Unlocking the plant growth-promoting potential of yeast spp.: exploring species from the Moroccan extremophilic environment for enhanced plant growth and sustainable farming

Abstract In this study, we successfully isolated two distinct yeasts from Moroccan extreme environments. These yeasts were subjected to molecular characterization by analyzing their Internal Transcribed spacer (ITS) regions. Our research thoroughly characterizes plant growth-promoting abilities and their drought and salt stress tolerance. In a greenhouse assay, we examined the impact of selected yeasts on Medicago sativa’s growth. Four treatments were employed: (i) control without inoculation (NI), (ii) inoculation with L1, (iii) inoculation with L2, and (iv) inoculation with the mixture L1 + L2. L1 isolated from Toubkal Mountain shared 99.83% sequence similarity to Rhodotorula mucilaginosa. Meanwhile, L2, thriving in the arid Merzouga desert, displayed a similar identity to Naganishia albida (99.84%). Yeast strains were tolerant to NaCl (2 M) and 60% PEG (polyethylene glycol P6000) in case of drought. Both strains could solubilize phsphorus, with L2 additionally demonstrating potassium solubilization. In addition, both strains produce indole acetic acid (up to 135 µl ml−1), have siderophore ability, and produce aminocyclopropane-1-carboxylic acid deaminase. Isolates L1 and L2, and their consortium showed that the single or combined strain inoculation of M. sativa improved plant growth, development, and nutrient assimilation. These findings pave the way for harnessing yeast-based solutions in agricultural practices, contributing to enhanced crop productivity and environmental sustainability.


Introduction
Extr eme envir onments (EEs) host an extensiv e arr ay of micr obial species, man y inter acting m utualisticall y with plants, and pr evious studies have characterized these extremophilic microbeplant interactions (Nafis et al. 2018, 2019, Devi et al. 2020, Tapia-Vázquez et al. 2020 ).Latel y, ther e has been a sur ge of inter est in the isolation, identification, and exploitation of extremophile microbes .T heir distinctive abilities, metabolic capabilities, and activities have made them exceptionally appealing to industries acr oss div erse biotec hnological fields (Tsuji et al. 2018, Ta pia-Vázquez et al. 2020, Loeto et al. 2021, Martínez-Ávila et al. 2021 ).For instance, Tapia-Vázquez et al. ( 2020 ) isolated and characterized four yeast strains from rhizospheric soil collected from the Xinantécatl volcano in Mexico.Their r esearc h highlighted the plant gr owth-pr omoting (PGP) abilities of these extr emophilic yeasts, whic h further r einforced the significance of such microbial resources in sustainable agriculture.
Consider able r esearc h efforts hav e been dir ected to w ar d using specific micr oor ganisms, mainl y plant gr owth-pr omoting rhizobacteria (PGPR), within a gricultur e .T hese PGPR serve as biofer-tilizers or biostimulants, contributing to impr ov ed soil fertility, enhanced plant gr owth, incr eased yield, and, ultimatel y, a better use of chemical fertilizer under diverse environmental conditions (Raklami et al. 2019, Anli et al. 2020, Mokabel et al. 2022, Sedri et al. 2022 ).Yeasts hav e r eceiv ed less attention among micr obial species due to their r elativ el y low population density.Astonishingl y, e v en to this day, < 1% of the yeast species in nature have been isolated, identified, and discov er ed (Kurtzman and Piškur 2006, Starmer and Lachance 2011, Segal-Kischinevzky et al. 2022 ).
As a prominent eukaryotic organism, yeast is renowned for its extr aordinary biodiv ersity and finds widespread utility in various biotec hnological a pplications, including a gricultur e (Ampr ayn et al. 2012 , Nand y and Sri v astav a 2018 ).A k e y aspect of their positive impact could be attributed to their PGP properties, including phytohormones (Ignatova et al. 2015, Kumla et al. 2020 ), phosphate, potassium, and zinc solubilization (Mirabal Alonso et al. 2008, Tapia-Vázquez et al. 2020, Srinivasan et al. 2022 ), nitrogen fixation, sulfur oxidation (Srinivasan et al. 2022 ), and siderophore production (Sansone et al. 2005, Silva et al. 2020, Tapia-Vázquez et al. 2020 ).Furthermore, certain species were recognized for their exopol ysacc haride secr etion (Hamidi et al. 2020 ), accelerating the decomposition of organic materials into usable nutrients, enhancing soil water-holding capacity, and improving the ov er all soil health (Ramya et al. 2021 ).Additionall y, yeasts r elease a spectrum of bioactive compounds, including vitamins, hormones, and enzymes .T hese are vital in stimulating plant gr owth, mana ging soil-borne pathogens, and making the crop stress tolerant (Tsuji et al. 2018, Loeto et al. 2021, Srinivasan et al. 2022 ).These characteristics are paramount in sustainable agriculture, as they are firml y r ooted in biological pr ocesses that activ el y bolster plant growth and producti vity.Moreover, the y contribute to the maintenance of soil fertility.Harnessing these attributes of extremophilic yeast presents a promising avenue for enhancing agricultural sustainability, reducing the reliance on chemical inputs, and nurturing the long-term health of our soils.Some species belonging to the genera Dothideomycetes , Pseudozyma , Sporidiobolus , Hanseniaspora , and Candida were able to promote plant growth (Amprayn et al. 2012, Fu et al. 2016, Jaiboon et al. 2016, Fernandez-San Millan et al. 2020 ).
While widespread studies have propagated arbuscular mycorrhizal fungi and PGPR in various agricultural contexts, as exemplified by studies such as those by Uzoh and Babalola ( 2018 ), Igiehon and Babalola ( 2021 ), Agbodjato et al. ( 2022 ), Xin et al. ( 2022 ), andRaklami et al. ( 2023 ), the potential to use yeasts as PGP agents r emain under exploited.Hence , using yeasts , especially the non-Sacc harom yces species, r epr esents a poorl y explor ed field with gr eat potential.This offers a promising and sustainable a gricultur e solution to combat nutrient deficiencies.Notably, despite a few existing reports on PGP yeast, our study marks the pioneering effort in exploring Moroccan yeast biodiversity and evaluating their impact on plant growth.
The primary objective of our study was to isolate and c har acterize the gr owth-pr omoting str ess-toler ant yeast isolated fr om two unexplored Moroccan extreme environments (MEEs).These envir onments ar e c har acterized by long-term harsh conditions.Specially, we aimed at the following: (1) Isolate non-Sacc harom yces yeast and compr ehensiv el y c haracterize the str ess-toler ant yeast strains in these EEs.(2) Assess and compare the plant growth-promoting traits of these isolated yeast strains.(3) Determine the parctical benefits of these yeast strains in stimulating the growth of Medicago sativa , a important agricultur al cr op.

Moroccan extremophilic sites description and soil sampling
Soil samples were procured from two distinct geographical locations, the Toubkal Mountain (TM) and the Merzouga desert (MD).The area is known for low water activity and intense radiation (Gommeaux et al. 2010 , Manni andFilali-Maltouf 2022 ).

Isolation of the stress-tolerant yeasts
The two used yeasts were isolated from soil collected from distinct parts of the TM region (31.05917N-7.91583 W) (for isolate L1) and the desert soil from Merzouga (31.147643N, −3.974280 W) (for isolate L2).The isolation was performed using the standard method.Ten grams of Toubkal and Mezrouga soils were meticulously homogenized in 90 ml of sterile physiological water (PW).Subsequentl y, we pr epar ed dilutions of eac h soil using the serial 10-fold dilution technique .T hen, 0.1 ml from each dilution was spread onto Sabouraud agar supplemented with chloramphenicol (100 mg ml −1 ) to pr e v ent the growth of bacteria.The Petri dishes were incubated at 30 • C for at least one week.The de v eloped colonies were isolated and purified on the same medium and stored in 25% (w/v) gl ycer ol at −20 • C for subsequent investigations.

Identification of culti va ble yeast
Molecular identification of the internal transcribed spacer (ITS) r egion of eac h of the isolated str ains was performed using the forw ar d primer ITS1 (5 -TCCGTA GGTGAA CCTGCGG-3 ) and the r e v erse primer ITS4 (5 -TCCTCCGCTT A TTGA T A TGC-3 ) and sequenced at Macrogen company, Korea ( http://dna.macrogen.com ).The ITS region, situated between the 18S and 5.8S rRNA region genes in the eukaryotic ribosomal RNA gene cluster, tends to exhibit sufficient genetic variability among different yeast species, making it a valuable target for species-level identification.The sequences obtained were classified using an essential local alignment search tool (BLAST) to compare our sequences with the deposited copies in the National Center for Biotechnology Information (NCBI eucaryotic database) (Kim et al. 2012 ).The sequences were aligned in the Molecular Evolution Genetics Analysis (MEGA) software (v12.0)using ClustalW (Larkin et al. 2007, Hall 2013 ).The phylogenetic tree was constructed based on the Tam ur a-Nei model using neighbor-joining analyses (bootstrap of 1000) (Tam ur a et al. 2011 ).

Scr eening f or salt and osmotic tolerance
The e v aluation of salt and osmotic str ess toler ance was carried out by monitoring the growth on Sabouraud medium amended with v arying concentr ations of NaCl (r anging fr om 0.092 to 4 mM) and polyethylene glycol P6000 (from 1.25% to 60%) using micr otiter plates.Subsequentl y, the plates wer e incubated at 28 • C for 96 h.After incubation, we measured the optical density at 600 nm using a microtiter plate reader.

Phosphate and potassium solubilizing capacity
We conducted the phosphate solubilizing activity using NBRIY broth, supplemented with 5 g l −1 tricalcium phosphate or Moroccan phosphate r oc k as the exclusive inorganic phosphate source (Nafis et al. 2019 ).The medium was inoculated with a 200-μl yeast solution, washed three times with PW, and adjusted to an optical density (OD 600 ) of 0.1.The medium was then incubated at 28 • C on a rotatory shaker, agitating at 140 rpm, for 96 h.After incubation, the yeast cells were separated by centrifugation (6000 rpm) for 10 min.In the supernatant, we measured the pH and soluble phosphate as described by Nagul et al. ( 2015 ).
Potassium solubilization capacity was determined onto Aleksandr ov a gar medium supplemented with 5 g l −1 Mica powder (Meena et al. 2015 ).The a gar plates wer e inoculated following the drop-on-plate method described by Alikhani et al. ( 2006 ) with a bacterial solution washed and adjusted to OD 600 = 0.8.The plates were incubated for 7 days at 30 • C, and the results were displayed as the ratio of the clear zone surrounding the colonies and the diameter of the colony.

Indole acetic acid and sider ophor e pr oduction
To e v aluate the ability of isolated yeasts to pr oduce indole acetic acid (IAA), strains were inoculated into 100 ml of Luria Bertani broth (Kumla et al. 2020 ) supplemented with 1.02 g l −1 of ltryptophan as IAA precursor.The yeast cell density was adjusted to 0.8 optical density (OD 600 = 0.8).After incubation at 28 • C on a r otatory shaker, a gitating at 140 r pm, 1 ml of the supernatant was mixed with 2 ml of Salkowski's reagent and two drops of phosphoric acid.This solution was immediately incubated in the dark for 30 min.The intensity of the red color formed was quantitativ el y determined by measuring the absorbance at 530 nm (Rodrigues et al. 2018 ).Concerning the sider ophor e estimation, the test was carried out using the standard Chrome Azurol-S (CAS) test in a solid medium.The sider ophor e pr oduction w as indicated b y measuring the orange-y ello w halo caused b y the change of the blue of the CAS medium (Louden et al. 2011 ).

Aminoc yclopr opane-1-carboxylic acid deaminase production
The isolated yeast str ains wer e e v aluated for their ca pacity to pr oduce aminocyclopropane-1-carboxylic acid (ACC) deaminase on the sterile minimal DF (Dworkin and Foster) salts media (4.0 g KH 2 PO 4 , 6.0 g Na 2 HPO 4 , 0.2 g MgSO 4 •7H 2 O, 2.0 g glucose, 2.0 g gluconic acid, and 2.0 g citric acid with 1 mg FeSO 4 O, 10 mg MoO 3 , pH 7.2) amended with 3 mM ACC instead of (NH 4 ) 2 SO 4 as sole nitrogen sour ce (Dw orkin and Foster 1958 ).The yeast str ains wer e inoculated to minimal DF and incubated at 28 • C for 24 h.Then, the ACC deaminase activity was quantified spectr ophotometricall y by α-ketobutyr ate pr oduction at 540 nm by comparing with the standard curve of αketobutyr ate, whic h r anged fr om 0.1 to 1.0 μmol (Honma and Smmom ur a 1978 ).The protein estimation was conducted using Bradford's pr ocedur e (Br adford 1976 ).This activity was expressed as nmol of α-ketobutyrate produced per milligram of cellular protein per hour, and it served as a critical indicator of the enzyme's efficiency.

Biological material, experimental design, and plant bioassay
To e v aluate the in-vivo impact of the isolated strains, we selected M. sativa (Demnate landscape variety) as the plant model.The experiment design emplo y ed in this study follo w ed a randomized complete block (RCDB) with four treatments (Trt) and four r eplications, eac h containing 12 seeds.The first Trt (NI) was the contr ol and involv ed noninoculated plants, the second Trt (L1) was inoculated with Rhodotorula sp.L1, the third Trt (L2) r eceiv ed inoculation with Naganishia sp.L2, and the fourth Trt (AM) was inoculated with the mixture of the two strains (L1 + L2).The y east inoculum w as pr epar ed by culturing the selected strains in Sabouraud broth (for 3-4 days at 28 • C).Following this incu-bation, y east cells w er e harv ested by centrifugation (6000 rpm, 10 min), washed once with sterile distilled water (DW), and then resuspended in DW to the final concentration (OD 600 = 1).Each pot r eceiv ed 10 ml of the a ppr opriate yeast str ains, and the mixed solution was ac hie v ed by mixing an equal volume of each strain.
Medicago sativa seeds were disinfected by immersion in sodium hypochlorite diluted 1/5 (v/v) for 5 min.Afterw ar d, the seeds were placed on wet filter paper in Petri dishes and germinated in the dark at 25 • C for 24 h.Once the seeds sprouted, they were transplanted into 2-l plastic pots filled with a peat and perlite mixture with a 1:1 r atio, whic h had been pr e viousl y sterilized.All pots were individually placed within trays in a controlled greenhouse, where the y recei ved natural daylight ranging from 250 to 1000 μmol m 2-1 sec −1 .The temper atur e was maintained at 25/21 • C day/night with a r elativ e humidity of 40%-60% r elativ e humidity.Plants wer e irrigated with 250 ml DW twice weekly to ensure regular water availability through treatments.
After 60 days from sowing, both the shoot and r oots wer e harv ested.The r oots wer e separ ated fr om the shoots and car efull y rinsed, with excess water r emov ed using a paper towel.Subsequently, the length of the shoot and root w as recor ded.The dry weight of the shoots and roots was measured by subjecting them to oven drying for 72 h at 70 • C.
Determining mineral elements (N, P, K) was performed after the mineralization of plant materials (shoot).Total nitrogen content was measured according to the method described by Rodier ( 1984 ).Phosphorus was determined according to the protocol described by Olsen and Sommers ( 1982 ) .Meanwhile, K was determined using a flame photometer (AFP 100 flame photometer).

Sta tistical anal ysis
We emplo y ed a Statistical Analysis System softw are for the data analyses, using the JMP module from 2019.One-way analyses of variance (ANOVAs) were conducted to evaluate significant differences among treatments.To compare means and determine specific differ ences, we a pplied Tuk e y's test method.Significant differences at P < 0.05 were denoted by different letters.Values sharing the same letter wer e consider ed not significantl y differ ent at P < 0.05.

Physico-c hemical c haracteristics of the soil samples
The soil properties of TM and MD demonstrate distinctive attributes reflecting the diverse environmental conditions present in these two Moroccan sites.In TM, the soil is c har acterized by a pH le v el of 7.9, indicating a slightl y alkaline envir onment.The electrical conductivity (EC) is measured at 2.14, suggesting moderate salinity levels .Furthermore , the total organic carbon (TOC) content is 1.02, indicating a r elativ el y enric hed or ganic matter status .Con v ersel y, the sandy soil properties found in the arid expanse of MD differ significantly.The pH is slightly higher at 8.04, r eflecting a mor e alkaline natur e than TM.The EC v alue is lo w er at 0.95, signaling lo w er salinity le v els .T he T OC content is 0.96, indicating a reduced organic matter presence compared to the mountainous counterpart.These variations in soil c har acteristics highlight the impact of climate, vegetation, and geological factors on the soil composition, illustrating the contrasting EE of the highaltitude TM and the arid MD.

Identity of culti va ble yeast strains
Using a culture-dependent method, two yeast strains were isolated, purified, and identified based on ITS sequencing.The BLAST homology searc h r e v ealed that the r ed-str ain isolate L1 belonged to the genus Rhodorula and exhibited a striking 99.83% sequence similarity to Rhodotorula mucilaginosa.In contrast , the isolate L2 was assigned to Naganishia genus and displayed 99.84% sequence identity with the Naganishia albida r efer ence str ains .To further understand the e volutionary r elationships of the isolated yeasts and their close r elativ es, w e emplo y ed the neighbor-joining method based on the ITS1/ITS4 (Fig. 1 ).

Scr eening f or salt and osmotic tolerance
We expand our r esearc h to e v aluate the ability of the extremophilic yeasts to withstand abiotic stress, such as those induced by salt and drought (Table 1 ).Concerning salt tolerance, all extremophilic y east sho w ed their high resistance to salt stress and could tolerate 2 M of NaCl (Table 1 ).Regarding drought tolerance, extremophilic yeast strains could also tolerate 60% of P6000.

Characterizing the isolated yeasts from extremophilic environments
According to the results, the isolated yeast strains demonstrated the ability to solubilize both complex inorganic phosphate, specifically tricalcium and rock phosphate, as shown in Table 1 .The solubilization activity of tricalcium phosphate was the most significant in the case of Rhodotorula sp.L1, with a solubilization activity of 113 mg ml −1 .Conv ersel y, for r oc k phosphate, Naganishia sp.L2 exhibited the highest activity (182 mg ml −1 ) (Table 1 ).The solubilization activity observed in both sources was accompanied by a notable medium acidification, with pH le v els ar ound 4-5.This acidification pattern suggests that the solubilization process might be attributed to the production of organic acids.Furthermore, potassium solubilization was recorded in the case of the Naganishia sp.L2, with a 2.5 cm halo diameter.The assay for IAA pr oduction r e v ealed that both yeast tr ains could pr oduce IAA using l -tryptophan as a precursor, with a significant production (up to 130 mg l −1 ) observed in the case of Rhodotorula sp.L1.Investigation of sider ophor e pr oduction sho w ed that both strains could pr oduce sider ophor es to c helate ir on fr om the medium.Additionally, Rhodotorula sp.L1, and Naganishia sp.L2 exhibits ACC deaminase activity, up to 2000 μmol mg protein −1 h −1 (Table 1 ).The results of the PGP activities strongly support the potential use of these yeast strains as bioinoculants, particularly in semi-arid regions.

T he gr owth-pr omoting effect of yeast isolates on Medicago sativa growth
To elucidate the in-vivo effect of the isolated yeast on plant growth, we utilized M. sativa as our plant model.Our evaluation of the str ess-toler ant yeast, L1 and L2, demonstrated their capacity to enhance alfalfa shoot growth and de v elopment.Statistical analyses r e v ealed a significant impact of the tested strains on the shoot, root, and total biomass dry weight (Fig. 2 ).Inoculated plants sho w ed higher shoot, root dry weight, and dry matter biomass (Fig. 2 A).Inoculated plants exhibited notably higher shoot and root dry weight, as well as ov er all dry matter of biomass (Fig. 2 A).Inter estingl y, ther e was no specific difference in the consortium's effect compared to the application of L1 and L2 alone in terms of shoot, root, and total biomass, except in case of leaves number.Specificall y, a ppl ying L1 r esulted in a 53% increase in shoot dry weight, while L2 boosted it by 49% (Fig. 2 A).Inoculation with mixed strains (L1 + L2) led to an 84% increase in root dry weight.
Ta ble 1. Plant gro wth-promoting (PGP) traits of the isolated endophytic actinobacterial strains.Similarly, the shoot length, root length, and leaves number were significantl y impr ov ed by a ppl ying yeasts (Fig. 2 B).The shoot and root length were improved by 19% and 31% by the inoculation with Rhodotorula sp.L1 (Fig. 2 B).The number of leaves was increased by 85% by a ppl ying the str ess-toler ant yeast strains (L1 + L2).

Nitr ogen, phosphorus , and potassium assimilation
The impact of chosen yeast inoculation on the acquisition of nutrients in M. sativa has been investigated (Table 2 ).The mineral acquisition sho w ed varying distinctions betw een plants that w ere inoculated and noninoculated.A remarkable illustration of the impact of inoculation is the significant enhancement of nitrogen acquisition by 136% in plant that were inoculated with L1 + L2.Ne v ertheless, it is worth noting that phosphorus acquisition did not exhibit significant ( P < 0.05) impr ov ement by the biological tr eatment.Conv ersel y, the inoculation with L2 notably enhanced the potassium acquisition, resulting in a significant improvement of 12%.

Discussion
We hypothesized that in EEs, micr oor ganisms hav e e volv ed and de v eloped se v er al toler ance mec hanisms and distinctiv e PGP traits as a response to the selection pr essur e imposed by the harsh environment.Despite efforts, the understanding of yeast diversity and their functional capacities linked to extreme ecosystems r emains insufficientl y compr ehended.We consider ed TM and MD to constitute a MEE for isolating unexamined-e volv ed non-Sacc harom yces str ains with unique, distinctiv e c har acteristics because of the high altitude, low oxygen le v el, atmospheric pr essur e, high temper atur e, and oligotr ophic conditions.In this study, we examined the gr owth-pr omoting attributes of yeast strains isolated for MEEs and their impact on the growth of M. sativa .Two yeasts, L1 and l2, were isolated from Morocco's highest peak and the largest desert dune , r espectiv el y.The r esults of the BLAST homology indicated a similarity between the red yeast L1 and R. mucilaginosa.In contrast, the extremophilic yeast L2 display a 99% sequence identity to N. albida.This is the first stud y re porting the isolation of R. mucilaginosa and N. albida from MEE.Our findings support earlier research that sho w ed the wide pr e v alence of non-Sacc harom yces str ains fr om differ ent EEs.For instance, R. mucilaginosa was isolated from EE, such as the Peninsula (Troncoso et al. 2017 ), Antarctic sea (Wang et al. 2019 ), oilfield (Der guine-Mec heri et al. 2021 ), and Sua pan (Loeto et al. 2021 ).Similarly, N. albida has been isolated from Antarctic soil samples (Białkowska et al. 2017 ), snow from the south pole (Hayw ar d et al. 2021 ), and hypersaline coastal waters (Fotedar et al. 2022 ).
Yeast strains, being remarkably adaptable microorganisms, employ a variety of molecular and cellular mechanisms to endure and thrive in diverse EEs .T he stress adaptation mechanisms in yeast are diverse and interconnected, often involving intricate signaling pathwa ys , gene expr ession c hanges, and alter ations in cellular physiology.In the face of ele v ated temper atur es, yeasts adjust the concentration of saturated fatty acids (also reaching 30%-40%) occurring in lipids and maintain an optimal degree of fluidity (Buzzini et al. 2017 ).Indeed, yeast activates a robust heat shoc k r esponse c har acterized by the induction of heat shock pr oteins (HSPs), whic h facilitate the pr oper folding and stabilization of proteins under thermal stress .T he transcription factors Hsf1 and Msn2/Msn4 mediate the heat shock response.It involves the upregulation of several genes for HSPs that participate in  Means ( ±standard deviation) within the same column followed by different letters are significantly different at P < 0.05.
tr affic king and matur ation, as well as the genes for the protein degr adation mac hinery (Tr ott andMor ano 2002 , Mühlhofer et al. 2019 ).Conv ersel y, under cold temper atur es, yeast species exhibit a cold shock response, often involving the upregulation of specific cold shock proteins and physiological adaptations that decrease their growth rate and synthesize enzymes active at low temper atur es and cryopr otectiv e molecules to maintain membrane fluidity and cellular functions (Buzzini et al. 2017, Sanino et al. 2017 ).For instance, cold-adapted R. diobovatum possesses a notable attribute c har acterized by the ele v ated pr oduction of unsatur ated fatty acids, ther eby ensuring a heightened fluidity of the plasma membrane (Turk et al. 2011, Segal-Kischinevzky et al. 2022 ).In the case of R. frigidialcoholis , the response to low temperatures in the permafrost of the Antarctic dry valley is ac hie v ed thr ough man y mec hanisms .T hese mechanisms include o v er expression of the pentose phosphate pathwa y genes , increasing the production of carotenoids , sphingolipids , unsaturated fatty acid, and exopol ysacc harides while coupled with a r eduction in expr ession of gr owth, tr anscriptional, and tr anslational mac hinery genes (Touchette et al. 2022 ).Yeast derived from arid habitats can endure the hydrological strain and generate aridity-enduring formations such as ascospores , teliospores , and chlamydospores , whic h spr out under favor able circumstances.At the same time, in their v egetativ e form, they can synthesize pol ysacc haride ca psules that pr e v ent desiccation (Buzzini et al. 2017 ) The findings of our investigation suggest that the isolated extremophilic yeast may be implicated in a broad spectrum of biological processes, offering potential application in the promotion of plant growth.The isolated yeast strains demonstrated salt and osmotic stress resistance and exhibited active characteristics associated with plant growth promotion.These characteristics include the ability to solubilize tricalcium and r oc k phosphate, solubilize potassium, produce ACC deaminase, and produce IAA and sider ophor es.In a gr eement with these findings, it has been documented that yeast strains isolated from various environment can display se v er al PGP activities (Nassar et al. 2005, Amprayn et al. 2012, Nutaratat et al. 2014, Fu et al. 2016 ).Fu et al. ( 2016 ) have pr ovided e vidence that yeast str ains exhibit a div erse spectrum of IAA synthesis, with concentrations ranging from 8 μg ml −1 (as observed in the case of Kazachstania jiainicus JYC361) to 610 μg ml −1 (seen in the case of Aureobasidium pullulans JYC104) depending on the species .Furthermore , it has been observed that Candida tropicalis HY possesses the capacity to dissolve phosphate to a concentration of 119 mg ml −1 (Amprayn et al. 2012 ).In this study, the extremophilic yeast strain, R. mucilaginosa L1, produces a significant amount of IAA ( > 100 μg/ml), which may have a deleterious effect on plant growth (Tapia-Vázquez et al. 2020 ); ho w ever, this strain shows a biotechnological potential for the production of IAA that is used in a gricultur e as a rooter.As demonstrated by Sen et al. ( 2019 ), the genome of R. mucilaginosa encodes genes associated with PGP, including auxin biosynthesis, cytokinin metabolism, abscisic acid and gibberellin biosynthesis, and jasmonic acid production.On the other hand, Sen et al. ( 2019) also highlighted the presence of specific genes coding for stress tolerance, especially for cold adaptation.This evidence supports to our hypothesis, which asserts that yeast has under gone e volutionary ada ptations to dev elop mec hanisms of toler ance in EEs .In our specific case , these toler ance mec hanisms pertain to salinity and drought.In contr ast, ther e may be limited specific information on N. albida regar ding plant gro wth pr omotion tr aits due to the limited number of studies reporting on the plant gr owth pr omotion tr aits of the Naganishia genus.
The isolated extremophilic yeast has demonstrated considerable utility by effectively promoting the growth of M. sativa .Whether applied individually or in combination, these yeast species have significantly improved both shoot/root dry weight and number of lea ves .Moreo ver, our research has revealed that these extremophilic yeasts possess the ability to enhance nutrient uptake, as evidenced by their increased assimilation of essential nutrients such as N, P, and K. Numerous studies have also substantiated the capacity of yeast strains to promote the growth of Oryza sativa , Solanum lycopersicum , and Nicotiana benthamiana (Amprayn et al. 2012, Thais et al. 2019, Fernandez-San Millan et al. 2020, Tapia-Vázquez et al. 2020). Fernandez-San Millan et al. ( 2020 ) demonstrated the effectiveness of applying R. dairenensis in enhancing the growth and development of Nicotiana benthamiana .Similarl y, Ta pia-Vázquez et al. ( 2020) have confirmed the ability of psyc hr ophilic and psyc hr otoler ant yeasts (specifically Rhodotorula sp .and Naganishia sp .) to pr omote Solanum l ycopersicum .Ho w e v er, despite the extensive use of yeast as a biocontr ol a gent, r esearc h on the application of yeast as a biofertilizer has been r elativ el y limited compared to bacteria, actinobacteria, fungi, and mycorrhizae.Consequentl y, ther e exists a knowledge ga p r egarding the potential of yeast as PGP agents and the specific mechanisms by which they facilitate plant growth.Nevertheless, there has been a recent surge in scientific interest to explore the PGP potential of yeast.This newfound attention is primarily driven by their ability to produce phytohormone compounds, solubilize inorganic phosphate, excr ete sider ophor es, and possess ACC deaminase activity (Hamidi et al. 2020, Tapia-Vázquez et al. 2020, Ramya et al. 2021, Srinivasan et al. 2022 ).

Conclusion
While the untapped potential of yeasts as agents for promoting plant gr owth r emains unexploited, it is clear that yeast has a substantial opportunity to establish itself as an indispensable component of sustainable a gricultur e in the coming decades.In this in vestigation, we ha ve un veiled the potential of two yeast strains that are not non-Saccharomyces .T hese strains , isolated from TM and MD, have displayed v arious PGP tr aits and demonstr ated their ability to promote plant growth under greenhouse conditions.Expanding upon these discoveries and exploring the full potential of these two isolated strains, Rhodotorula sp.L1 and Naganishia sp.L2, is imper ativ e in pr omoting plant gr owth under differ ent str essed en vironments .

Figure 1 .
Figure 1.Maxim um-likelihood tr ee based on ITS gene sequence showing the r elations between the str ess-toler ant yeast str ains .T he numbers at the nodes indicate the le v els of bootstr a p support based on maximum-likelihood analyses of 1000 resampled data sets (only values > 50% are shown).

Figure 2 .
Figure 2. Shoot and root, and total biomass dry weight (A), and lengths of shoot and root, and numbers of leaves (B) of Medicago sativa submitted to differ ent tr eatments [contr ol without inoculation, Rhodotorula sp.L1; Naganishia sp.L2; and the consortia of A3 + A4].Means ( ±SD) within the same parameter follo w ed b y differ ent letters ar e significantl y differ ent at P < 0.05.Symbols ar e the number of r e plicate (10).The Tuk e y's test method was used to separate means that were different at P ≤ 0.05.Values in columns follo w ed b y the same letter ar e not significantl y differ ent at P < 0.05 (Tuk e y's test).