Effects of combined inoculation with Rhizophagus intraradices and Paenibacillus mucilaginosus on plant growth, root morphology, and physiological status of trifoliate orange (Poncirus trifoliata L. Raf.) seedlings under different levels of phosphorus

Scientia Horticulturae 205 (2016) 97–105

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Effects of combined inoculation with Rhizophagus intraradices and Paenibacillus mucilaginosus on plant growth, root morphology, and physiological status of trifoliate orange (Poncirus trifoliata L. Raf.) seedlings under different levels of phosphorus Peng Wang a,b,∗ , Shao-Hui Wu a , Ming-Xia Wen a , Yin Wang a , Qiang-Sheng Wu c a

Institute of Citrus Research, Zhejiang Academy of Agricultural Sciences, Taizhou 318026, China National Center for Citrus Variety Improvement, Zhejiang Branches, Taizhou 318026, China c Institute of Root Biology, Yangtze University, Jingzhou, 434025, China b

a r t i c l e

i n f o

Article history: Received 16 February 2016 Received in revised form 11 April 2016 Accepted 20 April 2016 Keywords: Poncirus trifoliata Arbuscular mycorrhizal fungi Plant growth promoting rhizobacteria Combined inoculation Phosphorus deficiency

a b s t r a c t The effects of combined inoculation with arbuscular mycorrhizal fungi (AMF) and plant growthpromoting rhizobacteria (PGPR) on the growth of citrus seedlings under phosphorus deficient conditions have not been extensively studied. A pot experiment was performed to compare growth, root morphology, and other physiological variables in trifoliate orange (Poncirus trifoliata L. Raf.) seedlings that had been inoculated with AMF (Rhizophagus intraradices, Ri), PGPR (Paenibacillus mucilaginosus, Pm), or both, under three phosphate fertilizer conditions by adding 0.00, 73.41, and 220.23 mg kg−1 Ca3 (PO4 )2 (P0, P1, and P3, respectively) in a low available phosphorus (Olsen-P 4.96 mg kg−1 ) culture medium. Plant growth and nitrogen and phosphorus uptake were significantly increased by inoculation with Ri and Pm at three phosphorus levels, and combined inoculation yielded the highest efficacy, particularly at P0 level. Furthermore, combined inoculation at each phosphorus level significantly increased the colonization ratios of root length with mycorrhizal hyphae and arbuscules, rhizospheric hyphal length, and Pm populations. Root morphology traits like projected area, total volume, and total root length were also considerably improved by inoculation with Ri and Pm; however, taproot length was notably reduced by mycorrhizal inoculation. At P0 level, seedlings inoculated with a combination of Ri and Pm yielded the greatest leaf chlorophyll concentrations and fine root activity, in comparison to those had either not been inoculated at all, or inoculated with just one of them. Although malondialdehyde contents and anti-oxidative enzymes activities increased in non-inoculated seedlings at P0 level, they were significantly reduced by inoculation, particularly with Ri. At P2 level, mycorrhizal seedlings exhibited markedly higher levels of anti-oxidative enzyme activity than non-mycorrhizal seedlings. Therefore, our results suggested that the efficacy of Ri inoculation was superior to that of Pm inoculation. Furthermore, combined inoculation yielded additive effects on the growth of trifoliate orange seedlings under conditions of phosphorus deficiency. © 2016 Published by Elsevier B.V.

1. Introduction Citrus fruits are among the most economically important fruit tree crops in countries with subtropical and tropical climates. In China, most citrus trees are cultivated in mountainous areas and are frequently challenged by several stresses, such as nutritional deficiencies, drought, and extremes in temperature. Among these

∗ Corresponding author at: Yushanping, Toutuo Town, Huangyan District, Taizhou City, Zhejiang Province, 318026, China. E-mail address: peter [email protected] (P. Wang). http://dx.doi.org/10.1016/j.scienta.2016.04.023 0304-4238/© 2016 Published by Elsevier B.V.

challenges, low-phosphorus (P) stress has been a major constraint to acquiring high yield and superior quality in citrus production (Quaggio et al., 2002). The application of phosphate fertilizers can temporarily alleviate low P stress in citrus. However, use of such fertilizers can also increase production costs and environmental pollution owing to the rapid accumulation of phosphates in soil. As the promotion of low-input agricultural practices has increased over the last few decades, it is important to enhance the efficiency of citrus trees for utilizing phosphorus that has been abundantly deposited in soils. Microbes play an important role in the acquisition and transfer of nutrients in soil (Richardson, 2001). Therefore, the utilization of soil microbes to activate minerals and enhance

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P. Wang et al. / Scientia Horticulturae 205 (2016) 97–105

nutrient uptake in plants has attracted increasing attention in sustainable agriculture (Fayez and Mahmoud, 2006). Arbuscular mycorrhizal fungi (AMF) (which establish mutualistic relationships with the root systems of more than 80% of all terrestrial plants, including citrus species) are able to promote host plant growth by increasing uptake of mineral nutrients, particularly phosphate; improving water status; and conferring protection against other microbial pathogens (Smith and Read, 2008; Wehner et al., 2010). The positive effects of inoculation with AMF on citrus growth have been reported in various studies (Shrestha et al., 1995; Wu et al., 2011, 2013). Generally, it has been accepted that the extensive arbuscular mycorrhizal (AM) mycelial network formed around the roots of host plants can increase the area of absorption and efficiently translocate nutrients via hyphae, under conditions of nutrient deficiency (Smith and Read, 2008). Paenibacillus mucilaginosus is considered an efficient plant growth-promoting rhizobacterium (PGPR) and is widely used as a bio-fertilizer in agriculture (Lu et al., 2014). In addition, it can degrade insoluble soil minerals to release water-soluble P and potassium (K) nutrients (Aleksandrov et al., 1967), and produce plant growth regulators, such as indole acetic acid (Goswami et al., 2015). Since the 1990s, tobacco, tomato, groundnut, and Sudangrass have been grown successfully using Pm preparations (Basak and Biswas 2009; Li et al., 2007; Sugumaran and Janarthanam, 2007). However, information concerning the effects of Pm inoculation on citrus growth is lacking. Because of their synergistic roles in promoting crop growth, combined inoculation using AMF and PGPR has viable potential for agroecosystems (Lambers et al., 2009). Many studies have demonstrated that combined inoculation with beneficial soil microbes can improve crop growth to a much greater extent than individual inoculation can (Barea et al., 2005). Misbahuzzaman and Newton (2006) reported that dual inoculation with Glomus clarum and Pisolithus tinctorius could contribute to a significantly higher total dry mass in Eucalyptus camaldulensis Dehnh. seedlings, when compared to a single treatment. Likewise, combined inoculation with AMF and Thiobacillus sp. yielded significantly highest values of nitrogen (N), P, K and sulfur (S) concentrations in the rhizosphere of onion (Allium cepa L.) and maize (Zea mays L.) (Mohamed et al., 2014). Han and Supanjani Lee (2006) found that dual inoculation with phosphate- and potassium-solubilizing bacteria, Bacillus megaterium var. phosphaticum and P. mucilaginosus, respectively, yielded not only consistently higher P and K availability in soil, but increased growth in pepper (Capsicum annum L.) and cucumber (Cucumis sativus L.) plants. Kapoor and Singh (1998) also reported that both the grain and straw yield of mungbean presented the most significant increases following inoculation with a combination of phosphate solubilizing microorganisms (PSM, Bacillus circulans and Cladosporium herbarum) and AMF (Glomus fasciculatum). In addition, PSM populations and AM levels generally increased after combined inoculation. These findings suggest that the practice of combined inoculation with soil microbes that possess various beneficial properties should be adopted for sustainable agricultural production. Nevertheless, little is known about the effects of combined inoculation of AMF and PGPR on the growth of citrus. The objective of the present work study therefore was to examine the effects of dual inoculation with AMF and PGPR on citrus rootstock seedlings grown under different levels of phosphate fertilizer in a green house.

2. Material and methods 2.1. Plants and growth medium Seeds of trifoliate orange (Poncirus trifoliata L. Raf.) plants were obtained from a citrus germplasm resource garden, Zhejiang Insti-

tute of Citrus Research, Taizhou, China. Soil samples from the topsoil layer of citrus orchards at the Zhejiang Institute of Citrus Research and sand samples from the riverbank of Toutuo town, Taizhou, respectively were collected. Fine sands (<2 mm in diameter) were washed thoroughly with tap water. Soil and sand (1:2, v/v) samples were mixed well, sterilized at 121 ◦ C for 2 h in an oven. The chemical characteristics of the autoclaved media were pH 6.7, 5.1 g kg−1 soil organic matter, 4.96 mg kg−1 Olsen-P, 34.82 mg kg−1 alkali hydrolysable N, and 60.43 mg kg−1 available K, which maintained relatively low soil nutrient levels. 2.2. Microorganism inoculums Native spores of Rhizophagus intraradices (Ri) were isolated from the soil of a citrus orchard soil in southern China (Wang et al., 2013), and propagated in pots containing sterilized sand, with Sorghum vulgare as the host plant. After 16 weeks, the infected root systems and growth medium were harvested to obtain the AM fungal inoculum. The growth medium contained about 13 spores g−1 , as determined by the wet sieving and decanting method (Gerdemann and Nicolson, 1963). For the PGPR strain of interest, we used P. mucilaginosus (Pm, Agricultural Culture Collection of China). The rhizobacteria strain maintained the induced streptomycin (150 mg/ml) resistant phenotypes during several successive subculturing transfers in selective liquid medium (1% sucrose, 0.02% yeast extract, 0.05% (NH4 )2 SO4 , 0.1% MgSO4 ·7H2 O, 0.01% KCl, 0.05% Na2 HPO4 ·12H2 O, 0.3% powder of rock phosphate powder, pH 7.0) and did not differ from their parent strains in growth rate. And then, the strain was suspended in enrichment medium (0.5% beef extract, 1% peptone, 0.1% NaCl, 0.1% MgSO4 ·7H2 O, 0.05% K2 HPO4 ·3H2 O, and 0.01% CaCO3 , pH 7.0) on a shaker at 180 rpm for 36 h at 30 ◦ C. Cell suspensions were adjusted to 109 cfu mL−1 and used as a standard inoculum. 2.3. Seed germination, planting, and inoculation Seeds of trifoliate orange were surfaced-sterilized with 70% alcohol for 15 min and placed on sterilized and moist filter paper for germination in the dark at 28 ◦ C. After 20 d, a part of seedlings were dipped in the bacterial suspensions for 30 min. Before planting, the AM fungal inoculum (40 g) was firstly placed at 5 cm below the surface of the growth medium in a pot, and 5 mL of bacterial suspension was then inoculated into a digged hole for each seedling. The non-AMF pots received the same amount of autoclaved AM fungal inoculum plus 2 mL filtrate (sieved through 25 ␮m) of AM fungal inoculum per root hole, and the non-PGPR pots were treated with 5 mL of autoclaved bacterial suspension per root hole, after which, five uniform seedlings were selected and planted in the pot containing 3.0 kg of autoclaved growth medium. Seedlings were acclimatized for 2 weeks with daily administration of 100 mL distilled water per pot at 25 ◦ C. 2.4. Experimental design Greenhouse assays were conducted at the Zhejiang Institute of Citrus Research between the months of March and August 2014, to evaluate the effects of the microorganisms on plant growth. The experiment consisted of a randomized block design with two factors: four inoculation treatments (Ri, Ri + Pm, Pm, and noninoculated) and three phosphate fertilizer levels by adding 0.00 (P0), 73.41 (P1), and 220.23 (P3) mg kg−1 Ca3 (PO4 )2 in the culture medium, respectively. Three replications per treatment were performed, leading to total 36 pots. Each pot was periodically watered with distilled water and received a total of 2000 mL (P-free, pH = 6.0) nutrient solution (Hoagland and Aron, 1938) to maintain

P. Wang et al. / Scientia Horticulturae 205 (2016) 97–105

the soil moisture at approximate field capacity during seedlings growth stage.

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activities of SOD, G-POD, and CAT were measured according to the methods described by Giannopolitis and Ries (1977), Li (2000), and Cakmak and Marschner (1992), respectively.

2.5. Plant growth, nutrition and root morphology measurements 2.8. Statistical analysis Plants were harvested after 180 days of inoculation, and plant height, stem diameter, and leaf numbers were recorded. Shoots were separated from the roots, and the fresh roots were washed free of all media, and scanned by the Microtek ScanMaker i800 Plus system (WSeen, China). Root morphological traits, including projected area, surface area, total volume, average diameter, and taproot and total root lengths were all derived using the with LA-S Root System Analysis software (WSeen, China). After scanning, the fresh shoot and root samples were averagely divided into two parts: one part was oven-dried at 75 ◦ C for 72 h, and then weights were measured; and total N and P in whole plant were analyzed using the methods of Thomas et al. (1967); another was stored at −20 ◦ C before determination of mycorrhizal and physiological status. 2.6. Mycorrhizal status and rhizobacteria Pm population assessments Roots were chopped into 1-cm-long pieces, and fixed in a formalin/acetic acid/ethanol (FAA, 13:5:200, v/v/v) solution for 24 h. AM fungal structures on citrus roots were stained with 0.05% trypan blue in lacto phenol and detected under a compound light microscope (Nikon-i90, Japan), according to Koske and Gemma (1989). The colonization ratios of root length with AM fungal hyphae, arbuscules, and vesicles were quantified, using the magnified intersection method by examination 200 intersections per sample (McGonigle et al., 1990). Spores were extracted from the medium by sucrose centrifugation and the wet sieving technique method and counted with the aid of a stereoscopic microscope (Nikon-30, Japan) (Gerdemann and Nicholson, 1963). Spore density was expressed as the number of spores and sporocarps per gram dry weight of medium. All Hyphae were extracted, stained, and measured as described by Bethlenfalvay and Ames (1987). Hyphal length was determined by the grid-line method with the aid of an ocular micrometer, and expressed as the length of hyphae per gram of dry weight of medium. Rhizobacteria Pm survival was tested in serial dilutions of rhizospheric soils. Briefly, Aliquots of soil (1.00 g) were homogenized with 9 mL sterilized distilled water, and shaken for 30 min. The suspension was grown on a selective solid medium at 28 ◦ C for 5–7 d, and single colonies with typical Pm properties were enumerated. 2.7. Physiological status determination Leaf chlorophyll (Chl) was extracted from 0.2 g of fresh leaves in 25 mL 80% (v/v) ethanol in the dark, at 25 ◦ C. Chl a and b was detected at 649 and 665 nm, respectively, with a UV-2800 spectrophotometer (Unico, Shanghai, China), and calculated according to the method of Wintermans and Mots (1965). Fine-root activity was evaluated by the modified triphenyl tetrazolium chloride (TTC) method, as described by Ruf and Brunner (2003). Malondialdehyde (MDA) was extracted from 0.4 g of frozen leaves or roots homogenized at 4 ◦ C in 5 mL 10% (w/v) trichloroacetic acid (TCA) and centrifuged at 4000g for 10 min. MDA contents were then determined by the thiobarbituric acid reaction, as described by Rhee and Watts (1966). Soluble protein, superoxide dismutase (SOD), guaiacol peroxidase (G-POD), and catalase (CAT) were extracted from 0.25 g of frozen leaves or roots at 4 ◦ C in 5 mL 0.1 M phosphate buffer (pH 7.8) containing 1% (w/v) polyvinylpyrrolidone (PVP), and centrifuged at 4000g for 15 min. The resulting supernatant used for assays. Soluble protein was evaluated according to the method of Bradford (1976), using bovine serum albumin as the standard. The

Data were subjected to ANOVA and means were compared with the least significant difference (LSD) test at the 0.05 level, using the SAS 8.1 software (SAS Institute Inc., Cary, NC, USA). Mycorrhizal percentage colonization and bacterial counts were transformed by acrsin (x1/2 ) and by log (x + 1), respectively, prior to statistical analysis. 3. Results 3.1. Inoculum establishment and mycorrhizal properties Mycorrhizal structures were not observed in the non-AMF treated seedling roots. In comparison to individual inoculation with Ri at each P level, particularly at P0 and P1 levels, combined inoculation promoted AM fungal colonization, as evidenced by greater hyphae and arbuscules conlonization in root. However, vesicles colonization showed the opposite trend at all P levels. With the increasing Ca3 (PO4 )2 levels, hyphae and arbuscules conlonization rates remarkably decreased in both individual and combined inoculation treatments, and ranged from 58.93% to 21.96% and 45.31% to 20.50%, respectively. Vesicles colonization yielded a maximum value of 33.80% in individual inoculation with Ri treatment at P1 level. Likewise, the spores and hyphae of AM fungal propagules in rhizosphere exhibited the similar status with the hyphae and arbuscules conlonization in root. The population of Pm significantly increased with combined inoculation at all P levels, and reached the highest number 16.3 × 103 cfu g−1 soil at P1 level (Table 1). The Multi-way ANOVA (MANOVA) showed that Pm inoculation and P addition could significantly influence the AM fungal colonization and propagules numbers excluding spore. However, the effects of interaction between Pm and P on the AM status were not remarkable. Similarly, Ri and P significantly affected Pm number, but no marked effects on Pm number were noted as a result of the interaction between Ri and P (Table 1). 3.2. Plant growth and nutrients uptake The growth of trifoliate orange seedlings showed improvement in all P levels, following inoculation with Ri, Pm, or both, In comparison to the corresponding control at each P level, the combined inoculation consistently presented the greatest positive effects. At P0 level, combined inoculation increased plant height, stem diameter, shoot dry weight, and root dry weight by 99.83%, 44.73%, 102.99%, and 80.31%, respectively. At P1 level, the same parameters were increased by 71.65%, 20.86%, 73.25%, and 53.70%, respectively; and at P2 level, by 36.10%, 16.14%, 37.52%, and 32.93%, respectively. Individual Ri inoculations exhibited increases in plant height, stem diameter, shoot and root dry weights by 83.89%, 38.75%, 81.96%, and 67.72, respectively at P0 level; 57.92%, 15.23%, 58.91%, and 46.36%, respectively, at P1 level; and 16.42%, 7.72%, 24.15%, and 22.37%, respectively, at P2 level. Individual Pm inoculations yielded increases in the same parameters by 48.18%, 30.06%, 44.80%, and 35.25%, respectively, at P0 level; 24.94%, 8.83%, 20.79%, and 18.46%, respectively, at P1 level; and 1.75%, 3.86%, 1.99%, and 3.25%, respectively, at P2 level (Table 2). In addition, total N and P concentrations and uptake in seedlings were substantially improved both by individual and combined inoculation with Ri and Pm at all P levels. Combined inoculation likewise worked best in promoting total N and P uptake. Compared with corresponding non-inoculation, dual inoculation resulted in a 2.72 and 2.37 fold increase, respectively,

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Table 1 AM fungal properties (with respect to colonization rate, spore density, and hyphae length) and PGPR survival numbers in the rhizosphere of trifoliate orange seedlings grown under three phosphorus conditions. P level

Inoculation treatment

Colonization rate (%) Hyphae

Arbuscules

Vesicles

Spore density(No. g−1 )

Hypal length(m g−1 )

Pm number(cfu g−1 soil)

P0

CK Pm Ri Ri + Pm

– – 50.64b 58.93a

– – 38.28b 45.31a

– – 26.13b 22.77b

– – 7.31b 5.37c

– – 0.34c 0.43b

– 7.3c – 13.3ab

P1

CK Pm Ri Ri + Pm

– – 40.80d 51.04c

– – 30.68c 38.57b

– – 33.80a 24.04b

– – 7.73a 6.62b

– – 0.46b 0.55a

– 10.3bc – 16.3a

P2

CK Pm Ri Ri + Pm

– – 21.96e 29.49d

– – 20.50d 21.85d

– – 16.29c 10.49d

– – 4.56d 4.39cd

– – 0.31c 0.41b

– 11.7b – 15.7a

– ** ** – NS

– ** ** – NS

– ** ** – NS

– ** ** – *

– ** ** – NS

** – * NS –

Significance Ri Pm P Ri × P Pm × P

Different letters within a given column indicate significant differences (P < 0.05) between treatments using the LSD test. “—”, not exist; NS, not significant; *, P < 0.05; **, P < 0.01. P, phosphorus; CK, control check; Pm, Paenibacillus mucilaginosus; Ri, Rhizophagus intraradices.

at P0 level; a 2.17 and 1.93 fold increase, respectively, at P1 level; and a 1.60 and 1.53 fold increase, respectively, at P2 level (Fig. 1). The efficacy of inoculation gradually declined with the increasing Ca3 (PO4 )2 levels, and the positive effects of individual Ri inoculation were superior to those of individual Pm inoculation at all P levels. Unlike the above growth parameters, the ratio of root/shoot revealed an inverse trend. Compared with corresponding control group, the ratio was distinctly reduced by inoculation, significantly by dual inoculation at P0 and P1 levels (Table 2). The MANOVA showed that Ri, Pm, and P all remarkably affected the plant growth traits. Significant interactions between Ri and P were noted for the root/shoot ratio; between Pm and P for root/shoot

ratio; and among Ri, Pm, and P for plant height and stem diameter (Table 2). 3.3. Plant root morphological properties In comparison to corresponding control group, the root projected area, surface area, total volume, and total length of the seedlings were all enhanced by individual and dual inoculation with Ri and Pm at three P levels. Furthermore, the values for these parameters were ranked as follows: Ri + Pm > Ri > Pm. However, the taproot length of the seedlings was significantly reduced by inoculation with Ri and Ri + Pm. The average root diameter did not change significantly across all treatments. With the increasing lev-

Table 2 Effects of individual and dual inoculation with AMF and PGPR on plant height, stem diameter, dry weight, and root/shoot ratio of trifoliate orange seedlings grown under three phosphorus levels. Plevel

Inoculationtreatment

Plant height(cm plant−1 )

Stem diameter(mm plant−1 )

Dry weight (g plant−1 ) Shoot

Root

Root/Shoot

P0

CK Pm Ri Ri + Pm

17.55f 26.00e 32.27d 35.06cd

2.34g 3.04ef 3.25def 3.39cd

0.40e 0.58d 0.73c 0.81c

0.37f 0.50e 0.62cd 0.67c

0.93a 0.86b 0.85bc 0.82d

P1

CK Pm Ri Ri + Pm

26.49e 33.09cd 41.83b 45.46ab

3.02f 3.29de 3.48bcd 3.65ab

0.62d 0.74c 0.98b 1.07ab

0.54ed 0.64c 0.79b 0.83ab

0.88b 0.86b 0.81de 0.78f

P2

CK Pm Ri Ri + Pm

36.24cd 36.87c 42.19b 49.32a

3.28cd 3.41bcd 3.54bc 3.81a

0.82c 0.83c 1.02ab 1.13a

0.67c 0.69c 0.82ab 0.89a

0.82de 0.82cd 0.80def 0.79ef

** ** ** NS NS NS *

** ** ** NS NS NS *

** ** ** NS NS NS NS

** ** ** NS NS NS NS

** ** ** NS * * NS

Significance Ri Pm P Ri × Pm Ri × P Pm × P Ri × Pm × P

Different letters within a given column indicate significant differences (P < 0.05) between treatments using the LSD test. NS, not significant; *, P < 0.05; **, P < 0.01. P, phosphorus; CK, control check; Pm, Paenibacillus mucilaginosus; Ri, Rhizophagus intraradices.

P. Wang et al. / Scientia Horticulturae 205 (2016) 97–105

Ri

A

90.0

a

-1

ef gh

fg

h

i 25.0 20.0 15.0 10.0

1.4 1.2

ef

gh

bcd

fg

def

cde

ab

c

cd

cd

i

1.0 0.8 0.6 0.4

c

d e

e

20.0

f

D

a b

b

3.0

h

ab

30.0

3.5 a

a

40.0

0.0

abc

Ri+Pm

b

50.0

10.0

ab

Ri

a

60.0

0.0

B

Pm

70.0

5.0

1.6

CK

C

80.0

ab

cde

def

Total N uptake (mg plant )

35.0

ab

abc

bcd

30.0

Ri+Pm

c

-1

-1

Pm

40.0

1.8 P concentration in whole plant (mg g )

CK

Total P uptake (mg plant )

-1

N concentration in whole plant (mg g )

45.0

101

2.5

d

d

g

g

1.5 1.0

de ef

f

2.0

h

0.5

0.2

0.0

0.0 P0

P1

P0

P2

P1

P2

Fig. 1. Total N and P concentrations and uptake in trifoliate orange seedlings subjected to individual and dual inoculation with AMF and PGPR under three phosphorus conditions. Different letters indicate significant differences (P < 0.05). P, phosphorus; CK, control check; Pm, Paenibacillus mucilaginosus; Ri, Rhizophagus intraradices.

els of Ca3 (PO4 )2 , the root projected area, surface area, total volume, and taproot and total lengths were considerably increased in noninoculated seedlings (Table 3). Statistical analysis revealed that Ri, Pm, and P all significantly influenced the root projected area, surface area, total volume, and total length, but did not notably affected the average diameter of the seedlings. The taproot length was markedly impacted by Ri and P, but not by Pm The average diameter was significantly influenced by the interaction between

Ri and P. No significant effects were observed as a result of interactions between Ri and Pm, or Pm and P, on plant root morphological traits. However, with the exception of projected area and taproot length, significant interactions were noted in other root morphological traits between Ri and P; and in root volume as a result of the interactions among Ri, Pm, and P (Table 3).

Table 3 Effects of individual or combined inoculation with AMF and PGPR on root projected area, surface area, volume, taproot and total length, and average diameter of trifoliate orange seedlings grown under three phosphorus conditions. Plevel

Inoculationtreatment

Projected area (cm2 )

Surface area (cm2 )

Volume (cm3 )

Taproot length (cm)

Total length (cm)

Average diameter (mm)

P0

CK Pm Ri Ri + Pm

16.54f 22.91de 23.49de 26.23cd

51.05h 67.53gh 73.02ef 90.01d

1.37i 1.58g 1.61g 1.81e

27.58bc 27.05bcd 21.10e 20.71e

296g 359ef 384ef 446cd

0.79a 0.77ab 0.75b 0.75b

P1

CK Pm Ri Ri + Pm

20.85ef 25.29cd 31.64ab 32.72ab

59.85gh 79.76de 128.68b 148.25a

1.47h 1.69f 2.25c 2.36b

30.73ab 28.47bc 23.81de 23.79de

335fg 415de 558ab 595ab

0.77ab 0.74b 0.77ab 0.77ab

P2

CK Pm Ri Ri + Pm

26.31cd 28.66bc 31.53ab 33.53a

85.24d 110.80c 123.62b 155.48a

1.78e 1.90d 2.21c 2.46a

32.78a 30.66ab 25.49cd 24.99cd

455cd 483c 542b 612a

0.75ab 0.77ab 0.74b 0.77ab

** ** ** NS NS NS NS

** ** ** NS ** NS NS

** ** ** NS ** NS **

** NS ** NS NS NS NS

** ** ** NS ** NS NS

NS NS NS NS * NS NS

Significance Ri Pm P Ri × Pm Ri × P Pm × P Ri × Pm × P

Different letters within a given column indicate significant differences (P < 0.05) between treatments using the LSD test. NS, not significant; *, P < 0.05; **, P < 0.01. P, phosphorus; CK, control check; Pm, Paenibacillus mucilaginosus; Ri, Rhizophagus intraradices.

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Table 4 Effects of individual and dual inoculation with AMF and PGPR on leaf Chlorophyll concentration, root activity, and soluble protein and MDA contents of trifoliate orange seedlings grown under three phosphorus conditions. P level

Inoculation treatment

Leaf Chlorophyll (mg g−1 FW)

Root activity(ug g−1 h−1 FW)

Soluble protein (mg g−1 FW)

MDA (␮mol g−1 FW)

Leaf

Root

Leaf

Root

P0

CK Pm Ri Ri + Pm

1.63g 1.78fg 2.41 cd 2.64b

70.93g 90.50f 128.80d 145.81c

19.71e 35.94d 62.67c 77.86b

7.36h 9.84fg 15.03bc 16.08abc

19.74a 12.65b 4.51d 3.61d

6.97a 4.93b 2.47d 1.77de

P1

CK Pm Ri Ri + Pm

1.84ef 2.26d 2.75ab 2.89a

99.39f 118.99de 161.09b 175.52a

31.10d 55.78c 91.69a 92.08a

9.02gh 11.69ef 18.05a 17.15a

13.64b 8.14c 2.91d 2.65d

4.17bc 3.35c 1.80de 2.03de

P2

CK Pm Ri Ri + Pm

1.98e 2.46c 2.87a 2.86a

113.13e 128.65d 146.59c 142.96c

39.25d 64.58c 90.06a 90.52a

12.43de 14.39cd 16.85ab 17.99a

2.43d 2.73d 2.90d 2.73d

1.73de 2.06de 1.31e 1.52e

** ** ** ** NS NS **

** ** ** NS ** NS NS

** ** ** ** NS NS *

** ** ** * ** NS NS

** ** ** * ** NS NS

** * ** * ** ** NS

Significance Ri Pm P Ri × Pm Ri × P Pm × P Ri × Pm × P

Different letters within a given column indicate significant differences (P < 0.05) between treatments using the LSD test. NS, not significant; *, P < 0.05; **, P < 0.01. P, phosphorus; CK, control check; Pm, Paenibacillus mucilaginosus; Ri, Rhizophagus intraradices.

3.4. Physiological variables The leaf Chl concentration, fine root activity, and soluble protein content were lowest in non-inoculated seedlings at all P levels. At each P level, seedlings inoculated with Ri showed significantly higher levels of leaf Chl concentration, fine root activity and soluble protein content than those inoculated with Pm. MDA contents in both leaves and roots were the highest in non-inoculated seedlings at P0 and P1 levels. Although the leaf and root MDA contents considerably decreased in seedlings inoculated with Pm, they were still significantly higher than those detected in seedlings inoculated with Ri and Ri + Pm at P0 and P1 levels. Nevertheless, no remarkable

variations were observed in MDA contents among all treatments at P2 level (Table 4). The levels of anti-oxidative enzyme activity of SOD, G-POD and CAT in the leaves and roots were significantly higher in the corresponding seedlings grown at P0 level than those grown at P2 level. In comparison to non-inoculated seedlings, inoculation with Ri and Pm significantly reduced the activity of SOD and G-POD at P0 and P1 levels. However, inoculation, particularly with Ri + Pm and single Ri, significantly increased the activity of SOD and G-POD at P2 level. CAT activity in the leaves and roots was always higher in inoculated seedling; especially those inoculated with Ri and Ri + Pm at all P levels (Table 5). Generally, the inoculation with Ri and Pm significantly improved the physiolog-

Table 5 Effects of individual and dual inoculation with AMF and PGPR on SOD, POD, and CAT activities in leaf and root of Poncirus trifoliata seedlings grown under three phosphorus conditions. SOD (U mg−1 protein)

POD (U mg−1 protein)

Leaf

Root

Leaf

Root

Leaf

Root

36.52a 32.17b 24.92c 19.45de

328.48a 287.98b 195.89d 154.62ef

223.79a 189.64b 126.74d 109.07e

2436.29a 2125.54b 1832.96c 1596.55de

32.81f 44.23e 72.64a 64.03b

35.71f 46.23e 97.06a 84.22b

CK Pm Ri Ri + Pm

24.81c 20.04d 16.20f 16.89ef

264.14c 197.31d 164.20e 143.45fg

165.61c 136.94d 94.66ef 100.54ef

2058.48b 1775.90cd 1623.47de 1608.23de

44.78e 47.53e 56.72cd 60.45bc

44.67e 51.89de 59.38cd 65.20c

CK Pm Ri Ri + Pm

10.47h 12.16gh 15.11f 14.41fg

70.21j 93.93i 132.32gh 113.77hi

65.86h 77.33gh 91.93f 86.08fg

1007.91h 1314.05g 1537.33ef 1389.93fg

24.21g 33.45f 57.59cd 53.73d

24.90g 35.62f 58.30cd 56.69cd

** ** ** NS ** ** *

** ** ** NS ** ** **

** ** ** NS ** ** **

** ** ** NS ** ** **

** * ** ** ** NS **

** NS ** ** ** NS *

Plevel

Inoculation treatment

P0

CK Pm Ri Ri + Pm

P1

P2

Significance Ri Pm P Ri × Pm Ri × P Pm × P Ri × Pm × P

CAT (U mg−1 protein)

Different letters within a given column indicate significant differences (P < 0.05) between treatments using the LSD test. NS, not significant; *, P < 0.05; **, P < 0.01. P, phosphorus; CK, control check; Pm, Paenibacillus mucilaginosus; Ri, Rhizophagus intraradices.

P. Wang et al. / Scientia Horticulturae 205 (2016) 97–105

ical properties of trifoliate orange seedlings, such as leaf Chl, root activity and anti-oxidative enzyme activity. In addition, significant interactions among Ri, Pm, and P influenced the activity of antioxidative enzymes SOD,G-POD and CAT, as revealed by MANOVA.

4. Discussions Phosphorus deficiency can restrict plant growth considerably. Trifoliate orange seedlings in the present study exhibited poor performance under lower P condition (P0); however, inoculation with Ri and Pm could yield great improvement, especially for combined inoculation. These findings are consistent with those of ChiquitoContreras et al. (2012) who reported that individual or combined inoculation with AMF and PGPR promotes the growth of Citrus volkameriana and Rangpur lime plants grafted with Tahiti lime that have been 50% fertilized, in comparison to the control group subjected to 100% fertilization in a nursery. However, our results also suggest that the positive effects of mycorrhizal inoculation are reduced with increasing levels of Ca3 (PO4 )2 , indicating that mass application of phosphate fertilizers could weaken mycorrhizal contributions to citrus growth (Hoeksema et al., 2010). In fact, the positive effects of a single application of Ca3 (PO4 )2 on growth at P2 level were not superior to those of combined inoculation with Ri and Pm at P0 level in the present study. Thus, the combined bio-fertilization with Ri and Pm has considerable potential as an alternative to inorganic fertilizers, as these two microbial species can dramatically improve citrus growth without causing damage to the soil environment (Van Der Heijden et al., 2006; Vessey, 2003). In the present study, the positive effects of dual inoculation on growth were considerably more superior to those of individual inoculation with Pm, and slightly superior to those of individual inoculation with Ri. However, the statistical analysis results showed that the interaction effect between Ri and Pm was not significant on some plant growth properties. On one hand, this might be because that Ri and Pm interactive performance was directly or indirectly regulated by the factor P in the present experiment, and indeed the significant interactions among Ri, Pm and P for plant growth and physiology parameters like plant height, stem diameter, root volume, SOD, G-POD, and CAT activities were revealed in our study. On the other hand, this result also implied that Ri and Pm did not suppressed each other’s stimulation efficacy in improving plant growth, thereby combined inoculation could in some extent exhibit synergy, rather than inhibition effect on plant growth, particularly on root length, root activity, and leaf Chl concentration in the present study. In our experiment, the significantly higher leaf Chl concentration in dually inoculated plants might indicate an enhancement in photosynthesis due to inoculation with Ri and Pm (Liu et al., 2015). Several studies have revealed higher Chl contents in mycorrhizal citrus plants than in non-mycorrhizal citrus plants under adverse environmental conditions like iron deficiency (Wang et al., 2007), drought (Wu and Xia, 2006), and high pH (Wang et al., 2008). Actually, in P-limited soils, the extraradical mycelial network of AM possesses higher P capture and translocation efficiency than the absorption system of roots of the host plant (Clark and Zeto, 2000). In addition, higher fine root activity conferred enhanced metabolic activity and absorbing ability of the roots, following inoculation in the present study. These findings could directly or indirectly contribute to the improve host plant photosynthetic capacity under P deficiency condition. In fact, total dry biomass of the seedling was markedly increased by inoculation with Ri and Pm. AMF is known to function as a metabolic sink, causing basipetal mobilization of photosynthate to the roots (Demir, 2004). The enhanced photosynthesis compensates, in part, for the carbon used by the Ri and Pm symbiont. Consequently, carbohydrate accumulation in the root

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system relatively reduced, likely resulting in decrease in the ratio of root biomass/shoot biomass in the present study. Except the host plant photosynthate production and allocation, citrus root morphology was significantly affected by inoculation with Ri and Pm, particularly with Ri in our study. AMF colonization can reportedly alter the morphology of host plant root system in a structural, spatial, quantitative, and temporal manner (Kapoor et al., 2008). In our experiment, the total root length, root volume, and root surface area were remarkably increased by inoculation with Ri and Pm. Furthermore, mycorrhizal trifoliate orange seedlings tend to have more fine roots and less coarse roots (Yao et al., 2009). Generally, mycorrhization enhanced root system morphology (RSM) traits, mainly in terms of the number of higher-order lateral roots, by mediating metabolic levels of plant growth regulators, such as endogenous indole-3-butyric acid and polyamines; however, not all enhancements were necessarily due to improved P nutrition (Berta et al., 1995; Kaldorf and Ludwig-Müller, 2000; Wu et al., 2012). In addition, the taproot length was significantly reduced following mycorrhizal colonization, but was not affected by Pm inoculation in our experiment. This finding might imply that Ri colonization could more efficiently regulate the RSM of trifoliate orange seedlings than Pm inoculation could. The root system of trifoliate orange is generally characterized by coarse roots and sparse feeder roots (Spiegel-Roy and Goldschmidt, 1996). Therefore, improvement in the RSM of citrus seedlings, induced by Ri and Pm inoculation, is of importance in enhancing nutrient uptake. Apart from morphological adaptations, a series of physiological indices such as SOD, G-POD, and CAT, might also contribute to plant resistance in conditions of the low P, since these enzymes could transform reactive oxygen species (ROS) from a more toxic species to less toxic ones under P deficiency (Fu et al., 2014; Siyiannis et al., 2012). In the present study, an increase in lipid peroxidation was noted, as evidenced by the maintenance of MDA contents at relatively higher levels in non-mycorrhizal seedlings at P0 and P1 levels, and accompanying with the enhanced activity of SOD, G-POD, and CAT in leaves and roots serving to alleviate ROS accumulation and protein damage. At P0 level, mycorrhization could lead to lower MDA content and SOD and POD activity than individual inoculation with Pm, which suggests that mycorrhizal seedlings suffered less harm due to ROS accumulation under low P stress. In addition, seedlings inoculated with Ri indeed had significantly higher levels of SOD, G-POD, and CAT activity than those inoculated with Pm at P2 level. However, we also noted that the levels of SOD, G-POD, and CAT activity showed no significant difference between inoculation with Pm and non-inoculation at P2 level. Therefore, we propose Ri inoculation was more efficient than Pm inoculation at relieving the impacts of low P stress on citrus seedlings growth by not only improving mineral nutrition but also positively regulating the activity of anti-oxidative enzymes (Wu and Xia, 2006; Wu et al., 2013). It is reported that some soil bacteria in relation to genera Bacillus, Pseudomonas, and Arthrobacter could stimulate mycelial growth of mycorrhizal fungi and enhance mycorrhizal formation in pot experiments (Lugo et al., 2008; Pivato et al., 2009). Our results also indicated that dual inoculation with Ri and Pm could remarkably mutually enhanced each other’s development and reproduction, and this synergy might play a vital role in assisting host plant to acquire minerals under nutrient limited soils. Sometimes, combination of inoculants will not necessarily produce an additive or synergic effect, but rather a competitive process (Trabelsi and Mhamdi, 2013). These complex changes could be the result of direct effects from trophic competitions or indirect effects mediated by enhanced host plant growth and exudation. In our study, we speculated that a part of Pm maybe adhered on the surface of Ri external mycelium. Thus, Ri could transfer Pm or act as media in the process of spread of Pm along roots where Pm might produce biologically

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active organic molecules such as vitamins or plant grow regulators that stimulated Ri hyphae branching and colonizing (Barea et al., 2005; Gryndler, 2000.). With the extension of Ri external mycelium, Pm could rapidly approach and solve minerals far from plant roots. Consequently, this cooperation significantly increases minerals uptake by AM hyphae and hence the symbiotic host plant (Smith and Read, 2008). As a return, trifoliate orange seedlings needed to afford abundant carbohydrates to maintain the synergic relationships among Plant-AMF-PGPR. However, the dual inoculation also likely produced an inhibitive effect on seedlings growth at the initial colonization stage due to their competition for carbohydrates. Much further work will be focused on these mentioned above in our future study. In conclusion, our results showed that inoculation with Ri and Pm could improve growth, root morphology, and physiological properties, such as leaf chlorophyll, root activity, and levels of antioxidative enzymes, but decreased the MDA contents in trifoliate orange seedlings under conditions of P deficiency, even after inorganic P fertilizer has been added to the soil. Furthermore, we found that the efficacy of Ri inoculation was superior to that of Pm inoculation, and dual inoculation had a better synergistic effect on the growth of trifoliate orange seedlings. Thereby, combined inoculation with AMF and PGPR may be a viable method to mitigate the stress of low P levels in sustainable citrus production. Acknowledgments This work was funded by the National Natural Science Foundation of China (No. 31301737), the General Program of Agricultural Science and Technology of Taizhou city (No. 121KY18), and the Science and Technology Innovation Ability Promotion Project of Zhejiang Academy of Agricultural Sciences (No. 2013R27Y01E01). References Aleksandrov, V., Blagodyr, R., Ilev, I., 1967. Liberation of phosphoric acid from apatite by silicate bacteria. Mikrobiol Zh (Kiev) 29, 111–114. Barea, J.M., Pozo, M.J., Azcón, R., Azcón-Aguilar, C., 2005. Microbial co-operation in the rhizosphere. J. Exp. Bot. 56, 1761–1778. Basak, B., Biswas, D., 2009. Influence of potassium solubilizing microorganism (Bacillus mucilaginosus) and waste mica on potassium uptake dynamics by sudan grass (Sorghum vulgare Pers.) grown under two Alfisols. Plant Soil 317, 235–255. Berta, G., Trotta, A., Fusconi, A., Hooker, J.E., Munro, M., Atkinson, D., Giovannetti, M., Morini, S., Fortuna, P., Tisserant, B., Gianinazzi-Pearson, V., Gianinazzi, S., 1995. Arbuscular mycorrhizal induced changes to plant growth and root system morphology in Prunus Cerasifera. Tree Physiol. 15, 281–293. Bethlenfalvay, G.J., Ames, R.N., 1987. Comparison of two methods for quantifying extraradical mycelium of vesicular arbuscular mycorrhizal fungi. Soil Sci. Soc. Am. J. 51, 834–837. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Cakmak, I., Marschner, H., 1992. Magnesium deficiency and high light intensity enhance activities of superoxide dismutase ascorbate peroxidase, and glutathione reductase in bean leaves. Plant Physiol. 98, 1222–1227. Chiquito-Contreras, R.G., Osorio-Acosta, F., García-Pérez, E., Villanueva-Jiménez, J.A., Zulueta-Rodríguez, R., Castillo-Rocha, D.G., 2012. Biofertilization with rhizobacteria and a consortium of arbuscular mycorrhizal fungi in citrus rootstocks. Trop. Subtrop. Agroecosyst. 15, S72–S81. Clark, R.B., Zeto, S.K., 2000. Mineral acquisition by arbuscular mycorrhizal plants. J. Plant Nutr. 23, 867–902. Demir, S., 2004. Influence of arbuscualr mycorrhiza on some physiological growth parameters of pepper. Turk. J. Biol. 28, 85–90. Fayez, R., Mahmoud, G., 2006. Interaction between phosphorus availability and an AM fungus (Glomus intraradices) and their effects on soil microbial respiration, biomass and enzymes activities in a calcareous soil. Pedobiologia 50, 413–425. Fu, Y.Q., Yang, X.J., Shen, H., 2014. The physiological mechanism of enhanced oxidizing capacity of rice (Oryza sativa L.) roots induced by phosphorus deficiency. Acta Physiol. Plant. 36, 179–190. Giannopolitis, C.N., Ries, S.K., 1977. Superoxide dismutase: I. Occurrence in higher plants. Plant Physiol. 59, 309–314. Goswami, D., Parmar, S., Vaghela, H., Dhandhukia, P., Thakker, J.N., 2015. Describing Paenibacillus mucilaginosus strain N3 as an efficient plant growth promoting rhizobacteria (PGPR). Cogent Food Agric. 1, 10000714.

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