WHITE MULBERRY (MORUS ALBA L.) FRUIT-ASSOCIATED BACTERIAL AND FUNGAL MICROBIOTA

Morus alba L. has been worldwide cultivated and commercially exploited plant with profound potential in environmental management, food and medicinal industries. Plant-associated microbial communities are playing an essential role in sustainable plant development. In the present study, the bacterial and fungal microorganism populations distributed on the white mulberry fruits harvested in the Czech Republic for the first time were characterized by metagenomics approach. A total of 62 bacterial and 37 fungal families were identified on white mulberry. Bacterial population was represented by the genera Tatumella, Leuconostoc, Frateuria and Pseudomonas, while fungal microorganisms – by Hanseniaspora, Cryptococcus, Cladosporium and Phoma. Both potentially beneficial, inducing resistance in the hosting plant, and pathogenic, responsible for disease development, microorganisms were detected. The information on the prevalence of bacterial and fungal microorganisms on the carposphere of M. alba is highly relevant for the development of strategies for environment-friendly plant cultivation, disease management and prevention.


Introduction
Mulberry trees (Morus L.) have been widely cultivated in temperate and subtropical regions of Africa, America, Asia and Europe; their planting steadily increases every year due to realized economic and ecological benefits Xu et al., 2019). Mulberry (Morus spp.) has been commercially exploited as the host of the silkworm (Bombyx mori). Mulberry leaves and fruits contain a variety of nutrient and nutraceutical substances. Fruits have been recognized as a kind of natural nutrition and functional food; they are usually eaten fresh or made into jam, juice and wine (Łochyńska, 2015). Due to the presence of anthocyanins, carotenoids and flavonoids, mulberry fruits possess potential pharmacological properties including populated species, however almost forgotten in Europe. The potential of Morus L. plants in agriculture, environmental protection, industry and human health is rather huge (Łochyńska, 2015;Ercisli & Orhan, 2007). M. alba leaves can be used for tea making and in the silkworm diet for the commercial production of silk (Mahboubi, 2019). Very fast growth of white mulberry tree produces significant amount of plant biomass, which may be used as the renewable source of biofuel (Łochyńska, 2015;Chinnaswamy & Harisparad, 1995). Due to natural dyeing properties, it is applicable in furniture industry (Łochyńska, 2015). The food industry increasingly uses white mulberry in food production to provide valuable bioactive substances (Łochyńska, 2015). The main active constituents of M. alba include flavonoids, alkaloids, stilbenes, and polysaccharides, providing health benefits through anti-inflammatory and immunomodulatory effects (Zhang et al., 2018). Fruits, roots, and leaves of M. alba are used also for the treatment of dizziness, insomnia, and premature aging (Rodrigues et al., 2019). The resistance to disease and pests, relatively low soil requirements as well as attractive bioremediation properties substantiate great potential of M. alba for landscaping and ecological environment management (Jiang et al., 2017).
Economic value of the mulberry fruit is constantly increasing, so is the need for plant cultivation and disease management to produce suitable for consumption berries. Among multiple biotic and abiotic factors affecting plant development, plant-associated microbial communities are playing one of the primary roles. It must be mentioned that very few works concentrated on the characterization of mulberryassociated microbial assemblages are published so far. Some studies are focused on the diversity of endophytic bacteria on white mulberry cultivars by revealing seasonal variation and emphasizing antimicrobial and plant growth-promoting activities of bacteria Xu et al., 2019). Only two studies are intended for characterization of soil fungal communities of different white mulberry genotypes and analysis of relationship with fruit sclerotiniosis Yu et al., 2016). To the best of our knowledge, the white mulberry carposphere-associated microbial populations remain undescribed in the scientific literature. The aim of the present study was to characterize the bacterial and fungal microorganism communities found on the M. alba fruit surface by applying metabarcoding and Next Generation Sequencing (NGS) technology.

White mulberry sample preparation
Morus alba L. fruits were aseptically sampled from 30-40 years-old planted trees growing in Ostrava city park (the Czech Republic, GPS coordinates: 49°50′1′′N, 18°17′40′′E) in July, 2016. The fruits (at medium ripeness stage, without blemish) were randomly collected from reachable branches of the five white mulberry trees growing in distance of more than 30 meters. M. alba fruits were combined into sterile plastic bags and within 2 hours after harvesting were transported on ice to the Ostrava University for further processing. Gathered fruits (~300 g) were washed with aseptic 0.05M phosphate buffer, pH 6.8 (500 mL) for 30 min at room temperature. Plant debris was removed by filtration through 420 μm filters. Samples were centrifuged at 12,000×g for 20 min. The collected pellet was transported on ice to Lithuania and kept in -20 ºC freezer until DNA extraction and amplification of marker genes (Department of Chemistry and Bioengineering, Vilnius Gediminas Technical University).

Sequence analysis
Bioinformatics analysis was conducted using FLASH v1.2.11 (Magoč & Salzberg, 2011) to filter sequences with a minimum Q score of 25 and merge paired-reads. OTUs were picked by the greedy heuristic clustering algorithm, CD-HIT-OTU v4.5.5 (Li et al., 2012). Alpha diversity was calculated (including Chao1, Shannon, Good coverage) and relative abundance summaries were conducted using QIIME 1.8 (Caporaso et al., 2010). UNITE (Kõljalg et al., 2013) and Ribosomal Database Project (RDP) (Cole et al., 2014) were used as the reference databases for the taxonomy assignments of most abundant sequences. Non-bacterial, non-fungal sequences and singletons were filtered out and taxonomic relative abundance at all classification levels was calculated. The taxonomic results were visualized in Krona (Ondov et al., 2011).

Bacterial and fungal diversity of M. alba fruits
The Illumina MiSeq sequencing generated 516,658 and 554,058 paired-end reads from ITS2 and V3-V4 target regions respectively. Following quality filtering and checking of chimera sequences, 302,705 (for ITS2) and 169,313 (for V3-V4) reads were obtained. 97% threshold of sequence similarity was used for clustering into operational taxonomic units (OTUs) ( Table 1). 365 bacterial and 206 fungal OTUs were represented by identified sequences. Rarefaction curves ( Figure 1) and calculated Goods coverage confirmed that the majority of the bacterial (coverage: 99.9%) and the fungal OTUs (coverage: 99.9%) were recovered. In agreement with obtained OTU data, the Chao1 and Shannon indexes demonstrated that white mulberry had higher bacterial community diversity than fungal microorganisms.

Discussion
So far, a few studies were focused on the characterization of bacterial community distributed on different Morus L. cultivars using culture-dependent  and metagenomics  approaches. It was demonstrated that the endophytic Morus L. microorganism assemblies are specific for the host plant and the climatic conditions . Several aspects aggravate comparative analysis of our data with previous Morus L. microbiome studies: different plant site were chosen (in our study -fruits, in in mentioned above studies -stems), the lack of information on exact mulberry species of tested cultivars, and microorganisms analyzed (ours -epiphytic, in mentioned above studies -endophytic bacteria). Nevertheless, analysis of taxonomic composition of prokaryotic microorganisms revealed that Actinobacteria, Firmicutes, and Proteobacteria were the three most abundant bacterial phyla on different Morus L. cultivars, mainly represented by Pantoea, Pseudomonas, and Methylobacterium genera Xu et al., 2019). In our study, about 90% of M. alba fruits were also covered by microorganisms belonging to these phyla but represented by different genera. At genus level, Leuconostoc was the most prevalent taxa on white mulberry fruits. Leuconostoc spp. are non-pathogenic bacteria frequently found on plants, advantageous in most foods because of their flavor development and preservation abilities, capacity to improve the nutritional and organoleptic quality, possessing beneficial effects as potential probiotics (Shin & Han, 2015;Holland & Liu, 2011). Some Leuconostoc species are also capable of causing uncommon human infections or wine ropiness, nevertheless the genus is generally recognized as safe (Holland & Liu, 2011). In our study, Tatumella and Frateuria were detected among dominating bacterial genera on M. alba fruits. Some representatives of Tatumella genus are foodborne opportunistic pathogens isolated from various food sources, pineapples and clinical specimens, able to cause numerous infections (Mardaneh et al., 2014). Frateuria bacteria, represented in our study by Frateuria aurantia, were previously documented as capable to improve the plant growth and control phytopathogens (Lidor et al., 2019). Bacteria from Pseudomonas and Pantoea genera, observed in high content on white mulberry plants, are reported as plant-associated pathogenic microorganisms (Coutinho & Venter, 2009;Ligon et al., 2000). Nevertheless, both genera contain species possessing antimicrobial features (Trotel-Aziz et al., 2008; Ligon et al., 2000).
Among those identified at species level in low frequency, bacteria with known plant growth promoting and biocontrol potential (Pseudomonas geniculate, Lysinibacillus sphaericus, Myxococcus virescens, Rhizobium leguminosarum bv. viciae) were observed. So far, only two studies using molecular techniques have described the fungal communities related to a M. alba tree, in the investigation conducted on soil samples Yu et al., 2016). It was demonstrated that representatives of six phyla were observed with three dominant phyla (Ascomycota, Mucoromycota and Basidiomycota) , while fruits were mainly covered by representatives of two phyla as revealed in our study. Based on mulberry rhizosphere-associated fungal community analysis, it has been shown that plant genotype has a significant influence on the abundance and composition of soil microbial population (Yu et al., 2016). In addition, the differences in the soil microbial assemblages may affect the plant resistance to mulberry fruit sclerotiniosis (Yu et al., 2016). As observed in our study, Hanseniaspora was the most prevalent mulberry fruit-associated taxa at the genus level, represented by Hanseniaspora uvarum species. Hanseniaspora spp. possess low fermentative activity and have been frequently Pl -Pleosporales. Minor OTUs were not labeled. Pie-charts were constructed using KRONA (Ondov et al., 2011) discovered on the surface of different fruits, e.g. apples, blackcurrants, grapes, strawberries and etc. Vepštaitė-Monstavičė et al., 2018;Graça et al., 2015). It is well-recognized that fungal microorganisms from this genus could be useful to hosting plant by executing antagonistic activity on the development of fruit spoilage-causing mold (Tilocca et al., 2020;Liu et al., 2010). Among other dominating fungal genera, our study on mulberry fruits detected Cryptococcus, Cladosporium, and Phoma. Ubiquitous fungi Cryptococcus and Cladosporium are typical members of the yeast population have been often detected on the surface of different plants Vadkertiová et al., 2012). Some representatives of these genenera enclose species producing antifungal agents against many pathogens (Freimoser et al., 2019;Hashem et al., 2014;Wang et al., 2013), and have been established as biocontrol agents for the management of the postharvest diseases (Liu et al., 2013). On the other hand, certain species can cause plant, human and animal diseases (Sandoval-Denis et al., 2016;Bernal-Martinez et al., 2010). Therefore, the potentially beneficial or pathogenic features of Cryptococcus laurentii and Cryptococcus magnus fungal microorganisms, identified in our study, could be determined only by culture isolation and further analysis. Fungal microorganisms belonging to the genus Phoma are known to be plant pathogens, characterized by parasitic relationships with their host (Aveskamp et al., 2008). Phoma spp. have been shown to contaminate seeds, fruits and vegetables (Bennett et al., 2018;Lukša et al., 2018;Vepštaitė-Monstavičė et al., 2018;Oliveira et al., 2017;Termorshuizen, 2007), produce cytotoxic metabolites causing infections for humans and animals (Bennett et al., 2018). Among identified at species level in low frequency, potentially beneficial (Torulaspora delbrueckii, Wickerhamomyces anomalus, Candida railenensis, etc) as well as pathogenic fungi (Auriculibuller fuscus, Bulleromyces albus, Sporobolomyces roseus, Sp. salicinus, Sp. gracilis, etc) were observed. The abundance and distribution of prokaryotic and eukaryotic microorganisms on fruits are dependent on the plant origin and ripening stage, geographic location and climatic conditions, application of agrochemicals as well as other biotic and abiotic factors Pinto et al., 2015). Considering the biogeographic effect, we compared the M. alba-associated microbial communities with ones identified previously on Malus pumila Mill. and Ribes nigrum L. collected in Czech Republic, Ostrava region and Lithuania (Vepstaite-Monstavičė et al., 2018) (Table 3). All tested fruits were sampled in the same season on July-August, 2016. The higher amount of bacteria belonging to Tatumella, Gluconobacter and Sphingomonas genera and fungal microorganisms from Hanseniaspora genus were detected on the surface of fruits sampled in Czech Republic, as compared to Lithuanian samples. These results indicate the importance of climatic conditions and geographical distribution  (Deveau et al., 2018;Lindow & Brandl, 2003). In the natural environment, different factors act cumulatively, therefore differentiation of the major and specific impacts driving the divergence of bacterial and fungal microorganism assemblages is rather complicated.
In conclusion, the results of this study demonstrated that M. alba carposphere possesses high bacterial and fungal microorganism variety. Among eukaryotic and prokaryotic microbiota, both potentially beneficial and pathogenic microorganisms were identified. The obtained information on the structure of white mulberry-associated fungal and bacterial assemblages, spreading of potentially beneficial and phytopathogenic microorganisms is important to develop effective plant disease control strategy. Due to clear potential of white mulberry berries in food industry, the metagenomics data appear to be significant for the evaluation of the influence of microbiota in general, and specific microorganisms in particular on food quality and human wellbeing.