Twenty-five grams (25 g) of each strawberry fruit sample were suspended in 225 ml of sterile buffered peptone water (BPW) solution and homogenized with a grinder (Stomacher^® 400 circulator) for 2 min. Dilution series were prepared from stock solution. A volume of 0.1 ml of each dilution was transferred to Petri dishes surface containing culture media suitable for each target microorganism. However, for the detection of L. monocytogenes, 25 g of each sample were homogenized in 225 ml of PALCAM broth and incubated at 30°C for 24 h.

Microbiological analyses were studied according to the International Organization for Standardization (ISO) methodologies. The bacteriological food safety and quality parameters studied were: E. coli (ISO, 2001), Salmonella spp. (ISO, 2002), L. monocytogenes (ISO, 1996; ISO, 1998), B. cereus (ISO, 2004), Coagulase-positive staphylococci (ISO, 1999), and Sulfite-reducing clostridia spores (IPQ, 1986).

Presumptive L. monocytogenes colonies on PALCAM agar were streaked on sheep blood agar to assess hemolysis, carbohydrate zymogram (rhamnose, xylose, and mannitol), and the Christie-Atkins-Munch-Petersen (CAMP) test (Ortiz-Solà et al., 2020). For suspected Salmonella spp. colonies, biochemical confirmation was performed with Triple Sugar Iron (TSI) agar, urea agar, and L-lysine decarboxylase medium (Uyttendaele, 2014). For the determination of E. coli sample was placed in Petri dish center, subsequently, 20 ml of violet red bile agar (BD Difco, heated to 45°C) was added. After solidification, agar was overlaid with 5 ml of violet red bile agar (double strength) supplemented with 4-Methylumbelliferyl-β-D-glucuronide. The inverted plates were incubated at 35°C for 24 h, and colonies fluoresced under UV light as E. coli bacteria (Cárdenas et al., 2013).

For detection of Sulfite-reducing clostridia spores, 10 ml of each sample in buffered peptone water (BPW) solution was heat-shocked at 80–85°C for 10 min before culture to inactivate vegetative bacteria and enhance sporulation phenomenon. Samples were then transferred to 10 ml of differential reinforced clostridium medium (DRCM) and incubated under anaerobic conditions in a jar at 37°C for 5 days. This method is based on ferric Sulfite reduction to iron sulfide and samples are considered positive when color changes (black dots) (Oliveira et al., 2019). Bacilli showing typical B. cereus group growth, i.e., rough, dry colonies with a pink-purple background surrounding an egg yolk precipitation, were counted, isolated, and selected for hemolytic activity, tested on sheep blood agar plates after 1 day of incubation at 30°C. Convex black colonies of staphylococci, with or without halo on Baird-Parker medium for 24–48 h at 37°C, were isolated and tested for catalase reaction and coagulase activity using rabbit plasma (Evêncio-Luz et al., 2012).

Analysis of mycoflora associated with strawberry fruits was performed using the modified Buvard method (Benkirane, 1995). Strawberries with lesions were disinfected with 1% sodium hypochlorite. Then, strawberries pieces were placed in Petri dishes containing Potato Sucrose Agar (PSA) and incubated for 7 days in the dark at 24°C. Optical microscopic examination of the different cultures allowed for the determination of fungal species using the keys identification of Gilman (1957), Tarr (1962), Ellis (1971), Chidambaram et al. (1974), Domsch et al. (1980), and Champion (1997). Contamination percentage by different fungal species is calculated according to Ponchet (1966) method which defines the isolation frequency of the different fungi from 100 lesions present on the studied fruits according to the following formula (Eq. 1):

Yeasts were isolated on YPG agar medium (Y: 1% yeast extract, P: 2% peptone, and G: 2% glucose). Three times a week for 5 weeks in June and July 2020, plastic trays each containing 500 g of strawberries were harvested during the harvest period. Each tray was placed in a sterile plastic bag at the farm and sampled at the laboratory 3 h later. Whole strawberries with calyxes (100 g) were mixed with 100 ml sterile chilled KH₂PO₄ (0.1 M) for 45 s at half speed, then 45 s at full speed. Four successive dilutions of 10 times were performed aseptically with KH₂PO₄ (0.1 M). An aliquot of 100 μl was put on Petri plates containing YPG agar supplemented with two antibiotics chloramphenicol and kanamycin at 5 μg/ml and incubated at 30°C for 2 days.

Yeast colonies were counted on all Petri dishes and at all dilution levels when possible, after the longest possible incubation before colonies coalesced. Counts were expressed as the number of yeast per g of strawberries. Yeast purification was performed by at least five successive streaking on YPG agar (pH 4) incubated at 30°C. The identification was done on Wikierham medium for the assimilation (Auxanogram) and fermentation (Zymogram) tests. The identification key is the one proposed by Kreger-van Rij (1984).

Optical microscopic examination of the different cultures allowed us to determine the fungal species using the identification keys and consultation of the fungal collections of the National School of Agriculture (ENA-Meknes). Table 4 groups the results obtained in the two localities targeted (Loukkos and Gharb) by this microbiological study.

From these preliminary results, we notice that Aspergillus niger, B. cinerea, Fusarium spp., Penicillium spp., Rhizopus spp., and Alternaria alternata species are dominant in both study areas with a priority of fungal contamination in strawberry samples from Loukkos locality. Indeed, a marked presence is noted for A. niger on strawberry samples, with six positive samples from the Loukkos region and three positive samples from the Gharb region. B. cinerea was also found on five samples from Loukkos and two samples from Gharb. Three samples from the Loukkos locality were contaminated with Fusarium spp. while only one strawberry sample from the Gharb locality was positive. The same results were reported for Penicillium spp. Moreover, the presence of Rhizopus spp. was revealed on three samples from the Loukkos region against two samples from the Gharb region. Two strawberry samples from Loukkos and one sample from Gharb were positive for the Alternaria alternate strain. However, Colletotrichum acutatum, Mucor spp., and Penicillium notatum were isolated only from the Loukkos samples. While Aspergillus nidulans and Fusarium oxysporum species were found in the samples from the Gharb region. In general, strawberry samples from the Loukkos area have a relatively high prevalence of these microorganisms with 26 positive samples in total against 11 positive samples in the Gharb area.

According to the molds study, the most common fungi found in strawberry samples from both regions (Loukkos and Gharb) were A. niger, B. cinerea, Fusarium spp., Penicillium spp., Rhizopus spp., and A. alternata with a significant predominance in samples from the Loukkos region. In a study done by Saleh and Al-Thani (2019), samples of cucumber, tomato, strawberry, and orange were tested to identify spoilage agents. Various fungal types were isolated from strawberry samples, including 5 Aspergillus spp., 8 Rhizopus spp., 3 Penicillium spp., and 5 Botrytis spp. In the same report, results indicate that strawberry samples contain the greatest fungi variety of all the fruits. B. cinerea causes gray mold, the most important disease of strawberries harvested worldwide (Petrasch et al., 2019; Weber and Hahn, 2019). A. niger is by far the most common Aspergillus species responsible for the post-harvest rot of fresh fruit, including apples, pears, peaches, citrus fruits, grapes, and strawberries (Pitt and Hocking, 2009).

Strawberries are fruits closer to the ground and therefore are more susceptible to soft rot mainly Rhizopus spp. and Mucor spp. (Feliziani and Romanazzi, 2016). In a study conducted by Agyare et al. (2020) in the United Kingdom, Mucor piriformis and Rhizopus spp. were isolated from decaying strawberry fruit. Tournas and Katsoudas (2005) revealed the presence of Penicillium spp. in all types of berries tested (strawberries, blackberries, and raspberries). Penicillium expansum and some other species of the genus Penicillium can cause significant post-harvest losses in strawberries (Feliziani and Romanazzi, 2016). A study carried out by Mouden et al. (2013) marked the presence of B. cinerea on strawberry samples, with a percentage of contamination ranging from 48.4 to 58.1%. In the same report, an attack by A. alternata was revealed on the samples analyzed with a percentage of contamination ranging from 19.8 to 23.4% depending on the varieties studied. A study done by El-araby et al. (2022) reported that the fungal strain A. niger was isolated from spoiled strawberries.

The analysis of the genus (nitrate assimilation, mycelium form, and sporulation) and the assimilation (Auxanogram) and fermentation (Zymogram) tests of the yeasts (Tables 5, 6) allowed us to establish Table 7.

In our study, the prevalence of yeast strains contaminating strawberry fruits from the Loukkos and Gharb localities was negligible, with a priority on fungal contamination for strawberries from the loukkos. Indeed, a marked presence is noted for Candida sake in strawberry samples, with four positive samples from the Loukkos region and one positive sample from the Gharb region. While Rhodotorula glutinis was found in only three samples from the Loukkos.

A study by Tournas and Katsoudas (2005) reported the presence of yeasts in only about 5% of raspberries and 3% of strawberry samples. The lower incidence of yeasts may be partly explained by the fact that these organisms cannot penetrate the epidermis of the fruit and infect the internal tissues. In addition, yeasts are probably more sensitive to chlorine and are therefore inactivated or washed out during the disinfection step. Isolation and identification of Rhodotorula spp. and Cryptococcus laurentii from strawberries could be considered in light of bio-control of spoilage molds (Rhizopus spp. and B. cinerea; Campaniello et al., 2008). Similar results were reported by Zhang et al. (2007) for Cryptococcus laurentii against Rhizopus spp. in strawberry fruit.

A study performed by Zhang et al. (2007) reported that R. glutinis was isolated from the surfaces of strawberries harvested in unsprayed orchards. Another report mentioned the presence of R. glutinis and Debaryomyces melissophilus in packaged strawberries of the Belgian variety Elsanta (Ragaert et al., 2006). The most frequently isolated yeasts on strawberries from conventional and organic growers belonged to the genus Candida, Cryptococcus, and Rhodotorula (Jensen et al., 2013). Geotrichum candidum was isolated from diseased strawberries by Ma et al. (2018).

In Gharb, summers are hot, arid, and generally clear and winters are cool and partly cloudy. Over the course of the year, the temperature generally varies between 8°C and 32°C and is rarely below 4°C or above 37°C. The very hot season in Gharb lasts for 3 months, from June 20th to September 20th, with an average daily maximum temperature above 29°C. The hottest month of the year in Gharb is August, with an average maximum temperature of 31°C and a minimum temperature of 20°C. The cool season in Gharb region lasts 3.5 months, from November 24th to March 7th, with an average daily maximum temperature below 20°C. The coldest month of the year in Gharb is January, with an average minimum temperature of 8°C and a maximum of 18°C.

In Loukkos, summers are hot, humid, and generally clear and winters are long, cool, wet, windy and partly cloudy. During the year, the temperature generally varies between 9°C and 29°C and it is rarely below 5°C or above 36°C. The very hot season in Loukkos region lasts 3 months, from June 22nd to September 22nd, with an average daily maximum temperature of over 27°C. The hottest month of the year in Loukkos is August, with an average maximum temperature of 29°C and a minimum of 20°C. The cool season in Loukkos lasts 3.8 months, from November 25 to March 17, with an average daily maximum temperature of <19°C. The coldest month of the year in Loukkos is January, with an average minimum temperature of 9°C and a maximum of 17°C.

In the Gharb region, the rainy period of the year lasts 8 months, from September 20th to May 21st, with rainfall of at least 13 mm over a sliding period of 31 days. The rainiest month in Gharb is November, with an average rainfall of 65 mm. The dry period of the year lasts 4 months, from May 21st to September 20th. The least rainy month in Gharb is July, with an average rainfall of 1 mm (Figure 2).

In the Loukkos region, the rainy period of the year lasts 8.4 months, from September 14th to May 27th, with rainfall of at least 13 mm over a sliding period of 31 days. The rainiest month in Loukkos is November, with an average rainfall of 94 mm. The dry period of the year lasts 3.6 months, from May 27th to September 14th. The least rainy month in Loukkos is July, with an average rainfall of 1 mm.

The Emberger bioclimatic quotient (Q₂) was calculated according to the three formulas mentioned above (Table 1). Table 8 represents the Q₂ values calculated in the two regions Gharb and Loukkos.

The climatic parameters allowed us to locate the two target regions Gharb and Loukkos on the Emberger diagram (Figure 3). According to the latter, the Gharb region (Rabat-Sale-Kenitra) is located on the semi-arid stage. While, the Loukkos (Tangier-Tetouan-Al Hoceima) is located on the sub-humid stage. In Gharb, the climate is clearly Mediterranean in the inland cities and Mediterranean with an oceanic influence in the coastal cities. In Loukkos, the climate is Mediterranean in the coasts and surroundings, rather continental and with periods of heavy snowfall in the inland cities of the region. Bioclimatic indices have a strong influence on plant growth as well as its contamination, which explains the differential effect on the prevalence of bacteria in strawberry samples from Gharb (Tables 2, 3) and on the prevalence of fungi in strawberry samples from Loukkos (Tables 4, 5).

Strawberries are perishable fruits that, during handling, can be attacked by certain microorganisms, especially bacteria. Also, the main causes of strawberry diseases during storage and shelf life are the development of rots caused by a series of fungi. Climatic conditions were suspected to have a differential effect on the prevalence of bacteria in the Gharb strawberry samples and on the prevalence of fungi in the Loukkos strawberry samples. A climatological study was necessary to explain this regional distribution.

Analysis of the association between microbial indicators and climatology demonstrates the plausibility of the idea that climatology can affect foodborne pathogens pre-harvest. Climatic conditions are an important factor to consider regarding microbial contamination of fresh products (Machado-Moreira et al., 2019). It is known that changes in climatic conditions determine the development of strawberry plants, affecting flowering, fruiting, and fruit quality, among other characteristics (Khammayom et al., 2022). However, it is pertinent to mention that the influence of climatic conditions on plant contamination may be specific to each microorganism/plant/climatic conditions combination, meaning that no clear trend can be established (Ward et al., 2015). Climatic conditions during the flowering and ripening of strawberry fruit influence fruit quality and microbial contamination as well as affect shelf life more than the irrigation method used (Parikka et al., 2009).

Climatic conditions, including precipitation, humidity, temperature, wind, and orchard management activities, have previously been shown to be correlated with the risk of post-harvest fungal disease development during apple storage (Dutot et al., 2013; Bui et al., 2021). Studies on plant pathogens have shown that high humidity and moderate temperatures promote fungal growth (Griffin, 1996; Rowan et al., 1999). B. cinerea infections are correlated positively with relative humidity and negatively with temperature (Feliziani and Romanazzi, 2016). Weather factors, particularly humidity and temperature, had a direct influence on fungal abundance and richness in the air and on vegetation leaf surfaces, showing large differences among habitats (Talley et al., 2002).

According to the bacteriological and mycological study, there is a difference in the contamination level of strawberry fruits according to the locality of origin. The Gharb region shows a prevalence of bacterial contamination and the Loukkos region reveals a prevalence of fungal contamination. The climatological analyses carried out confirm this hypothesis. Evidently, post-harvest management determines food quality and safety, market competitiveness, and producer profits. Temperature and humidity management is the most important factor to control in order to minimize spoilage of strawberry fruit and maximize its post-harvest life. This management must take into consideration the climatic conditions of the cultivated areas.