fmicb-07-01030 June 25, 2016 Time: 12:42 # 1

ORIGINAL RESEARCHpublished: 28 June 2016

doi: 10.3389/fmicb.2016.01030

Edited by:Suhelen Egan,

The University of New South Wales,Australia

Reviewed by:Ilana Kolodkin-Gal,

Weizmann Institute of Science, IsraelShengguo Zhao,

Chinese Academy of AgriculturalSciences, China

*Correspondence:Xiao-Hong Yang

yangxh2@swu.edu.cn;Xin-Hua He

xinhua.he@uwa.edu.au

†These authors have contributedequally to this work.

Specialty section:This article was submitted to

Microbial Symbioses,a section of the journal

Frontiers in Microbiology

Received: 15 December 2015Accepted: 17 June 2016Published: 28 June 2016

Citation:Shi S-M, Chen K, Gao Y, Liu B,Yang X-H, Huang X-Z, Liu G-X,

Zhu L-Q and He X-H (2016)Arbuscular Mycorrhizal Fungus

Species Dependency Governs BetterPlant Physiological Characteristics

and Leaf Quality of Mulberry (Morusalba L.) Seedlings.

Front. Microbiol. 7:1030.doi: 10.3389/fmicb.2016.01030

Arbuscular Mycorrhizal FungusSpecies Dependency Governs BetterPlant Physiological Characteristicsand Leaf Quality of Mulberry (Morusalba L.) SeedlingsSong-Mei Shi1†, Ke Chen1†, Yuan Gao1, Bei Liu1, Xiao-Hong Yang1,2*, Xian-Zhi Huang3,Gui-Xi Liu1, Li-Quan Zhu1 and Xin-Hua He2,4*

1 Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education/College of Horticultureand Landscape Architecture, Southwest University, Chongqing, China, 2 Centre of Excellence for Soil Biology, College ofResources and Environment, Southwest University, Chongqing, China, 3 State Key Laboratory of Silkworm Genome Biology,Southwest University, Chongqing, China, 4 School of Plant Biology, University of Western Australia, Crawley, WA, Australia

Understanding the synergic interactions between arbuscular mycorrhizal fungi (AMF)and its host mulberry (Morus alba L.), an important perennial multipurpose plant, hastheoretical and practical significance in mulberry plantation, silkworm cultivation, andrelevant textile industry. In a greenhouse study, we compared functional distinctionsof three genetically different AMF species (Acaulospora scrobiculata, Funneliformismosseae, and Rhizophagus intraradices) on physiological and growth characteristicsas well as leaf quality of 6-month-old mulberry seedlings. Results showed thatmulberry was AMF-species dependent, and AMF colonization significantly increasedshoot height and taproot length, stem base and taproot diameter, leaf and fibrousroot numbers, and shoot and root biomass production. Meanwhile, leaf chlorophylla or b and carotenoid concentrations, net photosynthetic rate, transpiration rateand stomatal conductance were generally significantly greater, while intercellular CO2

concentration was significantly lower in AMF-inoculated seedlings than in non-AMF-inoculated counterparts. These trends were also generally true for leaf moisture, totalnitrogen, all essential amino acids, histidine, proline, soluble protein, sugar, and fatty acidas they were significantly increased under mycorrhization. Among these three testedAMFs, significantly greater effects of AMF on above-mentioned mulberry physiologicaland growth characteristics ranked as F. mosseae > A. scrobiculata > R. intraradices,whilst on mulberry leaf quality (e.g., nutraceutical values) for better silkworm growthas F. mosseae ≈ A. scrobiculata > R. intraradices. In conclusion, our results showedthat greater mulberry biomass production, and nutritional quality varied with AMFspecies or was AMF-species dependent. Such improvements were mainly attributedto AMF-induced positive alterations of mulberry leaf photosynthetic pigments, netphotosynthetic rate, transpiration rate, and N-containing compounds (methionine,threonine, histidine, and proline). As a result, application of Funneliformis mosseae orA. scrobiculata in mulberry plantation could be a promising management strategy topromote silkworm cultivation and relevant textile industry.

Keywords: Acaulospra scrobiculata, amino acids, Funneliformis mosseae, Rhizophagus intraradices, net leafphotosynthesis rate, transpiration

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INTRODUCTION

As a multipurpose plant, mulberry (Morus alba L.) has becomean increasingly attractive plant for its economic value andrestoration function. It has been extensively cultivated in Chinafor the main purpose of rearing silkworm (Bombyx mori L.), aneconomically important insect as the primary producer of silk,and thus contributing to local farming and textile industries.The quality of mulberry leaves strongly associates with thequality and quantity of cocoon, especially their high contents ofcarbon, nitrogen (N), amino acids and proteins that contributegreatly to the silkworm development (Machii and Katagiri, 1991;Sujathamma and Dandin, 2000; Rao et al., 2007). Althoughmulberry can survive in poor soil (Kumar et al., 2003; Huanget al., 2013), but its growth is retarded under infertile soil.A number of biotic or abiotic factors, such as soil beneficialorganisms and fertilization, etc., do enhance the growth andleaf quality of mulberry (Jeffries et al., 2003; Setua et al., 2007;Chowdhury et al., 2013; Fernandez et al., 2014).

The symbiotic associations formed by arbuscular mycorrhizalfungi (AMF) are widely associated with >80% vascularplants including mulberry in terrestrial ecosystems (Smithand Read, 2008). Rajagopal et al. (1989) was probably thefirst to report that mulberry roots were highly colonized byAMF in natural conditions, and phosphorus (P) uptake washence increased to lead a better leaf growth and biomassproduction of mulberry (Katiyar et al., 1995; Setua et al.,1999a,b). Both the growth and yield of 10-year-old mulberryplants were similar between fertilizations under 100% P and50% P + Glomus (now Funneliformis) mosseae inoculation(Mamatha et al., 2002). Recently, for 90-day-old AMF inoculatedmulberry seedlings, either Funneliformis mosseae orGlomus (nowRhizophagus) intraradices had enhanced leaf numbers, plantheight, chlorophyll, N and P, both aboveground and belowgroundbiomass production, root length and root TTC (2,3,5-triphenyltetrazolium chloride) deoxidizing ability, although there wasno such growth benefits by their dual fungal inoculation (Luet al., 2015). Meanwhile, the combined application of mycorrhizalfungi and other beneficial microorganisms could also promotethe growth of mulberry. For instance, the co-inoculation oftwo AMF species (Glomus fasciculatum and F. mosseae) withother three beneficial microorganisms (N2-fixing Azotobactersp., phosphate solubilizing bacterium Bacillus megaterium andfungus Aspergillus awamori) did increase the uptake of N, P, K(potassium) and leaf chlorophyll in 1-year-old mulberry plants inIndia (Baqual and Das, 2006). In addition, a combined positiveeffect of F. mosseae and bacterial biofertilizer (Azotobacterchrococcum) on mulberry leaf quality and cocoon characters wasalso found under semiarid fields in India (Rao et al., 2007).However, limited information is available about the influence ofdifferent AMF species either within the same fungal genus ordifferent genera on the quantity and quality of mulberry leaves,which in turn will directly affect the growth and developmentof silkworm, the production of cocoon and the quality of silkproducts. Therefore, further studies will determine if differentAMF species in the same genus or different genera havedistinctive roles in the growth and leaf quality of mulberry.

We have found that a high diversity of AMF, includingAcaulospra scrobiculata, Funneliformis mosseae, and Rhizophagusintraradices, associates with field-growing mulberry plants, andthat such an individual AM fungus promotes the growth ofmulberry (Shu et al., 2011; Shi et al., 2013) and soil fertility(Chen et al., 2014) in southwest China. In this study, wehypothesized that the growth of mulberry and quality of mulberryleaves could have varied responses to different AMF species ortheir physiological characteristics of mulberry might be AMF-species dependent. Our objectives were to address the followingquestions: (1) If AM fungus could successfully colonize thehost mulberry, and what physiological alterations could thehost have after colonization? (2) Whether the growth andperformance of mulberry depend on its symbiont fungal speciescharacteristics or not? In doing so, 3-month-old mulberryseedlings grown in a greenhouse were inoculated with or withoutan individual AMF species (A. scrobiculata, F. mosseae, andR. intraradices) from three different AM genera, and some oftheir basic physiological characteristics were then comparedafter a further 3-month-old growth. Answers to these questionscould improve our understanding of the physiological responsesof mulberry to different AM fungi in order to employ betterfunctionally effective AM species for promoting sustainablemulberry plantation and silkworm industry in China.

MATERIALS AND METHODS

Experimental DesignThe greenhouse experiment set-up was a randomized completeblock design that had three AMF inoculation treatments(Acaulospra scrobiculata, Funneliformis mosseae, or Rhizophagusintraradices) and a non-AMF control with four replicates for eachtreatment.

Plants, Mycorrhizal Inoculums, and PlantGrowth MediaSeeds of mulberry (Morus alba var. Gui-you-sang 12, suppliedby the Institute of Sericulture and Systems Biology, SouthwestUniversity, Chongqing, China) were disinfected with 0.1% HgCl2for 5 min, rinsed thoroughly with sterilized water and thengerminated on sterilized moistened filter paper. The germinatedseeds were cultivated for 1 month in plastic trays, whichcontained autoclaved (121◦C, 0.1 MPa, 120 min) sand growthmedia (peat: decomposed rice chaff: sand= 2:2:1, v/v/v.

Three AMF inoculums [A. scrobiculata (BGC HK02A),Funneliformis mosseae (BGC NM04A) and R. intraradices(BGC AH01)] were purchased from the Bank of Glomalesin China (BGC), locating in the Institute of Plant Nutritionand Resources, Beijing Academy of Agriculture and Forestry.Inoculums consisted of spores (40–50 spores per 10 g drysoil), mycorrhizal mycelia, root segments, and sand. The growthmedium was a mixture of soil (Eutric Regosol, FAO SoilTaxonomic Classification), peat and rice chaff (2:1:1, v/v/v).With a pH 7.28, this soil growth medium had 71.17 g organicmatter/kg, 373.12 mg available nitrogen/kg, 14.72 mg availablephosphorus/kg, 132.57 mg available potassium/kg.

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Shi et al. AMF Governs Production of Mulberry

Plant Growth and HarvestFour seedbeds containing the above-mentioned soil growthmedium were used to grow mycorrhizal mulberry seedlings. Eachseedbed (28 m × 1.2 m) was fumigated with 0.5% formaldehydeand divided into four blocks or replicates (6 m × 1.2 m with1 m interblock space) for each treatment. A total of 600 1-month-old non-mycorrhizal mulberry seedlings from the sandgrowth media were transplanted and grown in seedbeds withthe autoclaved soil growth media. After 2 months, about 400uniform seedlings (100 for each block or replicate) were chosenfor mycorrhizal inoculation with 3 g AMF inoculum (autoclavedfor the control) close to each root rhizosphere. Plants werethen grown in a greenhouse under 26/15◦C (day/night), 70–80%relative humidity and natural light intensity. No-fertilization wasemployed for a further 3-month period till harvest (i.e., the totalgrowth period were 6 months).

After 3 months of mycorrhizal inoculation, 20 uniformseedlings out of the 100 seedlings from each replicate wererandomly harvested and a total of eighty seedlings werethus harvested from each treatment to determine growth andphysiological parameters. At harvest, plant height, stem diameter,leaf numbers, taproot length and diameter, and fibrous rootnumbers were recorded. Leaves, shoots and roots were then oven-dried at 70◦C and weighed. The fresh fifth fully expanded leavesfrom the top to the bottom of each seedling were harvested,and a total of 80 leaves from each treatment were dividedinto two sample groups. One sample group of leaves was cutinto pieces and mixed for the determination of leaf chlorophylland carotenoid, soluble protein and sugar (see below). Anothersample group of leaves was oven-dried at 70◦C till constantweight, pulverized to fine powder for the determination of aminoacid, total nitrogen, and fatty acid (see below). The leaf moisturewas recorded according to the method described by Gao (2000).

Determination of MycorrhizalColonization and DependencyRoot mycorrhizal colonization was measured according toPhillips and Hayman (1970). The fresh roots were cut into 2 cmsegments and cleared with 10% (w/v) KOH at 98◦C for 30 min,rinsed in water three times, and bleached with 10% H2O2 untilroot clear. The clear root segments were then soaked into 0.2 MHCl for 3 min and stained with 0.05% trypan blue. Five stainedsegments were mounted on one slide and a total of 50 rootsegments from each treatment were examined under microscope.The root mycorrhizal colonization was determined as the percent(%) of infected root segments out of the total observed rootsegments. Mycorrhizal dependency (MD) was calculated as thedry weight of mycorrhizal seedling out of the dry weight ofnon-mycorrhizal seedling (Menge and Johnson, 1978).

Photosynthesis MeasurementsThe fifth leaf from the top to the bottom of each plantgrowing in the greenhouse was selected for the measurementsof photosynthesis variables. Gas exchange parameters (includingnet photosynthetic rate, stomatal conductance, intercellular CO2concentration, and transpiration rate) were determined with

20 seedlings from each replicate using a portable infraredgas analyzer LI-COR 6400 (LI-COR, Inc., Lincoln, NE, USA)from 09:00 to 11:00 am on a sunny day after 3 months ofmycorrhizal inoculation. The photosynthetically active radiationwas 1000 ± 12 µmol m−2 s−1, CO2 concentration 350 ± 2 cm3

m−3, leaf temperature 28.0± 0.8◦C, and flow rate of atmosphere0.5 dm3 min−1. Meanwhile, the chlorophyll and carotenoid in thefresh leaves were extracted with 80% acetone and the absorbancewas read spectrophotometrically at 663 and 645 nm. Chlorophylland carotenoid concentration was estimated by the method ofHiscox and Israelstam (1979).

Determination of Leaf Soluble Sugar,Soluble Protein, Amino Acid, Fatty Acid,and Total NitrogenDetermination of soluble sugar (mg g−1 FW) in fresh leaveswas followed by the anthrone method (Yemm and Willis, 1954).Briefly, 0.5 g fresh leaves were grinded with 80% ethanol andthen centrifuged at 3,500 × g for 10 min. The supernatantwas collected. The total soluble sugar was determined byreacting 0.1 ml supernatant with 5 ml freshly prepared anthronesulphuric acid solution (75%, v/v), and incubated in boilingwater for 10 min. After cooled, the absorbance of the incubatedsupernatant was spectrophotometrically read at 620 nm.

Soluble proteins (mg g−1 FW) of fresh leaves were extractedwith 50 mM potassium phosphate buffer (pH 7.5) on ice. Theextracts were centrifuged at 12,000 × g for 10 min at 4◦C. Thesupernatant was then collected for soluble protein determinationaccording to the protein dye-binding method of Bradford (1976).For each extract, the absorbance was spectrophotometrically readat 595 nm and the bovine serum albumin (BSA) protein was usedas the standard.

For the measurement of leaf total amino acids (mg g−1 DW),230 mg leaf powder was put into a beaker containing 8 ml 6 MHCl, and hydrolyzed at 110◦C for 22 h in thermostat drier. Aftercooled, the solution was filtered and the filtrate was transferredinto a volumetric flask and dried at 60◦C. The dried hydrolysatewas dissolved in 0.02 M HCl and then analyzed for total aminoacids with the ninhydrin method (Lee and Takahashi, 1966) by anautomatic amino acid analyzer (L-8900; Hitachi, Tokyo, Japan).

Leaf fatty acid (mg g−1 DW) was based on the ChineseStandard Method (GB5009.6-85). Briefly, 3 g dried leaf powderwas placed in a filter cartridge that was dried at 105◦C for2 h. The cartridge was placed in a 50 mL anhydrous ether andrefluxed 12 h using a Soxhlet apparatus. After the ether wasfully evaporated from the extract, the cartridge containing theextract was oven-dried at 105◦C, cooled in a desiccator and thenweighed.

The leaf nitrogen was determined with the Kjeldahl procedure.Briefly, 0.5 g leaf powder was placed in a digestion tubecontaining 10 ml digestion reagent and then dissolved inconcentrated H2SO4. After gently mixed, the digestion tubewas placed in the heating block preheated at 300◦C. A smallglass funnel was inserted in the mouth of the digestion tubefor ensuring efficient refluxing of the digestion mixture andpreventing loss of H2SO4. The sample was digested at the boiling

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point of the mixture for 2.5 h, removed from the heating block,and cooled to room temperature. The digest was diluted withdistilled water to 100 ml and performed a steam distillationanalysis as described by Nelson and Sommers (1973).

All relevant variables were analyzed with a UV1000spectrophotometer and all analyses were performed threetimes.

Statistical AnalysesData were subjected to one-way ANOVA and significantdifferences among treatments were tested by the Duncan’smultiple range test at P < 0.05 using the SPSS 19.0 (SPSS Inc.,Chicago, IL, USA). To test correlations, a polynomial regressionanalysis was performed using the OriginPro 8.0 (OriginLabCorp., Northampton, MA, USA).

RESULTS

Effects of AMF Colonization on PlantGrowthRoot AMF colonization existed only in AMF inoculated mulberryplants, and neither AMF colonization nor AMF structures weremicroscopically observed in the control seedlings (Figure 1).Percentages of root colonization in mulberry seedlings wereover 50% and significantly highest with F. mosseae, greater withA. scrobiculata and least with R. intraradices (Figure 1). Themycelial numbers were similar among the three mycorrhizalinoculations, while the vesicle numbers were lower underthe R. intraradices treatment (Figure 1). Correspondingly, thegrowth performance of mulberry seedlings was affected bymycorrhizal fungal colonization (Figure 2). Compared withnon-mycorrhizal seedlings, all tested plant growth variables,including aboveground height and taproot length, stem base andtaproot diameter, shoot and root biomass, leaf and fibrous rootnumbers, were significantly increased by a range of 31 to 121% inAMF inoculated plants (Figure 3). In short, significantly greaterplant growth performance, as well as MD, generally ranked asF. mosseae > A. scrobiculata > R. intraradices > non-AMFcontrol (Figure 3).

Effects of AMF Colonization onPhotosynthetic VariablesConcentrations of leaf photosynthetic pigments (chlorophylla and b, and carotenoid) were significantly higher in allmycorrhizal plants than in non-mycorrhizal plants (Figure 4).Among AMF plants, the higher concentration order of testedphotosynthetic pigments ranked as F. mosseae > A. scrobiculata≈ R. intraradices for leaf chlorophyll a, leaf chlorophyll band carotenoid, but under F. mosseae > A. scrobiculata> R. intraradices for leaf chlorophyll (a+b) (Figure 4).

Meanwhile, AMF colonization significantly enhanced leafnet photosynthetic rate by 52–99%, stomatal conductance by24–56%, transpiration rate by 66–104%, while significantlydecreased intercellular CO2 concentration by 5–15%, comparedwith non-AMF mulberry plants (Figure 5). Among

FIGURE 1 | Root arbuscular mycorrhizal fungal colonization in6-month-old mulberry seedlings. Fifty mycorrhizal root segments fromeach treatment were examined under a microscope: (a) non-AMF treatment;(b) A. scrobiculata, Acaulospora scrobiculata; (c) F. mosseae, Funneliformismosseae; (d) R. intraradices, Rhizophagus intraradices.

FIGURE 2 | Plant growth status of 6-month-old mulberry seedlings asinfluenced by mycorrhizal fungal species. (A) Whole plant growth, and(B) the growth of the fifth fully expanded leaf.

AMF colonized mulberry plants, significantly greater leafphotosynthetic variables, ranked as F. mosseae > A. scrobiculata> R. intraradices for the net photosynthetic rate,R. intraradices > A. scrobiculata > F. mosseae for the stomatalconductance, F. mosseae ≈ R. intraradices > A. scrobiculatafor the transpiration rate, and R. intraradices > A. scrobiculata> F. mosseae for the intercellular CO2 concentrations (Figure 5).

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FIGURE 3 | Effects of mycorrhizal fungal species on growth performance of mulberry plants. Data (means ± SE, n = 4) followed by different letters indicatesignificant differences among AMF treatments at P < 0.05. Eighty plants from each treatment were randomly selected to detect growth performance. Abbreviations:Control, non-AMF treatment; A. scrobiculata, Acaulospora scrobiculata; F. mosseae, Funneliformis mosseae; R. intraradices, Rhizophagus intraradices.

FIGURE 4 | Effects of mycorrhizal fungal species on the concentrations of leaf chlorophyll a, b, (a+b) and carotenoid of mulberry plants. Data(means ± SE, n = 4) followed by different letters indicate significant differences between AMF treatments at P < 0.05. The fresh fifth expanded leaf of each plant wasused to determine concentrations of leaf chlorophyll a, b, (a+b) and carotenoid. Forty fresh leaves were used in each treatment. Abbreviations: Control, non-AMFtreatment; A. scrobiculata, Acaulospora scrobiculata; F. mosseae, Funneliformis mosseae; R. intraradices, Rhizophagus intraradices.

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FIGURE 5 | Effects of mycorrhizal fungal species on net photosynthetic rates, intercellular CO2 concentrations, stomatal conductance, andtranspiration rates. Data (means ± SE, n = 4) followed by different letters indicate significant differences between AMF treatments at P < 0.05. Eighty seedlingsfrom each treatment were randomly selected to detect gas exchange parameters. Abbreviations: Control, non-AMF treatment; A. scrobiculata, Acaulosporascrobiculata; F. mosseae, Funneliformis mosseae; R. intraradices, Rhizophagus intraradices.

Effects of AMF Colonization on LeafQuality VariablesLeaf quality including amino acids, total N, soluble sugarand protein, fatty acid of mulberry plants were significantlyimproved by AMF colonization (Table 1; Figure 6). Significantlyhigher concentrations of leaf individual or subtotal essentialamino acids, histidine, proline, and total amino acids rankedas F. mosseae ≥ A. scrobiculata > R. intraradices (Table 1).Significantly higher leaf total N concentrations and solubleprotein among AMF colonized mulberry plants ranked asF. mosseae > A. scrobiculata > R. intraradices (Figure 6).Significantly higher leaf soluble sugar among AMF colonizedmulberry plants ranked as A. scrobiculata > R. intraradices ≈F. mosseae, while significantly higher leaf fattyacid concentrations ranked as A. scrobiculata > F. mosseae >R. intraradices (Figure 6). In addition, significantly higherleaf moisture among AMF colonized mulberry plantspatterned as F. mosseae (80.56 ± 0.36%) > A. scrobiculata(79.11± 0.62%) > R. intraradices (76.19± 0.41%).

Correlations between leaf biomass production and leafquality were showed in Figure 7. Leaf biomass production wassignificantly related to leaf total nitrogen, soluble protein or

soluble sugar (R2= 0.94–0.98, P < 0.05) (Figures 7A,C,D).

Meanwhile, leaf total nitrogen concentrations were significantlyrelated to soluble protein or soluble sugar (R2

= 0.78–0.93,P < 0.05) (Figures 7E,G). In contrast, either leaf biomass or leaftotal nitrogen was not significantly related to total amino acids(R2= 0.75–0.78, P > 0.05, Figures 7B,F).

DISCUSSION

AMF Significantly Promotes Growth ofMulberry PlantsIn this study, the three AMF species showed different effectson growth of shoot and root in mulberry. Overall F. mosseaecolonized mulberry seedlings showed the best potential andconsistently performed better in respect to plant growthcharacteristics than other two AMF species of A. scrobiculataand R. intraradices (Figures 2 and 3). However, Lu et al. (2015)found that R. intraradices was more efficient than F. mosseaein colonizing mulberry roots under greenhouse conditions.Meanwhile, R. fasciculatus was more efficient than Glomusetunicatum (now Claroideoglomus etunicatum) and F. mosseae

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Shi et al. AMF Governs Production of Mulberry

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e.

to infect mulberry roots under rainfed lateritic soil conditions(Setua et al., 1999b). As a result, such differences of AMFfunctioning might be related to AMF species and abiotic factors.In general, the successfully competitive establishment of AMFcolonization not only depended on the indigenous mycorrhizalspecies, but also on the introduced species (Sharma et al., 2005)and its placement in the soil (Hepper et al., 1988). It is well-known that AMF could facilitated mulberry P uptake (Setuaet al., 1999a,b) and other macro- and micro-elements uptake(Baqual and Das, 2006; Lu et al., 2015). The uptake functioncould reduce 75% cost of phosphate fertilization under low soil Plevels (Katiyar et al., 1995). Among nine AMF tested (Acaulosporalaevis, G. mosseae, Gigaspora margarita, Glomus caledonicum, G.fasciculatum, G. leptotichum, G. macrocarpum, R. intraradices,and Scutellospora calospora), G. leptotichum is the best AMsymbiont for 50-day-old nursery teak (Tectona grandis) seedlingsin terms of P, Zn (zinc), and Cu (copper) nutrition and plantgrowth (Rajan et al., 2000). The improved nutritional status inmycorrhizal plants contributed to an enhanced plant biomassproduction (Treseder, 2013; Taylor et al., 2014), resistances ofroot disease (Al-Askar and Rashad, 2010), salt (Kashyap et al.,2004) and drought (Tang et al., 2013). Moreover, an enhancedroot system by mycorrhization could greatly increase theabsorption surface and thus nutrient uptake capacity (Figure 3).As a result, both AMF species and cultivation managementshould be taken into account in mulberry plantations.

AMF Significantly Promotes thePhotosynthetic Capacity of MulberryLeaf Photosynthetic Pigments Are Increased by AMFand Vary with AMF SpeciesSignificantly higher leaf chlorophyll and carotenoid, andphotosynthetic rate displayed in the three AMF mulberry plants,and F. mosseae was the best symbiotic associate with the hostmulberry (Figures 1 and 4). Higher leaf chlorophyll with higherphotosynthetic rates under AM associations made it possibleto have higher carbon fixation and carbohydrate accumulation(Figures 4–6). Concurrently, AMF colonization also enhancedsignificantly higher carotenoid accumulations, leading to betterleaf nutraceutical values of mulberry plants. Leaf carotenoidof mulberry not only acted as a growth-promoting factor forthe silkworm larvae (Shimizu et al., 1981), but also played animportant role in the larvae’s and adult’s phototactic responseand visual sensitivity (Shimizu and Kato, 1978). Meanwhile,the pigmentation development and brilliant color of cocoonswere mainly depended on the carotenoid concentration in themulberry leaves (Tabunoki et al., 2004). Studies also showed thatAMF colonization could result in higher concentrations of bothleaf chlorophyll and carotenoid in lettuces (Baslam et al., 2013).As a result, the application of AMF species could benefit notonly the growth of mulberry, but also the growth and quality ofsilkworms.

Leaf Gas Exchange Capacity Is Enhanced by AMFThe increment of photosynthetic pigments might be due to aneffective synergism between transpiration and photosynthesisunder mycorrhization (Sheng et al., 2008; Borde et al., 2011).

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FIGURE 6 | Effects of mycorrhizal fungal species on leaf total nitrogen (mg g−1 DW), total amino acid (mg g−1 DW), soluble protein and sugar (mgg−1 FW) and fatty acid (mg g−1 DW) of mulberry plants. Data (means ± SE, n = 4) followed by different letters indicate significant differences between AMFtreatments at P < 0.05. The fifth expanded leaf of each plant were used to detect leaf quality. Forty leaves (either fresh or dried) from each treatment were used forthe analysis. Abbreviations: Control, non-AMF treatment; A. scrobiculata, Acaulospora scrobiculata; F. mosseae, Funneliformis mosseae; R. intraradices,Rhizophagus intraradices.

FIGURE 7 | Relationships between leaf biomass production and soluble sugar (A), total amino acid (B), soluble protein (C) or leaf total nitrogen (D); orbetween total nitrogen and soluble protein (E), total amino acid (F) or soluble sugar concentration (G) in mulberry plants. Abbreviations: Control,non-AMF treatment; As, Acaulospora scrobiculata; Fm, Funneliformis mosseae; Ri, Rhizophagus intraradices. ∗∗ P < 0.05.

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In the present study, photosynthesis, transpiration, and stomatalconductance were significantly greater but intercellular CO2 wassignificantly lower under AMF-inoculated than under non-AMFinoculated mulberry plants. These results from mulberry wereconsistent with those from maize and alfalfa (Medicago sativaL.), regardless of water and salt stress conditions (Zhu et al.,2012; Campanelli et al., 2013), and even under karst rockydesertification area (Chen et al., 2014). The greater stomatalconductance of AM plants implied a lower leaf resistance tomoisture diffusion, and ultimately leading to a faster watertransportation (Talaat and Shawky, 2014). In addition, anincreased leaf area of AMF colonized plants was concomitantwith an increase in photosynthetic rate and transpiration rate(Figure 2B vs. Figure 5). Meanwhile, a potential increaseof leaf surfaces with an increase of leaf numbers undermycorrhization could promote plant sunlight capture and thusbetter photosynthetic production (Adolfsson et al., 2015).

AMF Significantly Enhances Leaf Qualityof MulberryAMF Inoculation Accelerates CarbohydrateFormation of Mulberry PlantsBombyx mori L. requires essential amino acids, proteins, sugars,and fatty acid from mulberry leaves for its normal growth and silkproduction (Sengupta et al., 1972). We observed that an increaseof leaf soluble sugar in AMF inoculated mulberry plants withhigher photosynthetic rates. This observation was consistent withresults from other studies, i.e., the inoculation with F. mosseaeand R. intraradices did stimulate soluble sugar production inPoncirus trifoliate L. (Wu et al., 2011), Cyclobalanopsis glauca(Zhang et al., 2014), and Jatropha curcas L. (Kumar et al.,2015). Meanwhile, when leaf moisture was improved and stomasopened wider in mycorrhizal mulberry leaves, greater CO2fixation was then enhanced and carbohydrate accumulation wasultimately increased (Augé et al., 1987). In addition, solublesugar is a precursor of carotenoids, and an enhanced solublesugar production could hence increase the carotenoid production(Figures 4 and 6; Baslam et al., 2011). Moreover, significantlygreater ingestion, digestibility and consumption index wereobserved in silkworms fed with 80–85% high moisture leavesthan with 55–60% low moisture leaves (Rahmathulla et al., 2004).As a result, a higher range of 76–81% leaf moisture underAMF inoculation certified that AMF colonization could improveleaf nutritional quality of mulberry plants. Consequently, thepalatability of silkworm to mulberry leaves could be alsoimproved.

AMF Inoculation Accelerates Nitrogen Metabolism ofMulberry PlantsThe quantity and quality of cocoon shell closely related withconcentrations of mulberry leaf N and amino acids, especiallymethionine, histidine, and threonine. For instance, methionineand histidine are essential for the growth of silkworms,and threonine is crucial for the synthesis of silk protein(Machii and Katagiri, 1991). Our results showed that AMFcolonization generally significantly increased total nitrogen,

soluble protein, total amino acid, and all seven essential aminoacids including methionine, histidine, and threonine, particularlyunder F. mosseae and A. scrobiculata (Figure 6; Table 1).Pentón et al. (2014) reported that inoculation with Glomuscubense improved soil N extraction capacity of mulberry andcomplemented with chemical fertilization. Singh et al. (2010)showed that leaf protein and total amino acid in tea treewere increased by AMF inoculation with an indigenous AMFconsortia containing nine AMF species from three genera ofAcaulospora, Funneliformis, and Glomus. Govindarajulu et al.(2005) revealed that inorganic N taken up by the AM funguswas incorporated into amino acids and N transportation wasfrom extraradical mycelium to intraradical mycelium and then toplants, which would be a great boost to the nutrition compositionand growth of host plants (Azcón et al., 1992). Azcón et al. (1996)also claimed that mycorrhizal colonization increased activityof nitrate reductase and glutamine synthetase involving in Nassimilation of the host plant. Therefore, further studies onnitrogen, perhaps also phosphorus nutrition, are guaranteed ifmulberry plants could positively response to genetic differentAMF species by improving the growth and quality of mulberryhosts.

CONCLUSION

Our results showed that inoculation with AM fungi hadimproved mulberry leaf biomass production and nutritionalquality through an enhanced photosynthesis and growthperformance. A significantly higher rank of such positiveresponses of mulberry to AMF species was as follows:Funneliformis mosseae > A. scrobiculata > R. intraradices forplant’s physiological and growth characteristics, and F. mosseae≈A. scrobiculata > R. intraradices for leaf quality. Suchimprovements were relevant to the AMF-induced alterationsof leaf carbohydrates and N-containing compounds, essentialamino acids and soluble protein in particular. As a result,application of Funneliformis mosseae or A. scrobiculata inmulberry plantation could be a promising management strategyto promote silkworm cultivation and relevant textile industry insouthwest China.

AUTHOR CONTRIBUTIONS

X-HY and X-HH conceived and designed the experiments;S-MS, KC, YG, and BL performed the experiments. X-ZH wasresponsible for the field experiment and provided fund support.G-XL analyzed amino acid data with the help of L-QZ. S-MS andKC wrote the paper. All authors approved the final manuscript.

ACKNOWLEDGMENTS

This study was supported by the earmarked fund for ModernAgro-industry Technology Research System (CARS-22-ZJ0503)and a Postgraduate Education Teaching Reform Project inChongqing (yjg143028), China.

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Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.

Copyright © 2016 Shi, Chen, Gao, Liu, Yang, Huang, Liu, Zhu and He. Thisis an open-access article distributed under the terms of the Creative CommonsAttribution License (CC BY). The use, distribution or reproduction in other forumsis permitted, provided the original author(s) or licensor are credited and that theoriginal publication in this journal is cited, in accordance with accepted academicpractice. No use, distribution or reproduction is permitted which does not complywith these terms.

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