Endophytic Yeasts in Apple Fruits of Cultivated and Wild Growth Forms: Total Diversity and Occurrence of Opportunistic Species

Research Article

J Bacteriol Mycol. 2022; 9(2): 1198.

Endophytic Yeasts in Apple Fruits of Cultivated and Wild Growth Forms: Total Diversity and Occurrence of Opportunistic Species

Kachalkin AV1,2, Glushakova AM1,3* and Venzhik AS1

1Department of Biology and Geology, M.V Lomonosov Moscow State University, Moscow, 119234

2Department of Soil Science, G.K Skryabin Institute of Biochemistry and Physiology of Microorganisms of RAS, Pushchino, 142290

3I.I Mechnikov Research Institute of Vaccines and Sera, Moscow, 105064

*Corresponding author: Anna M. Glushakova, M.V. Lomonosov Moscow State University, Moscow, 119234, Russia

Received: July 05, 2022; Accepted: August 04, 2022; Published: August 11, 2022


Background: Apples are widely distributed in different geographical zones and are an important part of human diet. They are a popular subject for research and development of new technologies for biological protection of fruits against phytopathogens. Among yeasts, true phytopathogens are practically unknown. But among them there is a group of clinically significant species of the genus Candida. The development of these species in fruits can affect human health. Primarily, this concerns people with a weakened immune system and a genetic predisposition to mycogenic allergies. The presence of opportunistic yeasts is particularly likely in natural substrates under anthropogenic impact. This study is devoted to the comparison of endophytic yeasts of apples grown industrially and growing wild in urban areas.

Results: Endophytic yeasts were detected in 80% of samples. The average yeast abundance depended (F=24.26; p < 0.01) on the sugar concentration in tissues. At a content of 7-12 °Bx, the average yeast abundance was 4.96±1.07 × 10³ CFU/g; at 12-18 °Bx, 9.61±1.09 × 10³ CFU/g. A total of 33 yeast species were isolated from apples. The greatest number of endophytes was observed in wild apples. Detection the opportunistic yeast Candida parapsilosis distinguished yeast complexes of wild apples from commercial industrial products. The relative abundance of C. parapsilosis in apples collected in the urban area exceeded 30%.

Conclusion: The data on the high abundance of C. parapsilosis in endophytic yeast communities of apples collected in urban areas allow us to make a preliminary suggestion to avoid the consumption of such fruits.

Keywords: Malus domestica; Apples; Fruit; Endophytic yeasts; Candida parapsilosis


The apple tree (Malus Mill.) is widely distributed in a variety of geographical zones and is one of the most important, popular, attractive and widely consumed fruit plants. Currently, there are more than 15,000 apple tree cultivars worldwide, and breeding of new cultivars continues unabated [1–5]. Cultivars of Malus domestica have been successfully used for commercial cultivation, home gardens, and landscaping in urban areas.

Apple fruit is of particular interest as a subject of study related to the development of new technologies for fruit storage and transportation [6]. Endophytic yeast strains with pronounced fungicidal activity (Aureobasidium pullulans, Debaryomyceshansenii, Metschnikowiapuicherrima, M. sinensis, Wickerhamonecesanomalus) against the most common phytopathogens of fruits (Aspergillus niger, Botrytis cinerea, Colletotrichum capsica, Moniliniafructicola, Rhizopus nigricans and others) have been repeatedly found in the pericarp of apples [7–11]. The internal tissue of apple fruits is also an interesting substrate for the isolation and description of new yeast species [12].

The most common yeast species observed both on the surface and inside apples and some other juicy fruits are Aureobasidium pullulans, Buckleyzyma aurantiaca, Curvibasidium cygneicollum, Cysto filobasidium infirmo miniatum, Debaryomyces hansenii, Galactomyces candidus, Hanseniaspora guilliermondii, H. uvarum, Metschnikowia pulcherrima, Naganishia albida, Pichia kluyveri, P. kudriavzevii, Papiliotrema laurentii, Rhodotorula minuta, Rh. glutinis, Saccharomyces cerevisiae, and Sporobolomyces roseus [13–14].

However, in urban ecosystems exposed to complex anthropogenic influence, changes in endophytic yeast communities may occur [15]. The opportunistic yeast species Candida parapsilosis was found in the internal tissues of Malus domestica and Pyrus communis fruits grown in Moscow throughout the formation and ripening period, reaching a maximum relative abundance in ripe fruits. The presence of opportunistic Candida yeasts is an indicator of environmental conditions and anthropogenic pressure [16].

In this study, we compared the abundance and taxonomic structure of endophytic yeast complexes associated with industrially grown apple fruits in different countries and those growing wild in private gardens in urban environments.

Materials and Methods

Study Location and Dampling

Apples for the study of the abundance and endophytic yeasts’ taxonomic structure were purchased from trade networks in the Moscow region (imports from Argentina, Belarus, China, Chile, Serbia, Turkey, and supplies from Russia), collected on the territory of the private gardens near Moscow city, and in the urban area along roadsides and in city parks in Moscow. The study was conducted in 2019. A total of 553 apple fruits were analyzed.

Microbiological Analyses and Species Identification

The abundance and taxonomic structure of yeasts were studied using a surface plating method on solid media.

To study endophytic yeast communities, fruits were treated according to the following scheme: 70% ethanol, 30 min; 2% sodium hypochlorite, 30 min; 70% ethanol, 30 s; and washing in sterile distilled water for 10 min [17,18]. After the exocarp was removed with a sterile scalpel, the internal tissues were cut out, homogenized, and poured with sterile water to obtain a 1:10 dilution. The suspensions were vortexed on a Multi Reax Vortexer (Heidolph Instruments, Germany) for 15 minutes at 2,000 rpm. Three suspensions were prepared for each apple. Thus, 1659 prepared suspensions were plated in two replicates each on glucose-peptone-yeast extract (GPY) agar (20 g/L glucose, 10 g/L peptone, 5 g/L yeast extract, 20 g/L agar) supplemented with chloramphenicol (500 mg/L) to prevent bacterial growth. A total of 3318 plates were incubated at 22°C for 5-7 days. The grown yeast colonies were classified into morphological types using a dissecting microscope, and the number of colonies of each type was counted. From each morphotype, 5 to 7 colonies were isolated into a pure culture.

Identification of yeast species was based on the ITS rDNA nucleotide sequence. DNA isolation and PCR were performed according to the previously described procedure [15]. DNA sequencing was performed using the Big Dye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems, USA) with subsequent analysis of the reaction products on an Applied Biosystems 3130xl Genetic Analyzer at the facilities of Evrogen (Moscow). For sequencing, the ITS5 primer (5’- GGA AGT AAA AGT CGT AAC AAG G) was used. For species identification, nucleotide sequences were compared with those in public databases using the BLAST NCBI (www.ncbi.nlm.nih. gov) and the MycoID (www.mycobank.org) tools.

Analysis of Sugar Content

Total sugar content (measured by soluble solids concentration (SSC)) in fruit juice was estimated using a Milwaukee MA871 refractometer.

Data Analyses

The relative abundances of species were calculated as share (%) of colonies that appeared on the plates. Statistical data processing and graphical presentation of the obtained results were carried out using Excel 2010 (Microsoft, USA) and Statistica 8.0 (StatSoft, USA) programs. The Fisher’s test was used for comparing average data on the yeasts abundance after determining normality of their distribution by the Shapiro-Wilk test. The bars on figure represent the standard deviations (SDs).


Yeast Abundance

Endophytic yeasts were detected in 80% of the samples tested. The average yeast abundance in the internal tissues of ripe apples depended (F criterion, 24.26; p < 0.01) on the sugar concentration in the internal tissues of apples. At a sugar content of 7-12 °Bx, the average yeast abundance was 4.96±1.07 × 10³ CFU/g; at 12-18 °Bx, it was 9.61±1.09 × 103 CFU/g (Figure 1). It should be noted that “peaks” in yeast abundance were also observed in apples with average sugar concentration (10 °Bx).