Glomus mosseae bioprotection against aster yellows phytoplasma (16srI-B) and Spiroplasma citri infection in Madagascar periwinkle

Physiological and Molecular Plant Pathology 88 (2014) 1e9

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Glomus mosseae bioprotection against aster yellows phytoplasma (16srI-B) and Spiroplasma citri infection in Madagascar periwinkle Monther Mohumad Tahat a, Naghmeh Nejat b, *, Kamaruzaman Sijam a a b

Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 4 August 2014 Available online 15 August 2014

The bio-control potential of arbuscular mycorrhizal fungus Glomus mosseae against two pathogenic microorganisms aster yellows (AY) phytoplasma and Spiroplasma citri has been examined in Madagascar periwinkle (Catharanthus roseus). G. mosseae had a positive influence on healthy C. roseus plants and S. citri infection. It provided bioprotection against S. citri pathogen and induced significant degree of resistance to spiroplasma infection. Besides, symptom expression significantly reduced and shoot height, leaves number, root fresh and dry weight increased in spiroplasma-infected plants treated with mycorrhiza fungus. Although, G. mosseae had no positive effect on phytoplasma disease. The root architectures were affected by the phytoplasma pathogen, and the root surface area dramatically decreased in G. mosseae treated AY-infected periwinkles compared with the control. Nitrogen and Phosphorus concentrations notably increased in spiroplasma þ G. mosseae compared with control plants. Potassium concentration did not differ significantly in all mycorrhizal treated and untreated infected plants except in G. mosseae treated healthy plants. The spore density and root colonization rate did not vary in both pathogen treatments G. mosseae þ spiroplasma and G. mosseae þ phytoplasma. To our knowledge, this is the first report showing the bioprotective effect of G. mosseae on S. citri. The possible mechanisms involved in complex interaction between plants, cell wall-less bacteria and arbuscular mycorrhizal fungi (AMF) are discussed and the underlying mechanisms for the functioning of AMF are hypothesized. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Arbuscular mycorrhiza fungi Madagascar periwinkle Spiroplasma citri Aster yellows phytoplasma

Introduction Madagascar periwinkle (Catharanthus roseus (L.) G. Don.), a member of the Dogbane family, is widely grown in tropical and subtropical regions of the world [1]. Madagascar periwinkle is much more than an appealing flowering ornament. This valuable plant species is rich source of antioxidant, terpenoid indole alkaloids (TIAs) and produces eminently important anticancer dimeric alkaloids vinblastine and vincristine, and an antihypertensive alkaloid Ajmalicine [2,3]. The well-known C. roseus is by far the most powerful medicinal breakthrough in the treatment of childhood leukemia, different types of cancer and diabetes with potent tranquilizing characteristics [4e6]. Due to these important pharmaceutical alkaloids, C. roseus is one of the most extensively studied medicinal plant species [7]. Unfortunately, periwinkle is a

* Corresponding author. E-mail address: [email protected] (N. Nejat). http://dx.doi.org/10.1016/j.pmpp.2014.08.002 0885-5765/© 2014 Elsevier Ltd. All rights reserved.

poisonous plant and the natural medicinal alkaloids have neurotoxic activity in the human body [8]. Besides, periwinkle is used as a model host plant in plant pathology to study mollicutes because it is highly susceptible to phytoplasma and spiroplasma infection from different crops [9]. Phytoplasmas as obligate plant pathogens are wall-less prokaryotes belonging to the class Mollicutes. They are phloeminhabiting and naturally transmitted by insects mainly leafhoppers and planthoppers [10]. Phytoplasmas are systemic pathogens causing economic yield losses in more than 700 of plant species worldwide. They cause a wide variety of symptoms on infected plants that range from mild yellowing to death [11]. Aster yellows phytoplasma associated with the periwinkle proliferation has been isolated from periwinkle in Malaysia. Naturally infected periwinkles show phyllody (leaf-like petals), virescence (green flowers), proliferation and chlorosis symptoms [12]. Helical filamentous spiroplasmas are motile culturable mollicutes that lack a true cell wall. Spiroplasma citri is an obligate parasite with a wide host range, surviving in the phloem sieve

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elements of infected plants [13]. Lethal yellows disease caused by S. citri is the most destructive disease of periwinkle in Malaysia. The most obvious symptoms of infected periwinkles are rapid decline in the size and number of plant flowers, reduction in leaf size, chlorosis of leaf tips and margins, stunting and death [14]. Arbuscular mycorrhizal fungi (AMF) are ubiquitous microorganisms that form a beneficial symbiosis with more than 80% of all terrestrial plants. Most agricultural, horticultural and crop species are able to establish this mutualistic association [15,16]. Mycorrhizal root colonization provides a bio-protective effect against a broad range of soil-borne fungal pathogens [17e20]. Mechanisms involved in mycorrhizal fungi against disease organisms include enhancement of nutrient uptake [21,22], hormone production [23], phytochemical production [24], increasing host tolerance to pathogens [25e28], competition for host photosynthesis and colonization sites [29], root architectural changes [30,31], modification of rhizosphere microbial population and activation of plant defense mechanisms [16]. The genus Glomus mosseae is a frequent component of arbuscular mycorrhizal fungi associated with numerous plants from different regions of the world. G. mosseae has extensively been used to suppress several plant diseases and it has been very effective in the plant development and growth especially when associated with other beneficial microbes [32]. The protective effect of AMF has been investigated against different phytoplasmas in plants such as stolbur phytoplasma (16Sr XII) in tomato (Lycopersicon esculentum Mill.) [33], Candidatus Phytoplasma asteris (16SrI) strain CY in daisy (Chrysanthemum carinatum Schousboe) [34], two different strains of Ca. Ph. asteris in tobacco (Nicotiana tobacum L.) [35], and Ca. Ph. prunorum (16SrX-B) in Japanese plum (Prunus salicina L.) [36]. The reported results revealed that AMF has a positive effect on symptom expression and life span of infected plants. G. mosseae imperceptibly reduced the number of aster yellows-infected plants and extended their life span [34]. Furthermore, symptom expression significantly reduced in phytoplasma-infected tobacco and tomato plants treated with G. mosseae BEG 12 [33,35]. Likewise, Glomus intraradices BEG 72 increased tolerance of Japanese plum to European stone fruit yellows disease [36]. Lingua et al. [33], reported that AMF had a positive influence on morphological parameters (shoot and root fresh weight, Internode length, shoot height, leaf number and adventitious root diameter) of treated phytoplasma-infected tomato plants, reduced symptom expression and induced callose accumulation on the sieve pores. While, mycorrhiza inoculation had no any effect on shoot growth and weight of infected tobacco [35]. To date, there has been no report available on a bioprotective effect of arbuscular mycorrhizal fungi against S. citri. The current study was aimed to investigate the potential of G. mosseae as bio-control agent against the periwinkle proliferation and lethal yellows diseases caused by Candidatus Phytoplasma asteris (16SrI-B) and S. citri, respectively under glasshouse conditions. To the best of our knowledge, the present study is the first to examine the biocontrol effect of mycorrhization on S. citri.

0.023%), (K 0.30%), (Ca 0.063%), (Mg 0.034%), (S 0.063%), (Fe 1.52%), (Mn 0.0034%), (Zn 0.0057%), (Mo 0.00064%), (B 0.0003%) and (Cu 0.0015%). The soil mixture was autoclaved at 121  C for 1 h and 15 lb/inch2. Clean plastic pots (25 cm diameter) were filled with 4 kg of an autoclaved dry soil mixture. The plants were watered by tap water as needed without any fertilizer. Biological materials Seedlings of C. roseus cv. Pacifica Red (two weeks old) were obtained from certified nurseries in Serdang. They were kept under glasshouse conditions with minemax temperature 25 ± 5  C, 70e80% humidity and 13-h photo-period. The primary sources of inocula were aster yellows phytoplasma of the subgroup 16SrI-B (Accession no. FJ008869) [12,37] and S. citri (Accession no. HM015669) [14] isolated from natural infected C. roseus cv. rosea. These isolates have been transmitted to C. roseus cv. rosea by side grafting and confirmed as positive by polymerase chain reaction (PCR). They maintained in the glasshouse of Department of Plant Protection, University of Putra, Malaysia. Mycorrhiza preparation and evaluation G. mosseae was obtained from the laboratory of soil microbiology, faculty of agriculture, University of Putra, Malaysia [38]. The G. mosseae spores were cultured in pots using sorghum as a host in the glasshouse for 3 months and stored under laboratory conditions at 24e28  C. The wet sieves technique was used to isolate and purify the AMF spores [39]. Mature and healthy spores were isolated from the pot culture. Hundred spores/10 g dry soils were added to the pots and mixed well with the soil before planting of periwinkle seedlings. Healthy roots from the central part of the pot were collected (10 segments), washed thoroughly in distilled water and cut into 1 cm long segments. Then, the roots were cleaned with hot (90e100  C) 10% KOH and rinsed three times with distilled water. Roots segments were immersed in 10% HCl for 20 min and then stained with 0.05% trypan blue in lactophenol 90e100  C for 3 min [39]. The percentage of root colonization of lateral roots was measured using the technique designed by Giovannetti and Mosse [40], and the following formula was used to calculate the root colonization rate.

Root colonization rate ¼

number of colonized segments total number of segments examined  100

Materials and methods

AMF spores were separated from the soil by wet sieving and decanting technique. Ten grams soil from each replication were collected and mixed very well with tap water. The mixture was poured through three sieves sizes (250, 63, 45 mM). After several times of sieve washing, the liquid was collected in Petri dish, then the healthy and mature spores were counted under binocularmicroscope [41].

Soil preparation

CandidatusPhytoplasma asteris (16SrI-B) and S. citri Inoculation

Serdang series soil (fine sandy loam to fine sandy clay loam) was used in this experiment. The soil was collected from the campus of University of Putra, Malaysia. It was sieved using 5 mm pore sieve. The sieved soil was mixed with sand in the ratio of 3:1 (v/v soil: sand). Nutrient analysis of soil was performed using auto analyzer machine [20]. It showed the following results: (pH 6.5), (N 0.13%), (P

By 8 weeks after mycorrhizal inoculations, the main stems of periwinkle plants were graft-inoculated with scions of AY-infected periwinkle showing symptoms of phyllody, virescence, proliferation, little leaf and flower abortion, and S. citri-infected periwinkles showing symptoms of chlorosis of leaf tips and margins, little leaves and flowers, separately. The graft-inoculated area, in which

M.M. Tahat et al. / Physiological and Molecular Plant Pathology 88 (2014) 1e9

the diseased scions were inserted, was wrapped tightly with parafilm and grafted shoots were covered with a plastic bag for 1 week to minimize dehydration. Control plants were grafted with scions from healthy periwinkle (mock inoculation). DNA extraction and pathogen detection by PCR Total nucleic acids were extracted from approximately 0.15 g of fresh leaves using CTAB buffer by the small scale DNA extraction method [37]. DNA extracts were analyzed by direct PCR using the phytoplasma universal primer pair P1 (50 -AAG AGT TTG ATC CTG GCT CAG GAT T-30 )/P7 (50 -CGT CCT TCA TCG GCT CTT-30 ) [42]. Primer pair ScR16F1/ScR16R1 (50 -AGGATGAACGCTGGCGGCAT-30 / 50 -GTAGTCACGTCCTTCATCGT-30 ) [43] were used to detect S. citri in the periwinkle samples. All reactions were used in 20-ml volumes containing 1 ml of DNA template, 1 ml of each primer (10 pmols each), 0.2 mM of 10 mM dNTP, 0.75 mM of 25 mM MgCl2, 2 ml of 10 Taq polymerase buffer and 0.3 ml (0.5 units) Taq DNA polymerase (Fermentas, Inc.). The following PCR program was used to amplify 16S/23S rRNA genes of aster yellows phytoplasma using P1/P7 primer pair: 35 cycles of 1 min at 94  C, 1 min at 57  C and 90 s at 72  C with a final extension of 10 min at 72  C in a termocycler (iCycler, Bio Rad) [41]. Conditions for PCR with primer pair ScR16F1/ScR16R1 were 94  C for 2 min (an initial denaturation step), followed by 35 cycles of denaturation at 94  C for 1 min, annealing for 2 min at 61  C, and primer extension for 3 min (10 min in the final cycle) at 72  C [42]. Aliquots of 7 ml of each final reaction mixture were electrophoresed through 1% agarose gels in 1 TBE (90 mM Tris-borate, 2 mM EDTA, pH 8.0) as running buffer. DNA products in gels were stained with ethidium bromide, visualized by UV transillumination, and photographed. Severity assessment Disease symptoms appeared one month after inoculation in both phytoplasma and spiroplasma graft-inoculated plants.

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Spiroplasma and phytoplasma symptom expression was visually assessed in grafted C. roseus based on the degree of infected shoots following the scale of Hortfall and Barratt [44]. The scale based on foliar symptoms comprising 0: asymptomatic plant to 4: severe chlorosis and 5: nearly dead or dead plants. Based on the following formula, the percentage of disease severity was calculated.

Disease severityð%Þ ¼ R1 þ R2 …Rn =Y þ maximum rating scale  100 where, R ¼ score of disease severity of each plant, and Y ¼ total number of tested plant. Morphometry and root architecture Plant height was recorded monthly starting from the 8 weekse16 weeks of plant growth. Root and shoot fresh and dry weights were recorded. Root dry weight was measured for each single plant after oven drying (75  C) for 24 h. Flowers number, leaves number and branch number were documented for the periwinkle shoot. The root analyzer machine was used for the measurement of root tips, root volume, root total length. The root colonization rate and soil spore density were measured as mentioned in Section 2.3. The sequential order of the experiment is shown in Fig. 1. Macro-nutrients analysis (N, P, K) The wet ashing method described by Sharifuddin [45] was followed to measure the N, P, K (Nitrogen, Phosphorus, Potassium) concentrations in periwinkle shoots. Dried plant shoot (0.25 g) was grounded in a conical flask. Five ml of 100% sulfuric acid (H2SO4) was added to each sample. The samples were heated on a hot plate

Fig. 1. Chronological set up of the experiment.

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at 450  C for 15 min for sample digestion. Then, 10 ml of concentrated H2O2 was added to each sample and heated at 450  C for 7 min. The N, P, and K contents were measured using an auto analyzer machine. Experimental design and data analysis The experiment was arranged in CRD with 6 treatments and 4 replications. Six treatments comprising control plants without any inoculation, G. mosseae inoculated plants, AY phytoplasma-infected plants, S. citri-infected plants, AY phytoplasma-infected plants treated with mycorrhiza and S. citri-infected plants treated with mycorrhiza. The data were subjected to an analysis of variance using SPSS 15.0 software (SPSS Inc. Chicago, USA). Tukeyʼs HSD was used for the means separation. The percentage changes (increase/decrease) from x1 to x2 were calculated using the following formula:

Fig. 3. Disease severity after 16 weeks of mycorrhizal fungi treatment and 8 weeks of plant inoculation with the pathogens. GM: G. mosseae; Sc: Spiroplasma citri; AY: aster yellows phytoplasma.

Percentage change ¼ ððx2  x1Þ=x1Þ  100 where, x1 ¼ control treatment (treatment without Glomus mosseae), and x2 ¼ Glomus mosseae treatment.

untreated infected plants were strongly influenced by aster yellows phytoplasma infection (Fig. 5). Plant morphometry

Results AY phytoplasma and spiroplasma detection by PCR DNA prepared from the grafted periwinkles was examined for phytoplasma and spiroplasma infection by PCR with universal phytoplasma primer P1/P7 and S. citri primer pair ScR16F1/ ScR16R1. DNA was amplified from all diseased periwinkle plants, whereas no product was obtained from the control healthy periwinkles (Fig. 2). Disease severity assessments All AMF treated and untreated aster yellows phytoplasma (AY) and spiroplasma (Sc) grafted periwinkle developed disease symptoms by one month post-inoculation with infected scions. The disease severity and symptom expression reduced significantly in S. citri-infected plants treated with G. mosseae (Scþ/ AMFþ) compared with spiroplasma infected C. roseus without AMF (Scþ/AMF) (Figs. 3 and 4). AY phytoplasma-infected plants with AMF (AYþ/AMFþ) and without AMF treatment (AYþ/AMF) showed similar phytoplasma symptoms with no significant difference. Both treated and

The differences among treatments appeared at the end of 8th weeks. Plant growth was evaluated monthly 8 weeks after AM fungus treatment for the 3 months period. G. mosseae significantly increased the shoot height of the treated healthy plants after 8, 12, and 16 weeks of plant growth compared with untreated plants (26.31%, 55.2%, 74% respectively). The data analysis of plant shoot height illustrated that the Scþ/AMFþ treated plants did not vary statistically compared with the control plants, whilst they were significantly different from the Scþ/AMF treatment. Shoot height did not vary statistically in the AYþ/AMF and AYþ/AMFþ treatments though, these two treatments differ significantly from that of the control (Table 1). The statistical analysis showed that G. mosseae treated plants had the highest number of leaves followed by the control plants. The number of leaves was increased (51.2%) in the Scþ/AMFþ treatment compared with S. citri-infected periwinkles, while it was not different from the control plants. It was strongly and significantly reduced in the phytoplasma-infected and AYþ/AMFþ treated periwinkles compared with the control. A similar pattern was observed for the flowers number. The number of branches was higher significantly in G. mosseae treated and control plants compared with the other treatments. The Sc and Scþ/AMFþ

Fig. 2. Polymerase chain reaction (PCR) amplification of grafted-periwinkle samples. (A) M: 1 kb ladder; lanes 1e4: phytoplasma amplification using P1/P7 primers; lane 1: AYþ/ AMF infected sample; lanes 2e4: AYþ/AMFþ infected periwinkle plants; lane 5: mock-inoculated (healthy) periwinkle sample (B) M: 2-Log DNA ladder; lane 1: mock-inoculated periwinkle sample; lanes 2e4: amplification of spiroplasma using ScR16F1/ScR16R1 primer set, lane 2: Scþ/AMF infected periwinkle; Lanes 3 & 4: Scþ/AMFþ infected samples.

M.M. Tahat et al. / Physiological and Molecular Plant Pathology 88 (2014) 1e9

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Fig. 4. Spiroplasma symptoms on grafted periwinkles including severe chlorosis, little leaves and death, 2 months post-inoculation. A) Spiroplasma-infected C. roseus treated with G. mosseae, B) spiroplasma infected C. roseus without AMF.

Fig. 5. Aster yellows phytoplasma symptoms on grafted periwinkles including virescence, phyllody and chlorosis, 2 months post-inoculation. A) AY phytoplasma-infected C. roseus treated with G. mosseae, B) AY phytoplasma-infected C. roseus without AMF.

treatments and the AY and AYþ/AMFþ inoculated plants did not differ significantly (Table 2). Root growth, root fresh and dry weights were significantly higher in G. mosseae treated healthy periwinkles compared with the other treatments followed by Scþ/AMFþ treatment. They significantly raised in the Scþ/AMFþ treatment compared with the Scþ/AMF. While, root fresh and dry weights in the Scþ/AMFþ were similar to the control. Root fresh weight did not vary statistically in the Scþ/AMF, AYþ/AMF and AYþ/AMFþ treatments notwithstanding, it dramatically decreased in the latter treatments compared with the control plants. Root dry weight was the lowest in the AYþ/AMFþ treatment compared with the all other treatments. The shoot fresh and dry Table 1 Plant height of control plants, spiroplasma and phytoplasma-infected periwinkles untreated and treated with Glomus mosseae. Plant height (cm) Treatment

8th week

12th week

Gm. Spiroplasma Phytoplasma Gm. þ spiroplasma Gm. þ phytoplasma Control

43.2 a ± 0.8 28.4 b ± 1.3 31.0 bc ± 0.7 35.8 c ± 1.7 32.8 bc ± 0.8 34.2 c ± 1.8

62.4 a 30.2 b 32.4 b 44.6 d 34.2 bc 40.2 cd

± ± ± ± ± ±

2.6 1.2 0.7 1.6 0.6 2.0

weights of the symbiotic AMF treatment were the highest compared with the all treatments. While, Scþ/AMF, Scþ/AMFþ, AYþ/AMF and AYþ/AMFþ treatments did not vary compared to the control periwinkle plants (Table 3). Root architecture The root analysis indicated that the root tips, root length and root volume were significantly (P  0.05) higher in G. mosseae treated healthy periwinkles. The number of root tips of control plants varied significantly compared with all the other treatments. It was not statistically different in Scþ/AMF and Scþ/AMFþ treatments, although it reduced compared with the control. Whilst,

Table 2 Leaves number, flowers number and shoots number of periwinkle inoculated or not with Glomus mosseae and spiroplasma and/or phytoplasma. Treatment

Leaves number/plant

Flowers number/plant

Shoots number/plant

Gm Spiroplasma Phytoplasma Gm þ spiroplasma Gm þ phytoplasma Control

115.0 a ± 2.2 48.0 c ± 4.6 35.0 bc ± 1.5 72.6 d ± 3.5 30.6 b ± 2.6 75.8 d ± 2.6

108.8a ± 9.2 19.2 b ± 0.8 12.4 b ± 1.5 65.0 c ± 1.4 18.4 b ± 1.5 62.2 c ± 2.0

9.8 a ± 0.6 5.2 b ± 0.5 4.8 b ± 0.7 6.6 b ± 0.6 5.8 b ± 0.3 7.0 a ± 0.3

16th week 87.0 a 32.0 b 33.0 b 60.2 c 34.8 b 50.0 c

± ± ± ± ± ±

2.5 0.4 0.8 4.2 0.5 3.5

Means within columns followed by the same letter are not significantly different at P  0.05 level using Tukey test HSD. Gm: G. mosseae.

Means within columns followed by the same letter are not significantly different at P  0.05 level using Tukey HSD. Gm: G. mosseae.

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Table 3 Periwinkle root fresh weight (RFW), root dry weight (RDW), shoot fresh weight (SFW) and shoot dry weight (SDW) inoculated or not with Glomus mosseae, and spiroplasma and/or phytoplasma. Treatment

RFW g/plant

RDW g/plant SFW g/plant

SDW g/plant

Gm. 119.4 a ± 3.2 3.12 a ± 0.05 103.6 a ± 2.4 4.46 a ± 0.14 Spiroplasma 41.6 b ± 1.2 1.52 c ± 0.09 77.8 b ± 5.4 2.54 b ± 0.19 Phytoplasma 43.6 b ± 0.9 1.22 c ± 0.03 70.0 b ± 2.4 2.62 b ± 0.17 Gm þ spiroplasma 63.6 c ± 2.0 2.08 d ± 0.3 83.6 b ± 2.5 3.10 b ± 0.07 Gm þ phytoplasma 38.0 b ± 2.8 0.84 b ± 0.12 71.0 b ± 1.8 2.44 b ± 0.21 Control 71.0 c ± 2.4 2.16 d ± 0.06 82.8 b ± 3.6 3.00 b ± 0.08 Means within columns followed by the same letter are not significantly different at P  0.05 level using Tukey HSD. Gm: G. mosseae.

the lowest number of root tips was recorded in AY inoculated plants followed by AYþ/AMFþ. Root length was not different significantly within all treatments except in G. mosseae treated C. roseus plants which was the highest. The AMF treatment was able to increase the root volume significantly in healthy plants compared with the control and Scþ/AMF, Scþ/AMFþ, AYþ/AMF and AYþ/AMFþ inoculated plants. Root volume significantly increased in Scþ/ AMFþ compared with the Scþ/AMF treatment, and it was similar to the control. The root volume of AYþ/AMFþ did not vary compared to the pathogen treatments (Scþ/AMF and AYþ/AMF) (Table 4). The roots of healthy periwinkles were heavily colonized by G. mosseae spores. The Scþ/AMFþ and AYþ/AMFþ treatments were also colonized strongly but they did not differ statistically (Fig. 6).

Fig. 6. Spore colonization rate of the periwinkle roots inoculated with Glomus mosseae. Gm: G. mosseae; Sc: Spiroplasma citri; AY: aster yellows phytoplasma.

Spore density The rhizosphere of G. mosseae treated healthy plants produced the significant number of spores compared to the Scþ/AMFþ and AYþ/AMFþ treated plants (Fig. 7).

Fig. 7. Spores density of Glomus mosseae in the mycorrhizosphere of periwinkle plant. Gm: G. mosseae; Sc: Spiroplasma citri; AY: aster yellows phytoplasma.

N, P and K analysis The significant increase in N concentration was detected in the shoots of G. mosseae treated periwinkles. The data analysis has been revealed that the concentration of N was not different in the AY and AYþ/AMFþ treatments compared to the control plants. Whilst, N concentration significantly increased in the Scþ/AMFþ treatment (24.6%) compared with the Scþ/AMF. The highest significant concentration of P was detected in the mycorrhizal treated plants. Likewise, Scþ/AMFþ inoculated plants were significantly different and higher (130.5%, 97.6) compared with the Scþ/AMF and control plants, respectively. However, the AYþ/AMFþ treatment was not significantly different compared to the AYþ/AMF and control plants. The concentration of K was not different significantly within the double inoculated plants (AYþ/AMFþ), control, spiroplasma and phytoplasma treatments, whereas Scþ/AMFþ treated plants differed from the other treatments (Table 5). Table 4 Effect of Glomus mosseae, spiroplasma and phytoplasma on periwinkle roots growth characteristics [Root Tips (RT), Root Length (RL) and Root Volume (RV)] after 16 weeks.

Discussion AMF can increase plant growth, biomass, nutrient uptake and improve plant bioprotection against numerous of plant diseases through root colonization [16,28,46]. G. mosseae exhibited notable effect on healthy C. roseus. It significantly increased shoot and root dry weights, number of shoot branches, leaves and flowers, root tips, root length and root volume in healthy periwinkles. The AMF bioprotective effect depends on several factors such as the AMF isolate, the degree of mycorrhization, the genotype of pathogen, the host plant and host genotype, growing substrate and prevailing environmental conditions [47]. The mechanisms involved in the functioning and protective effects of AMF against soil-borne pathogens, viruses and cell wall-less bacteria are poorly understood. AMF confer the effective bioprotection and induce resistance against phytopathogens through Table 5 N, P and K concentrations in one gram of dried periwinkle shoots inoculated or not with Glomus mosseae and spiroplasma and/or phytoplasma.

Treatment

RT (no.)

RL (cm)

RV (cm3)

Treatment

N%

P%

K%

Gm Spiroplasma Phytoplasma Gm þ spiroplasma Gm þ phytoplasma Control

2177.0 a ± 58.4 912.2 c ± 37.9 525.2 b ± 43.9 1094.6 c ± 62.9 596.2 b ± 45.1 1330.6 d ± 58.5

1474.2 a ± 60.4 690.6 c ± 29.8 381.2 b ± 20.9 780.8c ± 22.1 357.2 b ± 35.9 700.2 c ± 33.1

13.0 a ± 0.51 5.9 b ± 0.41 6.0 b ± 0.23 8.8c ± 0.41 4.9 b ± 0.23 7.6 c ± 0.27

Gm Spiroplasma Phytoplasma Gm þ spiroplasma Gm þ phytoplasma Control

2.07 a ± 0.09 1.26 b ± 0.06 1.31 bc ± 0.05 1.57 c ± 0.06 1.32 c ± 0.05 1.33 bc ± 0.05

1.26 a ± 0.08 0.36 b ± 0.06 0.38 b ± 0.03 0.83 c ± 0.06 0.40 b ± 0.05 0.42 b ± 0.05

1.96 a ± 0.27 1.08 b ± 0.08 1.13 b ± 0.09 1.57 ba ± 0.06 1.07 b ± 0.10 1.20 b ± 0.12

Means within columns followed by the same letter are not significantly different at P  0.05 level using Tukey HSD. Gm: G. mosseae.

Means within columns followed by the same letter are not significantly different at P  0.05 level using Tukey HSD. Gm: G. mosseae.

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different mechanisms such as improvement in the mineral nutrient status of host plant, root damage compensation, morphological alterations of the root, competition for colonization sites, biomedical and molecular changes and activation of plant defense mechanisms in mycorrhizal plants [48]. There have been several reports on interaction among mycorrhiza fungi, plant, and phytoplasma. The obtained results regarding the protective effect of AMF on phytoplasma diseases are conflicting. Although, this finding did not concur with the previous results which AMF has a positive effect on phytoplasma infected tomatoes [33], and the number of plants infected with chrysanthemum yellows phytoplasma (CY) [49], reduced the severity of symptom expression and increased plant tolerance to the infection caused by CY [50] and decreased the phytoplasma infection in pear [51]. The current results suggest that G. mosseae colonization has no positive effect on AY phytoplasma infection. This finding is in
ska et al. (2010) findings which indicated agreement with Kamin that G. mosseae BEG 12 inoculation does not decrease the negative effect of two aster yellows phytoplasma strains in C. roseus plants [52]. Hence, these findings, while preliminary, suggest that the effects of AMF on phytoplasma infection are complex and likely due to the combination of host plant, AM fungus and phytoplasma isolate [53]. This study found that root fresh weight, root length and root volume were slightly reduced in AYþ/AMFþ compared with AYþ/
ska et al. (35) AMF. These findings are consistent with Kamin findings which showed significant differences in root fresh weight and total root length of aster yellows phytoplasma AY1 strain infected tobacco compared with AM AY1 treated tobacco plants. Phytoplasmas morphologically and histologically modify the roots of infected plants [35]. Therefore, the reduction of root weight in the presence of both microorganisms could be the consequence of competition between aster yellows phytoplasma and AM fungus for resources, and depends on phytoplasma isolate. Symbiotic mycorrhizal fungi need carbon to keep growing and helping plants take up more phosphorus. There is a functional linkage between C and P exchange [54,55], and C supply of the host plant triggers the uptake and transport of N in the AM symbiosis [56]. It can therefore be suggested that the biocontrol effect of mycorrhization depends on the carbon availability in mycorrhizal plants [47]. Phytoplasmas influence on soluble carbohydrates and starch in source and sink tissues of infected plants [57,58]. Soluble carbohydrates and starch accumulated in source leaves of infected periwinkle plants, whilst sugar markedly decreased in sink leaves and starch significantly reduced in periwinkle roots [59]. Hence, it can be hypothesized that direct strong competition exists between cell wall-less bacteria and AM fungus for resources, particularly carbon drain from the plant due to the carbohydrates used by the symbiotic AM fungus and phytopathogenic mollicutes. On the other hand, the results of this experiment revealed that G. mosseae was able to interact with S. citri and modify roots of infected plants, as the severity of disease symptoms reduced. Spiroplasma infected plants showed a large root system with different mycorrhizal colonization levels. The increase in the aerial part of periwinkle plant was supported by the large root system enhanced by the AMF colonization. The same denouement was reported by D'Amelio et al. [34], who found that Chrysanthemum carinatum infected by chrysanthemum yellows phytoplasma produced a larger root parameter in the combined inoculation of AMF and Pseudomonas putida S1PF1RiF. It was clear that the branching of the root seems to be the major general effect inducing by mycorrhizal fungi inoculation [60,61]. Mycorrhizal plants indicated tolerance to S. citri infection and conferred a higher P status than nonmycorrhizal infected

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periwinkle plants. The increased tolerance and bioprotection might have acquired through improvement in the mineral nutrient status and growth promoting of mycorrhizal spiroplasma infected plants. This result corroborates the findings of a great deal of the earlier studies, in which they have shown the positive effects of AM fungi in increasing tolerance to pathogen attacks via several changes in root architectures and features with different variations, enhancement of root growth and nutrient uptake particularly of P [17,46,62e64]. The reason behind that might be related to the pathogen and AM fungi interactions, particular the class of pathogen and AMF species involved, and thus may influence competition outcomes [28,30,64]. One of the mechanisms involved in the ability of AMF as a bioprotection microorganism is the fluctuation of the nutrient uptake, mainly N, P and K [65]. In the current finding the concentrations of N and P were correlated with the S. citri disease reduction. However, the N, P and K concentrations were not different in the AYþ/ AMF and AYþ/AMFþ treatments compared with the control plants. N and P nutrients shown positive responses to the combined inoculation treatments of G. mosseae and S. citri. The research conducted by McGonigle et al. [66] was partially confirmed the present finding. They reported that arbuscular mycorrhizal plants have more P uptake and growth when raised in undisturbed soil. The results obtained in the present study were matched with the results obtained by Tahat et al. [67] who found that the concentration of N, P and K in G. mosseae þ Ralstonia solanacearum (bacterial wilt) in tomato plant was significantly higher compared with single inoculation of the fungus G. mosseae. In this study the colonization level was 60% in G. mosseae þ spiroplasma and 55% in G. mosseae þ phytoplasma plants, which consider as a good level to help the plant to withstand the root disease infection and it can also influence directly the foliar infection. Nonetheless, phytoplasma was able to infect the plant and caused a clear symptoms. Whilst bioprotection of periwinkle against lethal yellows disease improved (Fig. 8). Hence, the plant was able to get benefits from the strong root colonization rate. This fact could explain the reduction of spiroplasma disease which was approved by Jaime et al. [68] who reported that G. intraradices propagules were able to reduce the disease incidence of white rot on onion when the colonization rate and the spore density were in high level. Moreover, in this experiment the number of spores was not affected by the disease infection because the spore manipulation in the soil was stable and the soil conditions were not disturbed by any other factors. The inoculum density

Fig. 8. The effect of sequential inoculation by G. mossese (Gm) on disease severity of spiroplasma (Sc) and aster yellows phytoplasma (AY) diseases.

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was an important factor to increase the tomato protection by G. mosseae against the bacterial wilt [67]. Some researchers found that plant colonization rate and host responses to AMF can be related to AMF species, soil conditions, and pathogen species as the main factors in colonization differences [16,21]. In the current study the AY phytoplasma pathogen was not affected by the colonization rate or the spore density. This study concluded that mycorrhization of periwinkle root can reduce the susceptibility of S. citri infection but not the AY phytoplasma pathogen using the same species of AMF. The results documented in this work depend on the pathogens behavior and carbon competition between AMF and cell wall-less bacteria, although the functional mechanisms behind the association between AMF and cell wall-less bacteria are still unclear. It is difficult to explain the results of complex interactions between periwinkle plant, cell wall-less bacteria and AM fungus. Further studies of these multifaceted interactions including transcriptomic and metabolomic analysis and new techniques like confocal laser scanning microscopy require to be undertaken to elucidate processes and signaling pathways involved in mycorrhization and bioprotection. Conclusion In the current study, G. mosseae appears to have different influences on the different pathogens. Phytoplasma (16SrI-B) was not affected by the AMF colonization. In contrast, S. citri pathogen was reduced significantly in some parameters. These findings revealed that differences between pathogens determine the interaction, although AMF isolate, host genotype, root colonization rate, soil and environmental conditions were same. Possibly more studies should be done to find out one or more of AMF species to be combined with another soil microbiological agent to compete strongly for nutrients and control or manage the systemic plant pathogenic bacteria such as phytoplasm and spiroplasma. Acknowledgments The authors would like to acknowledge University of Putra, Malaysia for supporting this project. References [1] Jaleel CA, Panneerselvam R. Variations in the antioxidative and indole alkaloid status in different parts of two varieties of Catharanthus roseus, an important folk herb. Chin J Pharm Tox 2007;21:487e94. [2] Zheng W, Wang SY. Antioxidant activity and phenolic compounds in selected herbs. J Agric Food Chem 2001;49:5165. [3] van der Heijden R, Jacobs DA, Snoeijer W, Hallard D, Verpoorte R. The Catharanthus alkaloids: pharmacognosy and biotechnology. Curr Med Chem 2004;11:607e28. [4] Sottomayor M, Ros-Barcelo A. The Vinca alkaloids: from biosynthesis and accumulation in plant cells, to uptake, activity and metabolism in animal cells. Stud Nat Prod Chem 2006;33:813e57. [5] El-Sayed A, Cordell GA. Catharanthus alkaloids. XXXIV. Catharanthamine, a new antitumor bisindole alkaloid from Catharanthus roseus. J Nat Prod 1981;44:289e93. [6] Ghosh RK, Gupta I. Effect of Vinca rosea and Ficus racemososus on hyperglycemia in rats. Indian J Anim Health 1980;19:145e8. [7] Roepke J, Salim V, Wu M, Thamm AMK, Murata J, Ploss K, et al. Vinca drug components accumulate exclusively in leaf exudates of Madagascar periwinkle. PNAS 2010;107:15287e92. ^que D, Jehl F. Molecular pharmacokinetics of Catharanthus (Vinca) alka[8] Leve loids. J Clin Pharmacol 2007;47:579e88. [9] Lee IM, Davis RE, Gundersen-Rindal DE. Phytoplasma: phytopathogenic mollicutes. Annu Rev Microbiol 2000;54:221e55. [10] Nejat N, Vadamalai G. Diagnostic techniques for detection of phytoplasma diseases: past and present. J Plant Dis Prot 2013;120:16e25. [11] Maejima K, Oshima K, Namba S. Exploring the phytoplasmas, plant pathogenic bacteria. J Gen Plant Pathol 2014;80:210e21.

[12] Nejat N, Sijam K, Abdullah SNA, Vadamalai G, Dickinson M. Phytoplasmas associated with disease of coconut in Malaysia: phylogenetic groups and host plant species. Plant Pathol 2009;58:1152e60. [13] Nejat N, Vadamalai G, Dickinson M. Spiroplasma citri: a wide host range phytopathogen. Plant Pathol J 2011;10:46e56. [14] Nejat N, Vadamalai G, Sijam K, Dickinson M. First report of Spiroplasma citri associated with periwinkle lethal yellows in Southeast Asia. Plant Dis 2011;95:1312. [15] Dickson S. The Arum-Paris continuum of mycorrhizal symbioses. New Phytol 2004;163:187e200. [16] Smith SE, Read DJ. Mycorrhizal symbiosis. London: Academic Press; 2008. [17] Harrier AL, Waston CA. The potential role of arbuscular mycorrhizal (AM) fungi in the bio-protection of plants against soil-borne pathogens in organic and/or other sustainable farming systems. Pest Manag Sci 2004;60:149e57. [18] Pal KK, Gardener BM. Biological control of plant pathogens. Plant Health Instr 2006:1e25. http://dx.doi.org/10.1094/PHI-A-2006-1117-02. [19] Sikes BA, Cottenie K, Klironomos JN. Plant and fungal identity determines pathogen protection of plant roots by arbuscular mycorrhizas. J Ecol 2009;97: 1274e80. [20] Tahat MM, Kamaruzaman S, Radziah O. Mycorrhizal fungi as a biological control agent. Plant Pathol J;9:198e207. [21] Lindermann RG. Role of VAM fungi in bio-control. In: Pfleger FL, Lindermann RG, editors. Mycorrhiza in plant health. St. Paul, Minn: APS; 1994. p. 1e25.
n-Aguilar C. Priming [22] Pozo MJ, Verhage A, García-Andrade J, García Jm, Azco
nplant defence against pathogens by arbuscular mycorrhizal fungi. In: Azco Aguilar C, Barea JM, Gianinazzi S, Gianinazzi-Pearson V, editors. Mycorrhizas e functional processes and ecological impact. Berlin: Springer Verlag; 2009. p. 123e36. [23] Dannenberg G, Latus C, Zimmer W, Hundeshagen B, SchneiderPoetsch H, Bothe H. Influence of vesicular-arbuscular mycorrhiza on phytohormone balance in maize (Zea mays L.). J Plant Physiol 1992;141: 33e9. [24] Toussaint JP, Smith A, Smith E. Arbuscular mycorrhizal fungi can induce the production of phytochemicals in sweet basil irrespective of phosphorus nutrition. Mycorrhiza 2007;17:291e7. [25] Demir S, Akkopru A. Using of arbuscular mycorrhizal fungi (AMF) for biocontrol of soil borne fungal plant pathogens. In: Chincholkar SB, Mukerji KG, editors. Biological control of plant diseases. USA: Haworth Press; 2007. p. 17e37. [26] Cordier C, Trouvelot A, Gianinazzi S, Gianinazzi-Pearson V. Arbuscular mycorrhizal technology applied to micropropagated Prunus avium and to protection against Phytophthora cinnamomi. Agronomie 1996;16: 679e88. [27] Azcon-Aguilar C, Barea Jm. Arbuscular mycorrhizas and biological control of soil borne plant pathogens e an overview of the mechanisms involved. Mycorrhiza 1996;6:457e64. [28] Borowicz VA. Do arbuscular mycorrhizal fungi alter plant-pathogen relations? Ecology 2001;82:3057e68. [29] Sylvia DM, Alagely A, Chellemi DO, Demchenko LW. Arbuscular mycorrhizal fungi influence tomatoes competition with bahiagrass. Biol Fert Soils 2001;34: 448e52. [30] Sikes BA. When do arbuscular mycorrhizal fungi protect plant roots from pathogens? Plant Signal Behav 2010;5:763e5. [31] Berta G, Fusconi A, Lingua G, Trotta A, Sgorbati S. Influence of arbuscular mycorrhizal infection on nuclear structure and activity during root morphogenesis. In: Azcon-Aguilar C, Barea JM, editors. Mycorrhizas in integrated systems: from genes to plant development, Proceedings of the Fourth European Symposium on Mycorrhizas; 1996. p. 174e7. [32] Morton JB. International culture collection of arbuscular and vesiculararbuscular mycorrhizal fungi. Ecosyst Environ 2000;74:77e93. [33] Lingua G, Giovanni D, Nadia M, Michele A, Graziella B. Mycorrhiza-induced differential response to a yellows disease in tomato. Mycorrhiza 2002;2: 191e8. [34] D'Amelio R, Berta G, Gamalero E, Massa N, Avidano L, Cantamessa S. Increased plant tolerance against chrysanthemum yellows photoplasma (Candidatus Pytoplasma asteris) following double inoculation with Glomus mosseae BEG12 and Pseudomonas putida S1Pf1Rif. Plant Pathol 2011;60:1014e22.
ska M, Klamkowski K, Berniak H, Treder W. Effect of arbuscular [35] Kamin mycorrhizal fungi inoculation on aster yellows phytoplasma-infected tobacco plants. Sci Hortic 2010;125:500e3. ~ a A, Sabate
J, Camprubí A, Estaún V, Calvet C. Tolerance increase [36] Batlle A, Lavin to ‘Candidatus phytoplasma prunorum’ in mycorrhizal plums fruit trees. B Insectol 2011;64:S199e200. [37] Nejat N, Sijam K, Abdullah SNA, Vadamalai G, Dickinson M. Molecular characterization of an aster yellows phytoplasma associated with proliferation of periwinkle in Malaysia. Afr J Biotechnol 2010;9:2305e15. [38] Azizah Chulan H. Propagation and maintenance of VAM cultures. Pertanika 1987;10:271e5. [39] Philips JM, Hayman DS. Improved procedures for clearing roots and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection. T Brit Mycol Soc 1970;55:158e61. [40] Giovannetti M, Mosse B. An evaluation of techniques to measure vesiculararbuscular infection in roots. New Phytol 1980;84:489e500.

M.M. Tahat et al. / Physiological and Molecular Plant Pathology 88 (2014) 1e9 [41] Gerdemann JW. Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. T Brit Mycol Soc 1963;46:235e44. [42] Nejat N, Vadamalai G. Phytoplasma detection in coconut palm and other tropical crops. Plant Pathol J 2010;9:101e10. [43] Lee IM, Bottner KD, Munyaneza JE, Davis RE, Crosslin Jm, du Toit LJ, et al. Carrot purple leaf: a new spiroplasmal disease associated with carrots in Washington State. Plant Dis 2006;90:989e93. [44] Horsfall JG, Barratt RW. An improved grading system for measuring plant disease. Phytopathology 1945;35:655. [45] Sharifuddin HA. Basic guide to soil and plant analysis. Universiti Putra Malaysia; 1981. p. 48. [46] Tahat MM, Sijam K, Radziah O, Kadir J, Masdek HN. Response of (Lycopersicum esculentum Mill.) to different arbuscular mycorrhizal fungi species. Asian J Plant Sci 2008;7:479e84. [47] Vierheilig H, Steinkellner S, Khaosaad T, Garcia-Garrido JM. The biocontrol effect of mycorrhization on soilborne fungal pathogens and the autoregulation of the AM symbiosis: one mechanisms, two effects? In: Varma A, editor. Mycorrhiza: state of the art, genetics and molecular biology, ecofunction, biotechnology, eco-physiology, structure and systematic. Berlin Heidelberg: Springer-Verlag; 2008. p. 307e20.
n-Aguilar C. Unraveling mycorrhiza-induced resistance. Curr [48] Pozo MJ, Azco Opin Plant Biol 2007;10:393e8.  S, Berta G, et al. Pre[49] D’amelio R, Massa N, Gamalero E, D’agostino G, Sampo liminary results on the evaluation of the effects of elicitors of plant resistance on chrysanthemum yellows phytoplasma infection. B Insectol 2007;60: 317e8.  A, Massa N, Cantamessa S, DʼAgostino U, Bosco D, Marzachì, et al. Ef[50] Sampo fects of two AM fungi on phytoplasma infection in the model plant Chrysanthemum carinatum. Agric Food Sci 2012;21:39e51. ~ a A, Camprubi A, Estaun V, Calvet C. Tolerance [51] Garcia-Chapa M, Batlle A, Lavin increase to pear decline phytoplasma in mycorrhizal ohf-333 pear rootstock. Acta Hortic 2004;657:437e41.
ska M, Klamkowski K, Berniak H, Sowik I. Response of mycorrhizal [52] Kamin periwinkle plants to aster yellows phytoplasma infection. Mycorrhiza 2010;20:161e6. [53] Romanazzi G, Musetti R, Marzachì C, Casati P. Induction of resistance in the control of phytoplasma diseases. Petria 2009;19:113e29. [54] Kiers ET, Duhamel M, Beesetty Y, Mensah JA, Franken O, Verbruggen E, et al. Reciprocal rewards stabilize cooperation in the mycorrhizal symbiosis. Science 2011;333:880e2. [55] Hammer EC, Pallon J, Wallander H, Olsson PA. Tit for tat? A mycorrhizal fungusaccumulates phosphorus under low plant carbon availability. FEMS Microbiol Ecol 2011;76:236e44.

9

[56] Fellabaum CR, Gachomo EW, Beesetty Y, Choudhari S, Strahan GD, Pfeffer PE, et al. Carbon availability triggers fungal nitrogen uptake and transport in arbuscular mycorrhizal symbiosis. PNAS 2012;109:2666e71. [57] Choi YH, Tapias EC, Kim HK, Lefeber AWM, Erkelens C, Verhoeven JTJ, et al. Metabolic discrimination of Catharanthus roseus leaves infected by phytoplasma using 1H-NMR spectroscopy and multivariate data analysis. Plant Physiol 2004;135:2398e410. [58] Santi S, Grisan S, Pierasco A, De Marco F, Musetti R. Laser microdissection of grapevine leaf phloem infected by stolbur reveals site-specific gene responses associated to sucrose transport and metabolism. Plant Cell Environ 2013;36: 343e55. [59] Lepka P, Stitt M, Moll E, Seemüller E. Effect of phytoplasmal infection on concentration and translocation of carbohydrates and amino acids in periwinkle and tobacco. Physiol Mol Plant P 1999;55:59e68. [60] Berta G, Sampo S, Gamalero E, Massa N, Lemanceau P. Suppression of rhizoctonia root-rot of tomato by Glomus mossae BEG12 and Pseudomonas fluorescens A6RI is associated with their effect on the pathogen growth and on the root morphogenesis. Eur J Plant Pathol 2005;111:279e88. [61] Hodge A, Berta G, Doussan C, Merchan F, Crespi M. Plant root growth, architecture and function. Plant Soil 2009;321:153e87. [62] Singh R, Adholeya A, Mukerji KG. Mycorrhiza in control of soil-borne pathogen. In: Mukerji KG, Chamola BP, Singh J, editors. Micorrhizal biology. New York: Kluwer; 2000. p. 173e96. [63] Lingua G, Sgorbati S, Citterio A, Fusconi A, Trotta A, Gnavi E, et al. Arbuscular mycorrhizal colonization delays nucleus senescence in leek root cortical cells. New Phytol 1999;141:161e9. [64] Lewandowski TJ, Dunfield KE, Antunes PM. Isolate identity determines plant tolerance to pathogen attack in assembled mycorrhizal communities. PLoS ONE 2013;8:e61329. [65] Whner J, Antunes PM, Powell JR, Mazukatow J, Rilling M. Plant pathogen protection by arbuscular mycorrhizas: a role for fungal diversity? Pedobiologia 2010;52:253e62. [66] McGonigle TP, Yano K, Shinhama T. Mycorrhizal phosphorus enhancement of plants in undisturbed soil differs from phosphorus uptake stimulation by arbuscular mycorrhizae over non-mycorrhizal controls. Biol Fert Soils 2003;37:268e73. [67] Tahat MM, Sijam K, Radziah O. The potential of endomycorrhizal fungi to control tomato bacterial wilt Ralstonia solanacearum under glass-house conditions. Afr J Biotechnol 2012;11:13085e94. [68] Jaime M, Hsiang T, McDonald MR. Effects of Glomus intraradices and onion cultivar on Allium white rot development in organic soils in Ontario. Can J Plant Pathol 2008;30:543e53.