al., 2011)Since several fungal species can be responsible for apple decay, the detection and an accurate identification of pathogens represent a preliminary step to take in order to control postharvest disease. Conventional methods for fungal identification involve the examination of morphological characteristics on standardized media such as color and texture of colonies, growth rate and size of colonies and microscopic structures. Phenotypic characterization may be influenced by various environmental factors such as temperature, humidity and oxygen level (Tiwari et al. 2011) leading to misidentification of the isolates.

Nowadays, fungal pathogens are identified using molecular methods alongside morphological characterization. The internal transcribed spacer rDNA sequence (ITS) is the most widely sequenced marker for fungi. Universal primers are available, and it is the official sequence for barcoding (Schoch et al. 2012). Different gene regions have been also used and showed potential to make a clear distinction between closely-related fungal species like Calmodulin (CaM) and Beta-tubulin (benA) (Visagie et al. 2014, Samson et al. 2014).

A comprehensive research on the diversity and pathogenicity of airspora associated with stored apple is needed in order to develop efficient control of fungal disease in storage units. However, there is a lack of information about the airspora profile in indoor warehouses of apple in Tunisia.

So, the aim of this study was to investigate the predominant airborne fungi occupying the atmosphere of cold storage stations of apples in Tunisia; to identify the obtained airspora using phenotypic and molecular tools and study their pathogenicity.

Sample collection

Sample collection was undertaken from two cold storage facilities of apples located in Ben Arous, Tunisia for three months (December 2016, February and April 2017). Station 1 (S1) and station 2 (S2) had three and seven refrigerated rooms, respectively. Airborne fungi were isolated using a calibrated impaction sampler MAS-100 Eco® (MBV, Switzerland). The MAS-100 Eco® was mounted in the center of each room, approximately one meter from the floor. Two agar plates were taken from each room and the viable particles were impacted on the surface of potato dextrose agar plates, amended with chloramphenicol. Samples were carried to the laboratory and incubated at 25°C for seven days (Despot and Klaric 2014). Fungal colonies were counted and converted to CFU/m³using Feller’s formula given by the manufacturer (MBV, Switzerland):

(Pr=N 1/N + 1/N-1 + 1/N-2 + 1/N-r+1), where Pr=probable statistical total; r=Number of CFU counted; N=total number of holes in the sampling head.

Isolates grouping and selection of representative isolates

Since the number of isolates was large, fungi were grouped based upon their micro- and macro-morphological characteristics using standard taxonomic keys (Dugan 2006; Johnston 2008). A number was assigned to each group and representative isolates were randomly chosen for molecular analysis.

Morphological identification

Spore suspension were made with a solution containing 0.2% agar and 0.05% tween 80 solution as recommended by Visagie et al. (2014) to obtain monosporic colonies. Macroscopic characteristics were studied on Czapek yeast autolysate agar (CYA), yeast extract sucrose agar (YES), malt extract agar (MEA) and potato dextrose agar (PDA) media (Rotem 1994; Samson et al. 2014; Visagie et al. 2014). The studied characteristics were the: colony size, colony color and texture. Microscopic observations were made from 7-10 days cultures grown on MEA media using light microscope (Leica).

DNA extraction

The genomic DNA was extracted from 7-days-old fungal cultures grown in culture plates. Using a sterilized scalpel, a piece of mycelia was transferred to a1.5 mL eppendrof tube containing 500 μl of lysis buffer (400 mM Tris-HCl [pH 8.0], 60 mM EDTA [pH 8.0], 150 mM NaCl, 1% sodium dodecyl sulfate). The tube was then maintained at room temperature for 10 min. After adding 150 μl of potassium acetate buffer (pH 4.8; 60 ml of 5 M potassium acetate, 11.5 ml of glacial acetic acid and 28.5 ml of distilled water), the tube was vortexed for 10 min. and spun at 12.000 rpm for 2 min. and 11.000 rpm for 5 min. The supernatant was transferred to another Eppendorf tube and centrifuged again at 12.000 rpm for 2 min. An equal volume of isopropanol was added to the supernatant, recovered and mixed together by inversion. After centrifuging at 12.000 for 2 min., the DNA pellet was washed in 300 μl of 70% ethanol and spun at 12.000 rpm for 2 min. After removing the supernatant, the DNA pellet was air dried and dissolved in 50 μl of 1× Tris-EDTA (Liu et al. 2000).

PCR amplifications

Calmodulin (Cam), Beta-tubulin (β-tub) genes were targeted in order to design a specific primer set for isolates belonging to Penicillium (PCAM 1, 5’-AGGACAAGGATGGCGATGG- 3’; PCAM 2 ,5’-TCGTCGGTCAGCTTCTC- 3’) and Aspergillus (ABEN 1, 5’-AGTCCGGTGCTGGTAACAACTGG- 3’; ABEN 2, 5’-TCCTGGTACTGCTGGTACTCG- 3’), respectively, using Vector NTI software.

These primers generated amplicon lengths of ≈500 pb and ≈ 900 pb. Specificity tests were conducted amplifying DNA from different fungal genera commonly associated with apple decay. All PCR amplifications were performed in a total reaction volume of 50 ml consisting of 1 µl DNA template, 5 µl of 10×PCR buffer, 1.5 ml of 50mM MgCl₂, 1 µl of dNTP (2.5 mM each), 3 µl of each primer, 1 µl of Taq DNA Polymerase and 34.5 µl of H₂O. The PCR cycle condition was performed in a thermal cycler (Biometra, Germany) with an initial denaturation step for 2 min. at 92°C, followed by 25 cycles (1 cycle consisting of denaturation for 30 s at 92°C, annealing for 30 min at 57°C and extension for 1 min at 72°C), with a final extension of 10 min at 72°C. An Internal Transcribed Spacer (ITS) gene was used in order to identify isolates belonging to Alternaria genus using universal primers (ITS4, 5’-TCCTCCGCTTATTGATATGC- 3’) and (ITS5, 5’-GGAAGTAAAAGTCGTAACAAGG- 3’) (White et al. 1990). The same PCR mix described previously was also used. Template DNA was initially denatured for 2 min. at 94°C followed by 25 cycles of amplification. Each cycle consisted of denaturation for 30 s at 94°C, primer annealing for 30 s at 56°C and extension for 1 min at 72°C. The last cycle was followed by a final extension at 72°C for 10 min. Amplicons were subjected to electrophoresis in 0.8% agarose gels.

Sequencing and phylogenetic analysis

The PCR products were purified using a PCR purification column (Macherey‐Nagel) and sent for sequencing. The sequences of each gene (i.e., Cam, β-tub and ITS) were, firstly, compared using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/) and they were deposited in the GenBank. Then, neighbour-joining phylogenetic trees were produced by MEGA v7.0 (Kumar et al. 2016) with each individual gene (CaM and benA). Aspergillus flavus and P. expansum were used as outgroups.

Pathogenicity tests

To verify the pathogenicity potential of detected isolates, three mature healthy apples (cv. Golden delicious) with uniform size and without physical injuries were used to perform a pathogenicity test as described by Louw and Korsten (2014). All fungal isolates were grown on MEA for seven days at 25℃. Conidia were scraped from the culture and spore concentration was adjusted to 10⁵ spores/ml. Apples were disinfected for two min in 2% sodium hypochloride solution, sterilized with 70° alcohol and rinsed three times with sterile distilled water. Next, two apples were wound-inoculated with 20 μl of conidial suspension on three sides per isolates and were placed on wet paper towels in plastic containers sealed with parafilm. Control fruit were inoculated with sterile distilled water and all the boxes were stored at 25°C for seven days in the dark. All experiments were repeated three times.

Cold storage trials

In order to describe pathogen behavior under storage conditions, mature healthy apples (cv. Golden delicious) were surface disinfested with 70° alcohol and then gently wounded with a sterile micropipette tips on three sides. A spore suspension (1 × 10⁵ conidia/ml), from each isolate, was prepared from 10 day-old culture then inoculated to apples while, controls were inoculated with sterile distilled water. Three replicates were put in sealed container and incubated under refrigerated conditions (2 ± 1.7°C) for 43 days in the dark. All the experiments were repeated three times. During the incubation period, the lesion diameters were recorded every second day starting from the third day after inoculation as described by Louw and Korsten (2014).

Statistical analysis

SPSS program v21 was used for descriptive statistics. Concentrations of airborne fungi are represented as mean ±SD of CFU/m³. The likelihood of statistically significant differences between the concentrations of airborne fungi measured in each sampling was assessed by one-way analysis of variance (ANOVA). Mean comparison of lesion diameter at storage conditions generated by 15 species was estimated according to Duncan test. Statistical significance was set at 0.001. Pathogenicity trials were conducted with two replications under a complete factorial design with two factors (species and incubation time).

Fungal concentration

The study showed that fungal spores were present throughout the storage period in all sampled cold rooms in both stations. Concentrations of airborne fungi in CFU/m³ found in both stations during three months of sampling are listed in Table 1.

In this study, the maximum measured concentration was 2228 CFU/m³and the lowest was 23.67 CFU/m³.

In S1, the highest spore incidence (847.16 CFU/m³) was recorded in N7 followed by N5 (643.5 CFU/m³) and N4 (563.33 CFU/m³). For S2, SF3 holds the lowest airborne fungal concentration with an average of 36.6 CFU/m³ followed by SF1 with 121.33 CFU/m³. As indicated in Fig. 1. Spore concentration in the atmosphere of all the sites peaked during the third month. Levels of airborne fungi in April were significantly higher than the first two months (p<0.001) reaching 1071.14 CFU/m³ and 177.33 CFU/m³ for S1 and S2 respectively.

Phenotypic characterization of collected airspora

Based on morphological features, 375 colonies were obtained from both locations. The obtained isolates were grouped into 21 morphologically similar groups. According to cultural and morphological characteristics, Penicillium, Aspergillus and Alternaria isolates presented 15, 4 and 2 different groups, respectively (Table 2).

The morphological characterization of representative isolates selected for identification is presented in Table 3, Table 4. In CYA, the diameter of the colonies ranged between 25- 53 mm for Penicillium spp. and 35-65 mm for Aspergillus spp. Conidia of all examined penicillia were colored in shades of grey green, blue-green or dull green. In the other hand, Aspergillus isolates produced yellowish to grey, green or well-marked black conidia (Table 4). After one week of incubation at 25ºC, Sporulation of Penicillium isolates were observed on YES. Thirteen samples have a strong sporulation on more than 90% of the colony. C5B1 and C7A29 presented a moderate sporulation; only in the center of the colony. MEA provided the growth of isolates with different colony textures. In this survey, Penicillium sp presented velutinous, fasciculate and floccose colony texture. Colour of conidium varied between green, grey green, blue green or dull green. Aspergillus spp. presented only velutinous and floccose colonies with diameters ranged between 24-56 mm. Microscopic examination showed that the conidiophores of

Penicillium isolates were one-stage branched (biverticillate) or two-stage branched (terverticillate) with metula length between 7.1 µm and 14µm. Examined Conidia presented nearby diameters (2.3 µm -3.7 µm) and were smooth walled except two isolates (S8 and D2) that were finely rough (Table 3). All of Aspergillus isolates had biseriate conidial heads. Conidia observed ranged between 3.5 µm-4.7 µm. Ornamentation of spores was rough in C7A16 and C6B6 echinulate in C7A13 and verruculose in C5B26 (Table 4). On PDA, Alternaria isolates presented olivaceous black and black colonies. Conidiophores were branched and conidia were formed in long ovoid chains.

Molecular identification and phylogenetic analysis

Partial amplification of Cam and β-tub was successful in all tested isolates and yielded a single amplicon band of approximately 500 bp and 900 bp in gel electrophoresis, respectively. Each primer set (PCAM 1-PCAM 2) and (ABEN 1-ABEN 2) were only able to amplify isolates belonging to Penicillium and Aspergillus genus, respectively, thus confirming their specificity. Using (ITS4-ITS5) primers, PCR generated amplicons around 600 pb (data not shown). BLAST analysis of the Calmodulin, the beta tubulin and ITS sequences showed that there was 100% homology of isolate C7A29 with Penicillium solitum CBS 424.89, isolate S7 with Penicillium chrysogenum CBS:109613, isolate C4A1 with Penicillium steckii CBS 139.45, isolate C6A1 with Penicillium sumatraense CBS 281.36, isolate C5A10 with Penicillium italicum CBS 339.48 and C5B1 with Penicillium citrinum CBS 139.45, isolate C7A16 with Aspergillus europeaus CCF 3365, isolate C7A13 with Aspergillus flavus NRRL3357, isolate C6B6 with Aspergillus niger CBS 513.88, isolate C5B26 with Aspergillus pulverulentus CBS 558.65.

Species including P. expansum (represented by three strains C4A4, C2B12 and C7A25), P. allii (represented by two strains C7B20 and C1A25), Penicillium viridicatum (represented by two strains D2 and S8) and P. polonicum (represented by two strains C2A8 and C7A25) were phylogenetically distinct from other species and sister to each other and their standard strains (CBS 325.48, AS3.6669, SZMC 23186 and CBS 222.28, respectively) (Fig. 3 and Fig. 4). Based on sequence analysis of the ITS, SPS1 and SPS2 were identified as Alternaria alternata species complexe (MT126617 and MT126618). Phylogenetic analysis was not conducted for Alternaria isolates because only two strains were amplified by ITS4 and ITS5. The Calmodulin, Beta tubulin and ITS sequences were deposited in the GenBank database and their accession numbers are listed in Table 5.

Biodiversity of aerospores in apple warehouses

The biodiversity of fungal species collected from the air of apple warehouses is listed in table 5. Fifteen fungal species attributed to three genera were isolated. Penicillium, Aspergillus and Alternaria were the most common genera collected with different frequencies (Table 2). In which Penicillium was represented by (10 species) followed by Aspergillus (4 species), one Alternaria specie.

In this survey, Penicillium was by far the most common genus detected with a frequency of 76.8%. P. expansum was the most abundant specie with 42 % followed by P. polonicum (16.7%), P. allii (12.8%), P. chrysogenum (8.7%), P. viridicatum (7.6%), P. solitum (4.9%), P. sumatraense (3.5%) and P.italicum (1.7%). P. citrinum and P. steckii occurred less frequently than the other species (1%).

Aspergillus was the second most common identified fungal genera with a frequency of (14.4%). Aspergillus niger was the most identified species with 53.7% followed by A. flavus (20.4%), A. pulverulentus (14.8%) and A. europaeus (11.1%).

Alternaria, represented by Alternaria alternata, ranked third in abundance in indoor atmosphere of apple warehouses with (5.6%).

In vivo experiments

Undescribed fungal pathogen to apple fruit

Ten species were found to be pathogenic at room temperature and under storage conditions. Among them, four undescribed fungal pathogen to apple fruit were observed and were able to cause decay in healthy apple fruit: A. europaeus, A. pulverulentus, P. allii and P. sumatraense. Lesions provoked by the later species varied in appearance and presented a light to dark brown color and wet to dry texture (Fig. 5).

Decayed area caused by P. allii and P. sumatraense began as water soaked lesions and enlarged gradually in diameter and developed into brown and soft lesions. The pathogens invaded apples tissue slowly in the early stage and blue-green spores were formed on lesion surface in the late stage, starting at the infection sites.

A. europaeus and A. pulverulentus provoked brown and medium-soft lesions on infected apples. As the decayed area ages, they turned to dark brown slowly. Infected apples have an earthy odor and no apparent sporulation was observed along experiments in the inoculation spots.

The inner decayed tissue of inoculated apples appeared yellowish brown. Cross section of mold lesion on apples showed a sharp margin between decayed and healthy tissue. Aspergillus europaeus presented a V-shaped lesions expanding towards the core of the apple.

Pathogenicity of Penicillium, Aspergillus and Alternaria isolates on apples

In ambient conditions, the most devastating species were P. expansum (C4A4) and P. polonicum (C2A8), generating a decay area of 37 mm after seven days post inoculation, followed by P. allii (C7B20) and A. flavus (C5B26) with 16 mm. A. alternata (SP1) and P. solitum (C7A29) had a moderate pathogenicity and presented diameters of 12 mm. Slightly brown lesions of 8 and 7 mm were produced by A. pulverulentus (C5B26) and by P. viridicatum (D2), respectively. Penicillium italicum (C5A10) and A. niger (C6B6) were weak pathogens and they generated the smallest decay area among all tested species (4mm).

Differences in terms of days to significant infection and diameters of lesions were observed among species, during cold storage trials. Decay began to develop five days after inoculation for some isolates. None of the inoculated apples presented sporulation or mycelia growth on injected wounds excluding P. expansum, which started to develop white mycelium on day 37. Pathogenicity varied within the investigated strains and five levels were described according to days where isolates presented a significant infection. P. expansum, P. polonicum, P. solitum, P. italicum and A. flavus showed the first significant lesions on the fifth and the seventh day, respectively, and presented a severe pathogenicity. P. viridicatum and P. pulverulentus started to provoke lesions that were classified as moderately severe on the ninth and eleventh day, respectively. After 13 and 15 days post inoculation, pathogenicity was described as moderate for A. europaeus, P. sumatraense and P. chrysogenum. Starting from the 17^th day, pathogenicity was considered mild for the four following species: A. niger, Alternaria alternata and P. allii (Table 6). P. citrinum and P. steckii were not pathogens to apples and did not present any lesions throughout the test period. According to the Duncan test, values of mean lesions diameters of tested species were significantly different (p<0.001) and presented eleven different groups of fungi over the storage period (Table 6). Penicillium expansum generated the highest mean lesion size (18.31 mm) followed by P. solitum (9.42 mm). The lowest decay area was recorded in A. niger and P. chrysogenum (2.77 mm and 2.82 mm, respectively). The experiment was stopped on the 43th day due to total decay of fruit by P. expansum. Among tested isolates, P. expansum was the only species able to provoke large lesions reaching 35 mm on apples. Penicillium solitum was able to generate lesions; however, they were smaller than P.expansum (15.6 mm) followed by P. sumatraense (11 mm).

Taxonomic description

Taxonomic description, photos of colonies, and fungal structures of P. allii, P. sumatraense, A. pulverulentus and A. europeaus which are undescribed pathogen to apple fruit and novel to Tunisia are given below and presented in Fig. 6 and Fig. 7.

Aspergillus europaeus Hubka, A. Nováková, Samson, Houbraken, Frisvad & M. Kolařík (Fig. 6 C, D)

Pl. Syst. Evol.:645 (2016)

Isolate that was examined was C7A16.

Colonies on MEA at 25°C, after 7 days: 28 mm, floccose, with raised colony centre, colony surface delicately granular, mycelium yellowish grey, sporulation on almost entire surface, reverse brilliant yellow with greyish yellow tint in the colony centre. Colonies on CYA at 25°C, after 7 days: 38 mm, floccose, yellowish white, sporulation yellow, poorly sporulate after 7 days, no exudate, no soluble pigment and reverse yellowish white colony.

Stipes are colourless, smooth, occasionally longer; vesicle biseriate; metulae broadening toward the top, 15 µm long, phialides ampulliform, 9.1×5 µm; conidia colourless, globose, coarsely roughened, 4.2 µm.

Aspergillus pulverulentus (McAlpine) Wehmer (Fig. 6 A, B)

Centralbl Bacteriol 2. Abth. 18: 394 (1907).

≡ Sterigmatocystis pulverulenta McAlpine, Agric Gaz New South Wales 7: 302 (1897).

Isolate that was examined was C5B26.

This strain develops quickly on CYA, produces colonies 35-40 mm in dimater after 7 days at 25°C. Texture is velvety, surface black, reverse white-cream. Basal mycelium is white, exudates are small and colourless. Conidial heads are blackish grey, characteristically radiate. Colonies on MEA developed rapidly and reached 50 mm in diameter at 25°C in 7 days. Texture is velvety, surface brown and black, reverse colour white; colourless exudates.

Stipes are smooth, colourless, upper parts are light brown, 15-20 µm in width, 1000-3000 µm sometimes 5000 µm in length. Vesicles are globose, 60-75 µm in diameter. Phialide biseriate, metulae 20-30 x 3.0-3.5 µm. Conidia are globose and 4.5-5.0 µm in diameter.

Penicillium allii Vincent and Pitt (Fig. 7 A, B)

Mycologia 81: 300, 1989

= Penicillium hirsutum var. allii (Vincent & Pitt) Frisvad, Mycologia 81: 856, 1989.

Isolate that was examined was C7B20

Penicillium allii produces colonies 38 mm in diameter on CYA after 1 week of incubation at 25°C. Colony texture is Granular to weakly fasciculate and only sulcate in center of colony. Conidium is Dull green. Exudate droplets are present, small and clear. Ehrlich reaction is Pink. On MEA, colonies are 36 mm in diameter with grey green conidium.

Conidiophores are Terverticillate, conidia 3.7 μm in diameter, smooth-walled and globose; phialides 9.7 μm x 3 μm; Metulae: 11.8 μm x 2.2-3.8 μm; stipes are short, rough-walled, 75-400 μm x 3.5-5 μm.

Penicillium sumatraense Szilvinyi (Fig. 7 C, D)

Archiv. Hydrobiol. 14, Suppl. 6: 535. 1936.

Isolate that was examined was C6A1

Colonies on CYA 33-42 mm in diameter after 7 days, velvety, conidia dull-green or dark-green, exudate absent or present as small or large (pale)-yellow droplets, occasionally clear or light brown, soluble pigments in most strains absent, in some isolates weakly produced and light brown coloured, margin entire.

Colonies on MEA 27–36 mm in diameter after 7 days, conidia blue-green, light green or grayish-green, floccose colony texture in fresh isolate. Ehrlich reaction is negative.

Conidiophores predominantly biverticillate, occasionally with an additional branch, stipes smooth or finely rough walls 2.0–3.0 µm wide. Metulae vesiculate (10–) 12–16 × 2.0–3.0 µm. Phialides ampulliform, 8.0–10 × 2–3.5 µm. Conidia subglobose or broadly ellipsoidal, finely roughened, occasionally smooth, 2.0–2.5 µm diam.

To our knowledge, this is the first aeromycological survey that provides an overview of fungi profile in the atmosphere of apple warehouses in Tunisia and a description of four previously unrecorded fungal pathogens of apple.

A good deal of the literature on airborne fungal spores of work places and industrial environments has been published (Barontini et al. 2014; Kozdrój et al., 2019; Kakde and Kakde 2012; Sawane and Saoji 2005; Şimşekli et al. 1999; Nova et al. 2015). However, there are only a few papers which deal with airspora collected from cold storage rooms and which have the potential to affect the quality of stored apple (Amiri and Bompeix 2005).

Quantitative analyses of fungal spores revealed that both warehouses had more spore concentration in April than the other two months (1016.24 CFU/m3 and 177.33 CFU/m3 in S1 and S2, respectively). The occurrence of fungal spores in cold chambers found in this study since the first month of sampling could be related to different factors such poor hygiene conditions. It has been suggested that lack of sophisticated storage rooms, poor handling practices by workers and poor sanitary conditions (Kibret and Abera 2012; Sharma et al. 2009) may highly contribute to spore dispersal, thus causing fruit decay. In addition, it has been reported that high spore counts in the air can be attributed to spore discharge mechanisms of the fungi, availability of substrates, temperature and humidity (Money 2015). Sufficient light and oxygen may also favor fungal growth in indoor environments (Khan and Karuppayil, 2012). In addition, moving and handling fruit crates have been suggested to be factors responsible for the increase of airborne fungal spore dispersal in refrigerated rooms (Grisoli et al. 2009; Lehtonen and Reponen 1993). Moreover, unclean fruit crates, rotten fruit and discarded leaves and stems could act as substrates for fungal growth and enhance spore dispersal in air (Arya and Arya 2007; Vermani et al. 2014). Also, the outdoor air is often reported as the dominant source of indoor fungi. Spores can usually enter a building through doors and ventilation systems, hence fungal growth and sporulation may occur (Shelton et al. 2002).

Three different fungal genera, Penicillium, Aspergillus, Alternaria were the most occurring fungi. In several aerobiological studies, the airborne spores belonging to Penicillium, Aspergillus and Alternaria have been demonstrated as the most widespread fungi detected (Dhruv et al. 2010; Khan and Karuppayil 2012; Jean Phellipe et al. 2019). Furthermore, these fungi were found to be responsible for considerable postharvest disease in a similar storage unit of fruit (Barontini et al. 2014; Oliveri et al. 2007). Our results revealed that among the total counts of viable fungi identified, Penicillium (76.8%) was the dominant genera followed by Aspergillus (14.4%) and Alternaria (5.6%). An abundance of Penicillium compared to the other fungi may be due to the amount of decay caused by this fungi found in these storage facilities. Amiri and Bompeix (2005) have confirmed that there is a correlation between the level of airborne Penicillium within the atmosphere of warehouses and the concentration of Penicillium spp. spores on apple surfaces. In such a manner, the decay occurring on apples in storage enhances the release of spores, thus increasing the level of airborne Penicillium. Among obtained isolates, we have determined 21 groups with distinct phenotypes. Penicillium and Aspergillus isolates were phenotypically diverse compared to Alternaria isolates. They were grouped into 15 and 5 morphologically different groups, respectively. Relying on sequencing of markers calmodulin and beta-tubulin allowed for accurate fungal identification of ten Penicillium species and four Aspergillus species. Using ITS4 and ITS5 primers, two Alternaria isolates were identified as A. alternata.

In this study, a rich diversity of airborne Penicillium spp. exists in the atmosphere of apple warehouses. These results were similar to that reported by (Amiri and Bompeix 2005) in a survey conducted in cold rooms in France. However, we reported the presence of P. polonicum, P. allii, P. viridicatum, P. sumatraense, P. italicum, P. citrinum and P. steckii, which were not identified in the later study. Similar Penicillium species were also reported in the pear fruit chain (Scholtz and Korsten 2016) except for P. allii, P. viridicatum, P. sumatraense and P. steckii. No other similar study could be found for comparative purposes.

P. expansum, P. polonicum, P. allii and P. chrysogenum represented approximately 80% of the total Penicillium species isolated. According to literature, these species are commonly associated with apple during the postharvest stage except for P. allii. Other Penicillium species identified in this study accounted for 20% of the total Penicillium population. Penicillium expansum scored the highest frequency and was the most dominant specie detected in the atmosphere. This is in agreement with that study that investigates airborne Penicillium from the atmosphere of cold rooms in France (Amiri et al. 2005). Several studies reported that P. expansum is considered among the most economically important postharvest spoilage fungal species that causes decay in cold-stored pome fruits and found mainly in warehouses (Gong et al. 2019; Grantina-Ievina 2015; Vico et al. 2014; Víctor et al. 2014; Ye et al. 2012). Penicillium polonicum was the second most dominant Penicillium specie found in this study. This specie was isolated by (Scholtz and Korsten 2016) from the pear export chain. Also, P. polonicum is common to cause decay in postharvest environment on stored onions (Duduk et al. 2014), stored yam (Kim et al. 2008) and stored cactus pear (Faedda et al. 2015). In this study, P. polonicum recorded a severe pathogenicity similar to P. expansum. This can be explained by apple cultivar sensitivity towards pathogens which plays an important role in pathogen invasion (Louw and Korsten 2014). Penicillium chrysogenum was also detected from the atmosphere of sampled cold rooms. This specie was isolated from the air of different environment and it was also detected on stored apple (Alwakeel 2013).

Penicillium chrysogenum is regarded as one of the most common representatives of its genus in indoor environments because of its ability to grow at low water activities. It is reported as an important fungal pathogen known to be found in temperate regions (Filtenborg et al. 1996) and to be associated with saprophytic colonization of wounded or decaying fruit.

P. viridicatum, P. solitum, P. sumatraense, P. italicumP. citrinum and P. steckii have been isolated at a frequency of less than 8%. Most of these species are ubiquitous in the food environment and are typical indoor environmental organisms. They were reported in several aeromycological studies (Scholtz and Korsten 2016; Johnston 2008; Altunatmaz et al. 2012).

Aspergillus was the second most common identified fungal genera from the atmosphere of cold rooms. Aspergillus niger and A. flavus represented approximately 75% of the total Aspergillus species isolated. Conidia of Aspergillus spp are commonly recovered from both indoors environments and outside (Gautam et al. 2011). It has been suggested that Aspergillus conidia released in the air may remain airborne for prolonged periods. So, spores found in the atmosphere could contaminate anything in contact with air (Richardson 1998). Aspergillus niger is most commonly found on decaying vegetation or soil and plants (Schuster et al. 2002). It causes rot in many fruits including apples (Liu et al. 2017; Zhang et al. 2018). Aspergillus flavus is a common constituent of airborne mycoflora. It can infect cotton seed, feed crops and apples under specific environmental conditions (Bock et al. 2004; Hasan 2000). The presence of A. flavus in indoor environment should be of concern because it has been reported that 65% of airborne A. flavus produces aflatoxin (Holtmeyer and Wailin 1980), a carcinogenic toxin that cause liver cancer. Then, Aflatoxin synthesis starts simultaneously with spore emergence, and culminates with maximum spore yield.

In this study, Alternaria, represented by Alternaria alternata, ranked third in abundance in indoor atmosphere of apple warehouses. It has been reported that the indoor level of Alternaria spores in the air is influenced by the activity in the room, changes in temperature and relative humidity and the ventilation rate (Woudenberg et al. 2015). Alternaria alternata is known to cause decay on apple fruit during storage (Jurick II et al. 2014) and Alternaria blotch in the orchard (Li et al. 2012).

The cold storage trial was performed in order to determine pathogenicity potential of collected airborne fungi on apples during storage. Thirteen of the fifteen species produced lesions on apples at cold storage temperature.

The ability of these species to cause disease symptoms under storage conditions makes them important pathogens to consider in apple warehouses. Among them, we found four undescribed fungal pathogen to apple; A. europaeus, A. pulverulentus, P. allii and P. sumatraense. This could be explained by cross-contamination phenome.

In this study, apart from apples, other fruits were also stored in sampled cold rooms. (Scholtz and Korsten 2016) reported that environmental isolates that originate from different areas (e.g. surfaces, air, other fruit sources) may cross‐infect different fruit types. The risk of cross‐contamination therefore exists in areas where various fruit types from diverse origins are received and stored.

Colony characteristics and micromorphology of MT452519 agreed well with the description of A. europaeus (Hubka et al. 2016). A. europaeus bears some resemblance to Aspergillus wentii and Aspergillus dimorphicus. But the later species have globose or subglobose vesicles in contrast to pyriform vesicles of A.europaeus. A. europaeus was described for the first by (Hubka et al., 2016) by re-identifying some Aspergillus isolates collected during surveys of the soil in Romania, Spain and Czech Republic, France and Tunisia. It has been reported from corn grain in Korea (Nguyen et al. 2020). To our knowledge, this is the first report of A. europaeus pathogenic to apple.

The strain MT580898 agreed with description of A. pulverulentus given by Thom (Thom 1926). Aspergillus pulverulentus is similar to A. niger in colony morphology and colouration. However, P. pulverulentus has globose to subglobose conidia, smooth to verruculose. It has been isolated from soil of wadi El Natraun, Egypt (Moubasher et al. 2015), from vineyards in Turkey (Eltem et al. 2004). In our study, it is the first record in Tunisia and pathogenic to apple.

The strain C7B20 reported under the accession number MT122170 agreed with the description of P. allii (Frisvad and Samson 2004; Vincent and Pitt 1989). P. allii Vincent & Pitt is a synonymous denomination of “Penicillium hirsutum Dierckx” and “Penicillium corymbiferum Westling” as used in earlier publications (Cavagnaro et al. 2005; Valdez et al. 2009; Dugan et al. 2007, Dugan et al. 2011). This fungus differs from P. hirsutum by fewer and more lightly coloured exudates droplets and from Penicillium crustosum by not becoming crustose on MEA. P. allii is the main postharvest pathogen of garlic (Allen 2009). The fungus has been reported in Argentina (Valdez et al. 2006) and many other countries (Frisvad and Samson 2004). This is the first record of P. allii in Tunisia and virulent to apple.The examined isolate MT224967 agreed well with the description of P. sumatraense (Houbraken et al. 2011). P. sumatraense was formally considered a synonym of Penicillium corylophilum (Pitt 1980), but Peterson (2000) and Houbraken and Samson (2011) showed that these two species are phylogenetically unrelated. Penicillium meleagrinum var. viridiflavum was described without a Latin diagnosis, making the description invalid. So, Pitt et al. (2000) synonymized this species with Penicillium janthinellum; however, Serra et al. (2008) showed that P. meleagrinum var. viridiflavum is genetically close to the type strain of P. sumatraense. Therefore they were maintained as one species. This fungus has been reported from marine environments (Malmstrøm et al. 2000), from cork (Serra et al. 2008), packaging material imported into the Netherlands, pomegranates and bromeliad leaf tissue, from grapes in Iran (Mahdian and Zafari, 2016). P. sumatraense has also been isolated from soil from Ras Rajel, in Tunisia (Houbraken et al. 2011). In our study, P. sumatraense was newly described as pathogenic to apple.

This finding confirms firstly, that elucidating which microorganisms are present in indoor air is fundamentally important to assess decay incidence in storage rooms. Secondly, airborne fungi in apple warehouses could persist, invade and infect apples during storage at low temperature. An increase of the number of storage facilities sampled, extending the sampling period and studying pathogenicity of the isolates on a large range of apple cultivars are required. Further studies must be undertaken on mycotoxin content and effect on apple, related to the four discovered species; A. europaeus, A. pulverulentus, P. allii and P. sumatraense.

Quantitative and qualitative analysis of air samples showed that apple warehouses in Tunisia were loaded with large quantities of airborne fungi and were contaminated by three major fungal spore type Penicillium, Aspergillus and Alternaria. Molecular identification using calmodulin, b-tubulin and ITS in conjunction with phenotypic characterization helped to identify 15 species of fungi. Thirteen species were confirmed to be associated with fruit decay at ambient temperature and cold storage trials. Among them, P. allii, A. pulverulentus, A. europaeus and P. sumatraense were described for the first time as pathogenic on apples. The presence of these pathogens affects the quality of indoor air and poses a potential threat to stored apple shelf life. Vigilant fruit handling, cold room sanitation and correct temperature management during storage are therefore essential in reducing fruit losses during storage.

Acknowledgements

This work was supported by funds from the VRR Project (n°.0310) and by the Ministry of Higher Education and Scientific Research of Tunisia (LR16ES05).

Declarations

Funding: This study was funded by Ministry of Higher Education and Scientific Research of Tunisia (LR16ES05) and by funds from the Project of Valorization of Results (VRR n°.0310).

Compliance with ethical standards

Conflict of interest: The authors declare that they have no conflict of interest.

Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent: All authors are consent to this submission.

Availability of data and material: all data generated or analyzed during this study are included in this published article.

Code availability (software application or custom code)

Authors' contributions: Marwa SMIRI carried out the experiment and wrote the manuscript with support from Najla SADFI, Eduardo Antonio Espeso, Mustapha Rouissi, and Mohamed Zouaoui. Najla SADFI supervised the Project. Amina Kheireddine helped in sampling. Eduardo Antonio Espeso contributed to molecular analysis. All authors read and approved the manuscript.

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Table 1 Mean total fungal spore concentration (CFU/m³) measured at two apple’s storage stations in Tunisia

Station	Room	December	February	April	Total (CFU/m³)
Station 1	Room N1	60.5 ± 6.36	404 ± 67.9	287 ± 4.24	250
	Room N2	50.5 ± 7.78	78.5 ± 9.19	303 ± 18.38	144
	Room N3	117.5 ± 7.78	50.5 ± 0.7	906.5 ± 833.68	358
	Room N4	19.5 ± 6.36	280 ± 7.07	1390.5 ± 1184.4	563
	Room N5	66.5 ± 9.19	72 ± 9.9	1792 ± 616.6	643
	Room N6	44 ± 4.24	78.5 ± 12.02	583 ± 149.9	235
	Room N7	157 ± 9.9	156.5 ± 7.78	2228 ± 0	847
Station 2	Room SF1	32 ± 3.6	42.33 ± 2.51	289.7 ± 6.11	121
	Rom SF2	124.67 ± 5.69	166.67 ± 4.93	182.33 ± 12.22	157
	Room SF3	23.67 ± 1.15	26 ± 2.64	60 ± 6.56	36

^[Mean^±SD]^{; All numbers are positive-hole transformed to account for multiple depositions of particles at single impaction sites.}

^{N1 to N7, 7 refrigerated rooms sampled belonging to station 1; SF1 to SF3, 3 refrigerated rooms sampled belonging to station 2.}

Table 2 Frequency of fungal genera and groups selected for identification

Fugal genera	Number of isolates	Frequency (%)	Phenotypic groups
Penicillium spp.	288	76.8	15
Aspergillus spp.	54	14.4	4
Alternaria spp.	21	5.6	2
Other fungi	12	3.2	_
Total	375	100	21

Table 3 Morphological characterization of Penicillium groups detected in indoor air of storage facilities

Group	Isolate code	CYA	YES	MEA	Phialide	Conidia	Metula	Branching pattern
1	C7B20	38 mm, dull green	Strong	Fasiculate, grey green	9.7 µm	3.7 µm, smooth	11.8 µm	Ter- verticillate
2	C5B1	40 mm, grey green	moderate	Velutinous, grey green	10 µm	2.7 µm, smooth	8.1 µm	Bi- verticillate
3	C1A25	30 mm, dull green	strong	Fasiculate, grey green	6 µm	2.5µm, smooth	11.5 µm	Ter- verticillate
4	C4A4	39 mm, blue green	strong	Floccose, green	7.7 µm	2.4 µm, smooth	10 µm	Ter- verticillate
5	C5A10	50 mm, grey green	strong	Velutinous, green	10.9 µm	4.9 µm, smooth	12.4 µm	Ter- verticillate
6	C6A1	30 mm, dark green	strong	Velutinous, blue green	8.1 µm	2.9 µm, smooth	9.2 µm	Bi- verticillate
7	C2B12	52 mm, green	strong	Floccose, green	7.7 µm	2.3 µm, smooth	14 µm	Ter- verticillate
8	D2	40 mm, Pure green	strong	Velutinous, green	8.3 µm	2.9 µm, finely rough	12.8 µm	Ter- verticillate
9	C2A8	38 mm, blue green	strong	Velutinous, blue green	8.9 µm	3.7 µm, smooth	11.9 µm	Ter- verticillate
10	C7A25	30 mm, dull green	strong	Velutinous, blue green	8 µm	3.3 µm, smooth	10.9 µm	Ter- verticillate
11	S7	38 mm, blue green	strong	Velutinous, blue green	6.4 µm	2.2 µm, smooth	7.8 µm	Bi- verticillate
12	C7A29	27 mm, blue green	moderate	Velutinous, blue green	9 µm	3.5 µm, smooth	11.6 µm	Ter- verticillate
13	C4A1	25 mm, grey green	strong	Velutinous, dull green	8.2 µm	2.4 µm, smooth	7.1 µm	Bi- verticillate
14	S8	18 mm, green	strong	Velutinous, green	8.2 µm	2.8 µm, smooth	12.6 µm	Ter- verticillate
15	C7A1	24 mm, green	strong	Fasiculate, green	7.8 µm	2.3 µm, smooth	14 µm	Ter- verticillate

^{CYA: Colony diameter, Colour of conidium;} ^{YES Degree of sporulation;} ^{MEA Colony texture, Colour of conidium}

Table 4 Morphological characterization of Aspergillus groups detected in indoor air of apple storage facilities

Group	Isolate code	CYA	MEA	Conidia	Vesicules
1A	C7A16	35 mm, yellow grey	24 mm, floccose	4.5 µm, rough	biseriate
2B	C7A13	58 mm, yellow green	56 mm, velutinous	3.5 µm, echinulate	biseriate
3C	C6B6	65 mm, dark brown	50 mm, velutinous	4 µm, rough	biseriate
4D	C5B26	63 mm, dark brown	49 mm, velutinous	4.7 µm, verruculose	biseriate

^{CYA: Colony diameter, Colour;}^{MEA: Colony diameter, texture}

Table 5 Sequence identification results for phenotypic groups analyzed in this study

Groups	Sequence identification	Code isolate	Accession number	Frequency (%)
(1-3)	Penicillium allii	C7B20	MT122170	12.8
		C1A25	MT157405
2	Penicillium citrinum	C5B1	MT157406	1
(4-7-15)	Penicillium expansum	C4A4	MN971604	42
		C2B12	MN793321
		C7A1	MN971600
5	Penicillium italicum	C5A10	MT224966	1.7
6	Penicillium sumatraense	C6A1	MT224967	3.5
(8-14)	Penicillium viridicatum	D2	MT224968	7.6
		S8	MT224969
(9-10)	Penicillium polonicum	C2A8	MN971603	16.7
		C7A25	MN885799
11	Penicillium chrysogenum	S7	MT247677	8.7
12	Penicillium solitum	C7A29	MT224965	4.9
13	Penicillium steckii	C4A1	MT247678	1
1A	Aspergillus europaeus	C7A16	MT452519	11.1
2B	Aspergillus flavus	C7A13	MT863347	20.4
1C	Aspergillus niger	C6B6	MT452520	53.7
3D	Aspergillus pulverulentus	C5B26	MT580898	14.8
(1a-2b)	Alternaria alternata	SPS1	MT126617	5.6
		SPS2	MT126618

Table 6 Pathogenicity trials at storage conditions

Species	Days to significant infection	Pathogenicity		Mean Lesion size at day 43 (mm)		Mean Lesion size during incubation
*Penicillium citrinum*	_	-	0		0 a
*Penicillium steckii*	_	-	0		0 a
*Aspergillus niger*	17	+	6.33		2.77 b
*Penicillium chrysogenum*	15	++	5.5		2.82 b
*Penicillium viridicatum*	9	+++	4.67		3.28 cd
*Alternaria alternata*	17	+	8.83		3.33 cd
*Penicillium allii*	17	+	6.3		3.46 d
*Aspergillus pulverulentus*	11	+++	7.17		3.78 e
*Aspergillus europaeus*	13	++	9.17		4.14 f
*Penicillium sumatraense*	13	++	11.00		5.14 g
*Penicillium italicum*	7	++++	7.33		5.44 h
*Penicillium polonicum*	5	++++	9.00		5.87 i
*Aspergillus flavus*	7	++++	11.00		6.83 j
*Penicillium solitum*	5	++++	15.6		9.42 k
*Penicillium expansum*	5	++++	35.33		18.31 l

^{*Values with different letter are significantly different}^;

^{_ No lesion observed along test period}

^{Scale for pathogenicity: Severe (++++), moderately severe (+++), moderate (++), mild (+), (-) none pathogen}

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First submitted to journal
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An Assessement of the Air Quality in Apple Warehouses : New Record of Aspergillus Europaeus, Aspergillus Pulverulentus, Penicillium Allii and Penicllium Sumatraense as Decay Agents

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