Yeasts from Nanfeng mandarin plants: occurrence, diversity and capability to produce indole-3-acetic acid

Abstract Plant growth promoters produced by microorganisms can play a significant role in the induction of some important physiological responses in the growth and development of plants. In this study, we provided a first insight into revealing the diversity of cultivable yeasts associated with Nanfeng mandarin (Citrus reticulata cv. Blanco) in China. Their capability to produce indole-3-acetic acid (IAA) was analyzed. A total of 796 yeast strains were obtained by the enrichment isolation technique from citrus soil, citrus leaves, citrus peel and citrus pulp of Nanfeng mandarin samples. On the basis of the 26S rDNA partial sequence analysis, the strains were identified as 14 yeast species in 9 genera belonging to Hanseniaspora sp., Pichia sp., Candida sp., Sporidiobolus sp., Meyerozyma sp., Symmetrospora sp., Rhodotorula sp., Starmerella sp. and Aureobasidium sp. The most abundant species in citrus soil were Starmerella meliponinorum and Meyerozyma caribbica. The species prevailing in citrus peel was Hanseniaspora opuntiae. Citrus pulp was rich in Meyerozyma guilliermondii. Additionally, Aureobasidium pullulans and H. opuntiae were the dominant species in citrus leaves. All yeast species obtained were accessed for the capability to produce IAA: 26 strains in seven species showed the capability of producing IAA. Rhodotorula paludigena produced the highest IAA concentrations of 76.22 mg/L. Our study confirms the phylogenetic diversity of yeast associated with Nanfeng mandarin and highlights that these yeast strains are promising resources of microbial fertilizer.


Introduction
Nanfeng mandarin (Citrus reticulata cv. Blanco) is one of the most economically important fruit tree crops in the world [1]. Nanfeng mandarin, with a cultivation history of 1300 years, is well known in China and is produced in the Nanfeng (NF) and Nancheng (NC) counties of Jiangxi Province [2]. Nanfeng mandarin requires a temperature of 23-29 C, annual precipitation of 1500-2000 mm and humidity of 80-85% for optimum growth [3]. Nanfeng mandarin is one of the most important exported fruits and it is globally popular for its nutritive value, sweet and sour flavour and thin, tender skin [4]. Current researches on Nanfeng mandarin and other citrus fruit have focused on improving the unique flavour and excellent traits of tangerine; the methods to grow, pick and preserve the citrus fruit; the analysis of its chemical composition [5,6]; the biological activity of its chemical constituents, their effect on human health and some methods to enhance the beneficial constituents of the fruit by chemical or physical means [7][8][9]. However, there has been little research on the microbial diversity and specifically, yeast diversity, associated with Nanfeng mandarin.
Yeasts are widely distributed in natural environment including the leaf, flower, fruit and other organs of trees and the orchard soil. Some previous studies have focused on the yeast diversity of strawberry, grapes and so on [10,11]. However, there are few studies on the yeast diversity of Nanfeng mandarin. Bryschherzberg and Seidel [12] revealed the yeast diversity on grapes in two German wine-growing regions. The metabolites of yeast have a great influence for the aroma components, colour and texture of wine, as suggested by Tofalo et al. [13]. Based on previous studies and some of the characteristics of yeast, we speculate that the flavour and quality of Nanfeng mandarin may be dependent on a certain relationship between yeasts and Nanfeng mandarin. At the same time, it is known that plant-associated microorganisms are extremely rich and have various biological functions: some of them can produce beneficial biologically active compounds [14,15], reduce the availability of heavy metals [16], improve the resistance of host plants to abiotic stresses [17,18], promote the agricultural disease resistance and have a growth-promoting action on plants [19][20][21][22]. It is believed that many of the plant-associated yeasts, like other microbes, have a definite biological function [23,24].
Indole-3-acetic acid (IAA), an indole compound and a common source of endogenous growth hormone, is known to adjust various developmental and physiological processes, and stimulate the rapid and longterm response of plants [25]. It is a naturally occurring auxin with broad physiological effects. Plants and microorganisms including bacteria [26], yeasts [27,28], actinomycetes [29] and filamentous fungi [30] can produce IAA; therefore, these microbes have been recognized as good sources of biofertilizer [31]. Applications of IAA-producing yeasts, such as Candida valida, Rhodotorula glutinis, Trichosporon asahii, Lindera saturnus and Rhodotorula mucilaginosa, to promote plant growth have been reported [32]. However, there is little information on IAA-producing yeasts of the Nanfeng mandarin.
To the best of our knowledge, this study is the first to report the diversity of cultivable yeasts associated with Nanfeng mandarin in China and their ability to produce IAA. It is important to isolate and identify the yeast populations of NF and NC areas where Nanfeng mandarin is mainly produced, and assess the capacity of these yeasts to produce IAA in vitro. This assessment will provide the foundation for the development of biofertilizers using these yeasts.

Sample collection
The yeast isolates were obtained from different cultivars of citrus trees located in NF and NC County in October 2016 ( Figure 1). NF (N 27 12 0 48 00 and E116 31 0 31 00 ) and NC (N27 33 0 and E113 40 0 12 00 ) are located in Jiangxi Province, China. Seven samples each were obtained from the two places. Soil samples (about 10 g/sample) were taken from a depth of about 10 cm. Nanfeng mandarin samples, weighing around 500-1000 g each, were collected. Undamaged leaves were collected from each selected individual tree. All the collected samples were stored in sterile plastic tubes and transported immediately to the laboratory and stored at 4 C within 24 h of collection.
Isolation and identification of Nanfeng mandarinassociated yeasts from Citrus reticulate cv. Blanco Large impurities visible in the soil were removed. The soil samples were dried and fed through a 2-mm sieve in the sterile operation room in order to obtain 10-g samples. The leaves and Nanfeng mandarin samples were washed with sterile distilled water, after which the pulp and peel were separated from each other in a sterile condition. The treated samples (10 g) were then cut into 1-cm 3 pieces.
Yeasts were isolated by an enrichment technique carried out as described by Limtong et al. with certain modifications [33]. The treated samples were ground and placed in Erlenmeyer flasks containing 200 mL of YPD (1% yeast extract, 2% peptone and 2% glucose) and agitated on a rotary shaker at 150 rpm for 30 min. The solution was then diluted with sterile water and aliquots of 100 mL from these serial dilutions (1 Â 10 4 , 1 Â 10 5 and 1 Â 10 6 ) were plated on PDA culture medium (20% potato, 2% peptone, 2% glucose and 2% agar) supplemented with 100 mg/mL streptomycin or chloramphenicol. After incubation at 25 C for 3 days, the colonies were counted and the means and standard deviations of three replicates were calculated. Representative colonies were selected by dereplication based on their phenotypic and morphological characteristics such as colony colour (milky white, rose red, creamy white, orange), size (diameter size), surface (smooth or rough), border type (circle, radial) and growth rate and picked for pure culture [34]. Purified yeast strains were suspended in PDA broth supplemented with 20% v/v glycerol and maintained at À80 C for future use.
DNA extraction, amplification of yeast D1/D2 26S rDNA fragment sequence and its phylogenetic analysis Yeast strain identification was performed by sequence analysis of the region D1/D2 of the rDNA large subunit (LSU) as described by Barnett et al. [34]. Amplification of the referred region in the 26S rRNA gene was done with the primers NL1 (5 0 -GCATATCAATAAGCGGAGGAAAAG-3 0 ) and NL4 (5 0 -GGTCCGTGTTTCAAGACGG-3 0 ) as proposed by Kurtzman and Robnet [35]. DNA extraction and analysis was performed as described by Querol et al. [36]. Polymerase chain reaction (PCR) and amplifications were performed as described by Santo et al. [10].
The amplified products were sequenced by Sangon Biotech (Shanghai) Co. Ltd. The obtained sequences were compared for homology with the sequences of the described species available in the GenBank database at the National Center for Biotechnology Information (NCBI) using BLAST (http://www.ncbi.nlm.nih.gov/ BLAST/). The D1/D2 26S rDNA region sequence of the yeasts was initially aligned using the program package MEGA 6 [37]. Then, the sequence was used as a query to search for similar sequences from GenBank using the FASTA and BLAST programs for identification.
Afterwards, the resulting sequences were aligned with the ClustalX software with gaps treated as missing data [38]. The phylogenetic tree was constructed using the neighbour-joining method [39]. The bootstrap analysis was done for 1000 replicates to assess the reliable level of the nodes of the tree.

Determination of indole-3-acetic acid production
Production of IAA by the yeasts was quantitatively analyzed [32]. A yeast culture grown for 1-2 days on YM agar at 25 C was inoculated in 5 mL of yeast extract-peptone-dextrose (YPD) broth (10 g/L yeast extract, 2 g/L peptone and 2 g/L dextrose) supplemented with 1 g/L L-tryptophanin in a test tube and incubated for 7 days at 30 ± 2 C under shaker conditions at 150 rpm. After 7 days, an aliquot of 1.5 mL of the culture broth was centrifuged at 6791 Â g for 5 min and the supernatant was collected for determination of IAA concentration. Subsequently, 1 mL of supernatant was added to 1 mL of Salkowski reagent prepared in 12 g/L FeCl 3 and 7.9 mol/L H 2 SO 4 , and the intensity of pink colour developed in the mixture after 30 min was quantified with a spectrophotometer (UV-1800, Mapada) at a wave length of 530 nm against a calibration curve.

Statistical analysis
Yeasts were tabulated and summarized according to their isolation percentages. Graphics and tables were drawn by Origin. All measurements were performed in triplicates. One-way analysis of variance (ANOVA) was performed and differences were considered statistically significant when p < 0.05.

Isolation of yeasts
The diversity of yeast species in the orange pulp, leaves, soil and peel was investigated in this study. A total of 796 strains were isolated: 510 strains were recovered from the orchard soil, 72 strains were isolated from orange peel, 62 from orange pulp and 152 strains from orange leaves. If the sampling area is considered, 220 strains were isolated from NF and 576 strains were isolated from NC. A total of 796 yeast isolates were assigned to 24 morphotypes using dereplication. Among these, 14 isolates were representative, whereas 10 ones were different. Figure 2 and Table 1 Morphology show the phenotypic characters and morphological characteristics of the representative strains.
Further analysis of the sequenced genomes revealed that only one yeast strain (N12-1) showed a lower homology of 98% with R. paludigena (KU316709.1). Other strains were associated with the corresponding model strain with a higher homology. This confirms that the results of the sequence analysis of yeast 26S rDNA D1/D2 are credible.
Some of the isolated strains may have strong biochemical functions and application potential. Generally, the functional roles of yeast can be divided into three categories: biological agents, biological fertilizers and flavour enhancers. Many studies have explored the potential of yeast for biological control. For example, Hanseniaspora inhibits the grey mould decay and affects the postharvest quality parameters [44] as well as the postharvest diseases in strawberries [45]. Sporidiobolus is also known to be a biocontrol agent that is effective against postharvest diseases in table grapes [46,47]. Meyerozyma has antifungal activity [48]. Aureobasidium and Rhodotorula have potential as biocontrol agents: they compete for nutrients and space to inhibit the growth of plant pathogens [49]. In this sense, most yeasts exhibit effects of biological control. Among the yeasts isolated by us, M. guilliermondii is useful in controlling plant soft rot, whereas R. paludigena and A. pullulans have the potential to reduce citrus green mould and thereby, to promote plant growth.

Different yeast species isolated from different samples and different sampling regions
The yeast abundance in the citrus samples was estimated. H. uvarum was isolated from citrus soil and H. opuntiae was the prevalent species in orange peel   [50]. Byrsonima crassifolia and A. pullulans were isolated from orange leaves [51]. The isolation data indicated that most species of yeasts were isolated from citrus leaves. This included 12 species of yeasts, four out of which, namely Candida metapsilosis, Symmetrospora spp., Candida cf. azyma and P. kluyveri only, occurred in the leaves, and did not appear in the other samples. On the contrary, H. thailandica was obtained from the soil and peel, but it did not appear in the leaves and pulp. It is noteworthy that there were only three species of yeast isolated from peel, two of which were also obtained from the soil. More intriguingly, the number of yeast strains isolated from soil and peel were the same, but the compositions were very different, Starmerella meliponinorum being the only yeast strain common to both. It is generally recognized that different yeasts have different functions and therefore, the presence of different yeasts on different plant parts may produce different effects ( Figure 4). The samples were collected from two different counties (NF and NC counties) which are both Nanfeng mandarin production sites but the fruits from NF county taste more delicious and are more popular with the public. Different factors such as soil and climate may act alone or in combination to cause this phenomenon. Interestingly, comparison between the yeast samples from the two places ( Figure 5) showed that the yeast species are more abundant in NC. Although M. caribbica is the only common yeast to be isolated from both the places, its proportion is much higher in samples from NC than in samples from NF. Therefore, the taste of Nanfeng mandarin may be related to the diversity of yeasts. These results are in agreement with a similar report that the yeast diversity of Xinjiang grape, especially non-Saccharomyces, can affect the quality and flavour of grape wine [52].

Indole-3-acetic acid production
Previous research has revealed that some yeast strains are not only potential biocontrol agents, but also potential biological fertilizers owing to their ability to produce beneficial substances. Pichia spp. can be used as an expression vector for the effective expression of useful substances [53,54]. Production of bio-alcohols by Candida has also been carried out [55]. The yeast C. maltosa can produce IAA, thereby promoting the growth of plants [56]. Cloete et al. found evidence of symbiosis between the soil yeast Cryptococcus laurentii and a sclerophyllous medicinal shrub, Agathosma betulina Pillans [57]. Each soil fraction has a distinct yeast assemblage depending on the soil nutrient availability and the physiological capacities of the yeast. Endophytic yeasts, like Pichia fermentans and Candida railenensis, play an important role in gall formation [58]. Rhodotorula acheniorum, R. mucilaginosa and R. glutinis produce some plant growth-promoting   compounds [59]. The yeasts that we obtained, such as H. uvarum, R. paludigena and A. pullulans, may also have a plant-growth promoting effect, although further experiments are needed to confirm this. Among the 26 strains of yeast, 9 strains demonstrated the ability to produce IAA when cultivated in YPD broth supplemented with 0.1% L-tryptophan ( Table 2). The other 17 strains grew in this medium, but no IAA was produced. R. paludigena produced relatively high concentrations (76.22 mg/L) of IAA. This result indicated that IAA production was strain dependent. Some strains of some species were able to produce IAA, whereas others were not. Other strains such as A. pullulans produced IAA. However, the concentration of IAA produced was relatively low.
Simultaneously, yeasts also play an important role in food fermentation. Their unique characteristics impart a unique taste to the food. C. tropicalis, Pichia membranifaciens, Candida boidinii and Saccharomyces cerevisiae are involved in the natural fermentation process of table olives causing a change in their taste [43,60]. S. cerevisiae can impact the fragrance of cherry wines [60]. Whether the yeasts obtained by us can be potentially applied in changing the flavour of tangerine-flavoured beverages still needs further study.

Conclusion
Our present study revealed the phylogenetic diversity of yeast associated with Nanfeng mandarin and highlights that the IAA-producing yeast strains could be used as promising resources of microbial fertilizers. The yeast species identified in this study have also been reported to be found in the soil, leaves and fruits of many other hosts from different biomes. Until date, the research on the use of yeasts as biofertilizer suggests that yeasts possess this effect. Moreover, further and more thorough studies of Nanfeng mandarin yeast are needed. The results of this study represent an important step in the phylogenetic analysis of yeast isolates collected from citrus trees of the Jiangxi Nanfeng. Moreover, they provide an insight into the eco-friendly use of yeast resources.

Disclosure statement
The authors declare there is no conflict of interest for their study.