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Molecular defense response of mycorrhizal bean plants infected with Rhizoctonia solani

Molecular defense response of mycorrhizal bean plants infected with Rhizoctonia solani Ann Microbiol (2013) 63:1195–1203 DOI 10.1007/s13213-012-0578-5 ORIGINAL ARTICLE Molecular defense response of mycorrhizal bean plants infected with Rhizoctonia solani Elsayed E. Hafez & Gamal M. Abdel-Fattah & Safwat A. El-Haddad & Younes M. Rashad Received: 14 August 2012 /Accepted: 21 November 2012 /Published online: 19 January 2013 Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract A time course study was conducted to investigate Keywords Arbuscular mycorrhizal fungi . . disease development and molecular defense response in common β-1,3-Endoglucanase Chitinase Differential display bean (Phaseolus vulgaris L.) plants colonized by a mixture of five technique Gene expression and root rot arbuscular mycorrhizal (AM) fungi, namely, Glomus mosseae, G. intraradices, G. clarum, Gigaspora gigantea,and Gigaspora margarita, and post-infected with the soil-borne pathogen Introduction Rhizoctonia solani. Results showed that pre-colonization of bean plants by AM fungi significantly reduced disease severity and Rhizoctonia solani Kühn is a common necrotrophic soil-borne disease incidence. DNA fingerprinting using the differential dis- fungus which can cause seed decay, damping-off, stem canker, play technique revealed a genetic polymorphism (86.8 %) in bean root rot, fruit decay, and foliage diseases in a wide range of plants that resulted from the colonization by AM fungi. Two plant species over a large part of the world (Tu et al. 1996). genetic mechanisms were recorded: (1) switching on of new This unlimited host range, combined with competitive sapro- genes and (2) induction of other active genes, including the phytic ability and lethal pathogenic potential, earns R. solani its defense genes chitinase and β-1,3-glucanase, to a highly status as formidable pathogen. expressed state. During recent years research has focused on identifying potential biocontrol agents to reduce the severity of root-rot disease caused by R. solani. A limited number of fungal E. E. Hafez antagonists against R. solani have been reported, among which City for Scientific Research and Technology Applications, antagonistic and plant growth-promoting yeasts (El-Tarabily Alexandria, Egypt e-mail: elsayed_hafez@yahoo.com 2004), Penicillium spp. (Ciavatta et al. 2006), Trichoderma koningii, T. pseudokoningii, T. viride, T. polysporum, T. aureo- G. M. Abdel-Fattah viride (Shalini and Kotasthane 2007), and Gliocladium roseum Botany Department, College of Science, Mansoura University, (Tarantino et al. 2007) are known to be very efficient. Bacterial Mansoura, Egypt e-mail: Abdelfattaham@yahoo.com antagonism has also been reported against R. solani (Kai et al. 2007). Among the potential biocontrol agents, the arbuscular S. A. El-Haddad mycorrhizal (AM) fungi have received special attention (Aly Mycological Research and Disease Survey Department, Plant and Manal 2009; Abdel-Fattah et al. 2011). Biological control Pathology Institute, Agricultural Research Center, Giza, Egypt using AM fungi is unique, being an eco-friendly and cost- e-mail: safwat@epq.gov.eg effective strategy for disease management that provides greater Y. M. Rashad (*) levels of protection and sustains plant yields, in addition to the Biology Department, Teachers College, King Saud University, positive effects on the plant growth and its nutrition. Riyadh, Saudi Arabia Common bean (Phaseolus vulgaris L.) is susceptible to e-mail: younesrashad@yahoo.com Rhizoctonia root-rot disease in most of the tropical, subtrop- Present Address: ical, and temperate areas of the world where it is grown. G. M. Abdel-Fattah Yield losses of 5–10 % are common, but 60 % yield losses Plant Production Department, College of Food and Agricultural have been reported in Brazil (Tu et al. 1996). Sciences, King Saud University, Riyadh, Saudi Arabia 1196 Ann Microbiol (2013) 63:1195–1203 Mechanisms that can account for the disease control ability Schenck, Gigaspora gigantea (Nicol. & Gerd.) Gerd. of AM fungi may include competition for infection sites and & Trappe, and Gigaspora margarita (Becker & Hall) 6 -1 host photosynthates, root damage compensation, enhance- in suspension at a concentration of 1×10 spores L ment of plant resistance through various physical and physi- (El-Haddad et al. 2004). ological mechanisms, such as increasing the cell-wall thickness of the host or the accumulation of some antimicro- Planting and growth conditions bial substances (Abdel-Fattah et al. 2011), or changes in the composition of the microbial communities in the mycorrhizo- Pots (diameter 25 cm) were each filled with 2.5 kg sterilized sphere (Singh et al. 2000). Several inducible defense-related soil (autoclaving at 121 °C for 2× 1 h). A sandy-loam soil genes, including those encoding isoflavonoid phytoalexins, (sand:silt:clay, 70:20:10 %) was used; soil characteristics such as phenylalanine ammonia lyase, chalcone synthase, and −1 were pH 8, electrical conductivity 270 μScm , total nitro- chalcone isomerase, have been reported to be induced during gen 1.05 %, total organic matter 1.64 %, and phosphorus mycorrhizal establishment (Guillon et al. 2002). The expres- −1 content 4.43 μgg . Five healthy seeds of common bean sion of genes encoding enzymes that synthesize phenolpropa- Phaseolus vulgaris L. (cv. Giza 3) were surface-disinfected noid compounds has also been detected in mycorrhizal roots with 2 % sodium hypochlorite for 2 min, washed with sterile (Garcia-Garrido and Ocampo 2002). Other defense-related distilled water, and sown in each pot. Half of the pots genes shown to be up-regulated in mycorrhizal symbioses received AM inoculum as a suspension at two time points: include genes involved in the metabolism of reactive oxygen in the bean seed bed at the beginning of the experiment and species, chitinase, and β-1,3-glucanase, and genes involved in as a soil drench 14 days after sowing) at a concentration of senescence, including glutathione-S-transferase (Zeng 2006). −1 5mLL water (El-Haddad et al. 2004). Plants were inoc- The role of AM colonization in sustaining bean plants ulated with Rhizobium leguminosarum one time with against R. solani is unclear. Depending on the time after infec- 1 mL of culture suspension at a concentration of 5×10 tion with R. solani and the tissue examined, responses varying −1 CFU mL and watered regularly to near field capacity with from stimulation to suppression to no change in transcript levels tap water. Pots did not receive any fertilizers in this study. All have been detected (Guillon et al. 2002). Therefore, the aim of pots were kept outdoors under natural conditions [day temper- our study was to investigate the molecular aspects of defense ature 25 °C, night temperature 20 °C, 16/8-h (light/dark) responses in mycorrhizal bean plants infected with R. solani. photoperiod]. At 4 weeks after inoculation with AM, the pathogen inoculum was mixed with the upper layer of the pot soil at Materials and methods a rate of 2 % (w/w) potential inoculum. Five pots were treated with tap water to serve as a negative control. Five Causal organism and bean cultivar pots were used as replicates for each treatment. The treat- ments applied in this study can be summarized as follows: Rhizoctonia solani (AG-2-2 IIIB) was isolated originally from untreated control (no mycorrhiza, no pathogen; CNM); my- naturally diseased common bean plants. Fungal identification corrhiza only (CM); pathogen only (no mycorrhiza; PNM); was based on cultural properties and morphological and mi- mycorrhiza + pathogen (PM). All pots were arranged in a croscopical characteristics, as described by Sneh et al. (1991). completely randomized design. Three plants from each The isolate was then assigned an AG designation according to treatment were harvested at 1, 3, 7, and 28 days after hyphal anastomosis with tester isolates from AG 1 to AG 10 pathogen infection for molecular analysis (Guillon et al. using the slide technique of Kronland and Stanghellini (1988). 2002; Mohr et al. 1998). Five plants of each treatment were The inoculum was prepared by growing the pathogen in bottles carefully harvested with their entire roots (2 weeks after containing sterilized sorghum grains for 15 days at 25±2 °C. inoculation with the pathogen), washed under running water The most common bean cultivar in Egypt (Giza 3) was used. to remove soil particles, and evaluated for shoot length, root length, leaf area, and shoot and root dry weights. Dry AM inoculum weights (in grams) were recorded after the samples had been dried at 80 °C for at least 48 h in a hot air oven until a A mixture of Egyptian formulated AM fungi (Multi-VAM) constant weight was reached. Disease severity (DS) and kindly provided by Dr. Safwat El-Haddad (Mycological disease incidence (DI) of the Rhizoctonia root rot were Research and Disease Survey Department, Plant Pathology assessed (2 weeks after inoculation with the pathogen) for Institute, Agricultural Research Center, Giza, Egypt) was each treatment. Disease severity was estimated as the used. This mixture consists of spores (in equal proportions) degree of root damage according to the scale of Carling of Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe, G. et al. (1999). intraradices Schenck & Smith, G. clarum Nicol. & Ann Microbiol (2013) 63:1195–1203 1197 Staining and estimation of mycorrhizal root colonization was visualized and photographed using a gel documen- tation system. Five plants of each treatment were carefully harvested with DNA fingerprints obtained using the differential display their entire roots and washed under running water to remove technique were first analyzed visually and a positive re- soil particles. The roots were separated and fixed in FAA sponse (a score ‘1’) was defined as the presence of a visible (formalin–acetic acid–alcohol) for evaluation of mycorrhi- band of a given size, while a negative response (a score zal root colonization. Roots fixed in FAA were rinsed re- ‘0’) was defined as the absence of any band of the peatedly in tap water, cut into small segments (0.5–1 cm), same size. These scores were then merged in a and stained with 0.05 % trypan blue (Sigma, St. Louis, MO) Microsoft Excel spreadsheet and then inserted in the according to Phillips and Hayman (1970). Fifty randomly SPSS ver. 13.0 computer software (SPSS 2004)for the selected stained root segments of each treatment were construction of the dendrogram using the genetic dis- mounted on slides in lactoglycerol and examined micro- tance method. scopically for estimation of mycorrhizal root colonization according to Trouvelot et al. (1986). Gene expression of two defense-related genes: chitinase and β-1,3-endoglucanase DNA fingerprinting using the differential display technique The expression of two defensin genes [chitinase (Chi) and Total RNA was extracted from treated common bean roots endoglucanase (EGase)] was examined using a gene-specific TM using a GStract RNA Isolation kit II (Maxim Biomedical, RT-PCR (Bishop et al. 2005): Rockville, MD). About 0.5 g of root sample was subjected Chitinase: primer CHI15 (5′-GGYGGYTGGAATG to RNA extraction, and the extracted RNA was dissolved in ATGG-3′) and anti-sense primer CHI25 (5′-GAYTT DEPC-treated water, quantified spectrophotometrically, and AGATTGGGAATAYCC-3′); the amplified fragment is analyzed on 1.2 % agarose gel. 560 bp. Reverse-transcription (RT)-PCR analyses were per- β-1,3-Endoglucanase: primer EGase forward (5′- formed in a total reaction volume of 25 μL. The reaction TCCGGGGTATGTTATGGAAGA-3′) and EGase re- mixture contained 2.5 μL of 5× buffer with MgCl ,2.5 μL verse (5′-GCCATCCACTCTCAGACACA-3′); the am- (2.5 mM) dNTPs, 1 μL (10 pmol) oligo dT primer plified fragment is 681 bp. (Promega, Madison, WI), 2.5 μLRNA,and 0.2 μLM- The PCR for each gene was performed in a total reaction MuLV reverse transcriptase (New England Biolabs, Ipswich, MA). RT-PCR amplification was performed in a volume of 25 μL containing 2.5 μL 5× Colorless GoTaq® Flexi Buffer, 2.5 μL 5× Green GoTaq® Flexi Buffer, with thermal cycler (Eppendorf, Thermo Fisher Scientific, 2.5 μLMgCl ,3 μLdNTPs,2 μL (10 pmol) primer Dublin, Ireland) programmed at 95 °C for 5 min, 42 °C for 1 h, and 72 °C for 10 min; the cDNA was then stored (forward), 2 μL (10 pmol) primer (reverse), 1.5 μL cDNA, -1 and 0.2 μL(5U μL ) GoTaq® Flexi DNA Polymerase at −20 °C (Chen et al. 2005). The differential display PCR reaction was carried out in a (Promega). PCR amplification was performed in a thermal cycler (Eppendorf, Thermo Fisher Scientific) programmed total reaction volume of 25 μLcontaining2.5 μL 5× Colorless GoTaq® Flexi Buffer, 2.5 μL 5× Green GoTaq® Flexi Buffer, for one cycle at 95 °C for 5 min, followed by 34 cycles of 1 min at 95 °C, 1 min at 41 °C for Chi and at 60 °C for 2.5 μL MgCl , 2.5 mM dNTPs, 5 μL (10 pmol) primer, −1 1.5 μLcDNA, and0.2 μL(5 U μL ) GoTaq® Flexi DNA Egase, and 1 min at 72 °C. The reaction was then incubated at 72 °C for 10 min for a final extension. The gel was Polymerase (Promega). PCR amplification was performed in visualized and photographed using a gel documentation a thermal cycler (Eppendorf, Thermo Fisher Scientific) system, and the gene expression was analyzed using PCR programmed for one cycle at 95 °C for 5 min, 40 cycles of Analyzer ver. 1.0 software (Smith et al. 2007). The reaction 1 min at 95 °C, 1 min at 30 °C, and 1 min at 72 °C, followed by one cycle at 72 °C for 10 min. The following primers were was repeated many times, and the concentration was calcu- lated as the mean of the replicates. used: A1A13 (5′-CAGGCCCTTCCAGCACCCAC-3′), Chi15 (5′-GGYGGYTGGAATGARGG-3′), F1 (5′- CCSCSCCGGATCAAYAAGTWYTAYATC-3′), and P2 (5′- Statistical analysis CGCTGTCGCC-3′). Loading dye (2 μL) was added prior to loading of 10 μL per gel slot. Electrophoresis was performed Data were analyzed with the statistical analysis system at 80 V with 0.5× TBE as running buffer in 1.5 % agarose/ (CoStat 2005). All multiple comparisons were first sub- 0.5× TBE gels. To visualize the electrophoresis products, we jected to analysis of variance (ANOVA). Comparisons -1 stained the gel in 0.5 μgmL (w/v) ethidium bromide solu- among means were made using Duncan’s multiple range tion and then destained it in deionized water. The gel test (Duncan 1955). 1198 Ann Microbiol (2013) 63:1195–1203 Results polymorphism (86.8 %) and the other five bands were monomorphic. The molecular weights of the 38 bands Effect of mycorrhizal colonization on plant growth ranged from 100 to 900 bp. Primers Chi15 and A1A13 were and disease incidence the most informative, allowing 100–91.7 % variation be- tween the examined samples, respectively. With primers F1 The growth parameters of mycorrhizal plants infected with and P2, the percentages of the genetic variation obtained R. solani were significantly enhanced when compared to were 88.9 and 62.5 %, respectively. those of the non-mycorrhizal plants infected with R. solani Most of bands obtained in the treated samples were novel (Table 1). However, AM colonization of pathogen-free bands compared with those of the control sample; this was plants significantly increased shoot and root length, shoot especially evident with primers (Chi15 and A1A13) (Fig. 2). and root dry weight, and leaf area when compared with the For primers F1 and P2, the most conspicuous findings was non-mycorrhizal control (CNM) (Table 1). that of the up-regulated bands (high expression of these bands) in all treated samples compared with the control Disease assessment sample (Fig. 2). Mycorrhizal plants infected with R. solani (PM) showed a Cluster analysis of the data significant decrease in both disease severity and disease incidence when compared with the non-mycorrhizal plants The cluster analysis was carried out using the SPSS (PNM) (Table 2). program. The dendrogram illustrated in Fig. 3 shows that the data analysis based on the DNA band pattern sepa- Mycorrhizal root colonization rated the examined samples into two main groups, with each group containing two subgroups and eight samples. The level of mycorrhizal root colonization continued to in- In the first group, subgroup I contained CM 28, PM 28, crease with increasing plant age in all treatments (Table 3). At and PM 7, while subgroup II contained CNM 7, CNM the same time, the level of mycorrhizal root colonization 28, PNM 28, CNM 1, and CNM 3. In the second group, (frequency and intensity of root colonization and frequency subgroup I contained PM 1, CM 7, and CM 1, while of arbuscules) in the PM treatment at 1 or 4 weeks after subgroup II contained PNM 1, PM 3, PNM 3, PNM 7, inoculation with the pathogen was significantly lower than that and CM 3. of the mycorrhyzal control (CM). No mycorrhizal colonization Based on this analysis, in subgroup I of the first group, was observed in the CNM and PNM treatments. Mycorrhizal samples CM 28 and PM 28 were very closely related to each colonizationinthe rootsofbeanplantsisshown in Fig. 1. other, while the third sample (PM 7) was not closely related to the other two samples. Subgroup II of the first group was DNA fingerprinting using the differential display technique divided into two sub-subgroups. The first contained three samples (CNM 7, CNM 28, PNM 28) and the other Total extracted RNA of 16 treated common bean plants was contained two samples (CNM 1 and CNM 3). In the first subjected to analysis using the differential display technique sub-subgroup, the same observation was obtained as in with four different arbitrary primers (Chi15, A1A13, F1, subgroup I; samples CNM 7 and CNM 28 were very closely P2). About 38 bands were obtained using these four differ- related to each other, while sample PNM 28 was poorly ent arbitrary primers (Table 4), of which 33 bands showed related to these two. The other sub-subgroup showed more Table 1 Effect of mycorrhizal colonization on growth parameters of common bean (Phaseolus vulgaris L.) plants infected with Rhizoctonia root-rot disease a 2 Treatment Shoot length (cm) Shoot dry weight (g) Root length (cm) Root dry weight (g) Leaf area (cm ) CNM 32.9 b 0.68 b 21.8 b 0.32 b 46.4 b CM 37.8 a 0.85 a 29.8 a 0.42 a 63.0 a PNM 26.5 d 0.41 c 18.1 c 0.23 d 34.9 d PM 31.6 bc 0.58 b 21.3 b 0.29 c 43.2 bc Data are presented as the mean of five replicates. Values in each column followed by the same letter(s) are not significantly different according to Duncan’s multiple range test (p00.05) CNM, Untreated control (not mycorrhiza, no pathogen); CM, mycorrhiza only; PNM, pathogen only (no mycorrhiza); PM, mycorrhiza + pathogen Ann Microbiol (2013) 63:1195–1203 1199 Table 2 Effect of mycorrhizal colonization on disease incidence and the induced gene (1, 3, 7, and 28 days post-inoculation) was disease severity of common bean Rhizoctonia root-rot disease -1 converted into DNA (gene) concentration (ng μL )using two- dimensional gel documentation software (Fig. 4b). The results Treatment Disease incidence (%) Disease severity (%) obtained on the first day of inoculation showed an increase in CNM 0c 0c Chi gene expression with all treatments (PM, PNM and CM) CM 0c 0c versus the untreated control (CNM). The highest gene expres- -1 PNM 100 a 84.5 a sion was recorded in the PM treatment (450 ng μL ). PM 73.9 b 67.4 b However, gene expression in the PNM treatment was more than that in the CM treatment when compared with the un- Data are presented as the mean of five replicates. Values in each treated control. In increase in gene expression was lower on column followed by the same letter(s) are not significantly different post-inoculation day 3 than on day 1, but the variation in gene according to Duncan’s multiple range test (p00.05) expression between the treatments remained the same as on the Disease severity was estimated according to Carling et al. (1999) first. On post-inoculation day 7 gene expression of all treat- ments decreased compared with that on post-inoculation day 3, difference, where this sub-subgroup contained only two but the same variation pattern remained. The decrease in gene samples which were poorly related to each other. In the expression continued up to post-inoculation day 28 with the second group, subgroup I showed the same behavior as same variation pattern. subgroup I of the first group, where samples PM 1 and CM 7 were closely related to each other more than the third EGase gene one (CM 1). Finally, subgroup II of the second group was divided into two sub-subgroups. One of these contained two Expression of the EGase gene in common bean plants is samples (PNM 1 and PM 3), which were moderately related shown in Fig. 5a. The band intensity for the differential to each other. The other sub-subgroup included three sam- expression of the induced gene was converted into digital -1 ples (PNM 3, PNM 7, CM 3); samples PNM 3 and PNM 7 DNA (gene) concentration (ng μL ) and is illustrated in were very closely related to each other, while sample CM 3 Fig. 5b. The results on the first day showed higher expres- was moderately related to the other two samples. sion levels in the pathogen-incorporated treatments (PNM and PM) than in the pathogen-free ones (CNM and CM). Gene expression of two defense-related genes However, the gene expression in the treatment (PM) was -1 higher than that in PNM) (400 vs. 200 ng μL , respective- Chi gene ly). On post-inoculation day 3, gene expression increased in -1 treatments PM and PNM (420 and 250 ng μL , respective- Expression of the Chi gene in common bean plants was pre- ly), while no variation was recorded in the CNM and CM sented in Fig. 4a. Band intensity for the different expression of treatments when compared with the first day. Gene expres- sion decreased for all treatments on post-inoculation day 7 Table 3 Effect of infection with Rhizoctonia root-rot disease on the in comparison with the third day, but the same variation levels of mycorrhizal colonization in common bean pattern remained. On post-inoculation day 28, gene expres- a b c sion continued to decrease compared with that on the sev- Treatment Weeks after inoculation F (%) M (%) A (%) with R. solani enth day, with gene expression remaining the highest in the PM treatment. CNM 1 0 d 0 e 0 e CM 92.5 b 43.7 b 15.0 bc PNM 0d 0 e 0e Discussion PM 73.8 c 12.4 d 3.3 d CNM 4 0 d 0 e 0 e Results of DNA fingerprinting using the differential display CM 100.0 a 71.5 a 59.3 a technique demonstrated a markedly genetic polymorphism PNM 0d 0 e 0e (86.8 %) in bean plants as a result of AM colonization. Most PM 93.3 b 34.8 c 15.9 bc of the bands obtained in the mycorrhizal samples were novel bands compared with the control sample, especially those Data are presented as the mean of three replicates. Values in each obtained with primers Chi15 and A1A13. In contrast, with column followed by the same letter(s) are not significantly different according to Duncan’s multiple range test (p00.05) primers F1 and P2, the most conspicuous observation was F (%), Frequency of root colonization, that of up-regulated bands (high expression of these bands) M(%), Intensity of cortical colonization in all treated samples compared with the control sample. A (%), Frequency of arbuscules The results obtained here reveal that the colonized cell made 1200 Ann Microbiol (2013) 63:1195–1203 Fig. 1 Arbuscular mycorrhizal (AM) colonization in the roots of common vesicles, d high magnification image of colonized root showing a vesicle, e bean (Phaseolus vulgaris L.) plants. a Non-mycorrhizal root, b heavily low magnification image of colonized root showing many arbuscules, f colonized root, c low magnification image of colonized root showing many high magnification image of colonized root showing arbuscules use of two mechanisms: (1) switching on of new genes; (2) This genetic variation was also supported by the cluster induction of other active genes to a higher expression level. analysis of the data. Table 4 Polymorphism among the bands obtained using the differential display technique No. Primer Sequence (5′–3′) length (bp) Total no. No. of polymorphic Polymorphism (%) of bands bands 1 Chi15 GGYGGYTGGAATG-ARGG 100–500 9 9 100 2 A1A13 CAGGCCCTTCCAG-CACCCAC 100–900 12 11 91.7 3 F1 CCSCSCCGGATCAA-YAAGTWYTAYATC 200–700 9 8 88.9 4 P2 CGCTGTCGCC 300–700 8 5 62.5 Total 38 33 86.8 Ann Microbiol (2013) 63:1195–1203 1201 Fig. 2 DNA fingerprinting using the differential display technique. Four different arbitrary primers were used: Chi15 (a), A1A13 (b), F1 (c), P2 (d). Treatment: CNM Untreated control (not mycorrhiza, no pathogen), CM mycorrhiza only, PNM pathogen only (no mycorrhiza), PM mycorrhiza + pathogen Our results show that infection with the pathogen singly and frequency of arbuscules), the expression of the two led to a higher induction in the expression of the two defense genes in mycorrhizal bean plants infected with the defense genes (Chi and EGase) than that which occurred pathogen was higher than that recorded in plants treated in the case of AM colonization alone (pathogen-free). with either one alone, indicating a synergistic effect when Despite the negative effects of pathogen infection on AM both AM and the pathogen were present. The work of colonization (frequency and intensity of root colonization Lambais and Mehdy (1998) lends support to our results. These authors described Chi and EGase coding mRNA accumulation in arbuscule-containing cells and adjacent CM 28 ones and the repression of EGase mRNA accumulation PM 28 some millimeters distant from the AM fungi colonized zone. PM 7 98 81 CNM 7 In an in situ hybridization study of common bean colonized CNM 28 roots using probes for PAL and Chi, Blee and Anderson PNM 28 (1996) showed that the accumulation of both transcripts CNM 1 CNM 3 occurred only in arbusculated cells. In addition, the accu- PM 1 92 mulation of both PAL and Chi mRNA was greater in cortical CM 7 cells containing young arbuscules than in cells containing CM 1 PNM 1 clumped arbuscules. These results may explain the decrease PM 3 in gene expression of Chi and EGase over time in the PNM 3 mycorrhizal roots in our study. In another study, Pozo et PNM 7 CM3 al. (1999) studied β-1,3-glucanases in tomato roots after AM colonization by Glomus mosseae and G. intraradices Fig. 3 Dendrogram using average linkage (between groups). Number and/or pathogenic infection by Phytophthora parasitica us- on bar Genetic similarity between samples, with genetic similarity increasing with increasing value ing polyacrylamide gel electrophoresis. In control roots, two 1202 Ann Microbiol (2013) 63:1195–1203 acidic EGase isoforms were constitutively expressed, and their activity was higher in that in mycorrhizal roots. Two additional acidic isoforms were detected in extracts from G. mosseae-colonized tomato roots, but not in G. intrara- dices-colonized roots. In addition, when tomato plants were pre-inoculated with G. mosseae and post-infected with P. parasitica, two additional basic isoforms were clearly revealed. The findings of Garmendia et al. (2006) are in agreement with these results. These authors studied the role of AM colonization in the induction of defense-related enzymatic activities in pepper roots before and after infec- tion with Verticillium dahliae. Their results show that the CNM CM PNM PM colonization of pepper roots by Glomus deserticola induced the appearance of new isoforms of acidic chitinases, super- oxide dismutase and, at early stages, peroxidases, but only 350 in mycorrhizal plants, and that the inoculation with V. dahliae slightly increased both phenylalanine ammonia- 200 lyase and peroxidase activities 2 weeks later. In this same context, El-Khallal (2007) also recorded an induction in these enzymes in infected pepper and tomato plants, re- spectively, when the plants were pre-colonized by AM 1 day 3 days 7 days 28 days fungi. These enzymes were found at low levels in healthy Time plants; however, their expression was induced during Fig. 4 Effect of AM colonization on gene expression of the chitinase pathogen attack. In contrast, Guenoune et al. (2001) found gene (a) and DNA concentration (b). Results are presented as the mean that defense responses of alfalfa roots to the pathogenic of three independent experiments fungus Rhizoctonia solani were reduced significantly in roots simultaneously colonized by the AM fungus G. intraradices. The production of chitinases elicits other plant responses, including the synthesis of antifungal phy- toalexins (Gooday 1999). In addition, the production of Chi and EGase are considered important in the biological control of soil-borne pathogens because of their ability to degrade major component of cell walls (chitin and β-1,3- glucan, respectively) (Cota et al. 2007). No effects of inoculation with Rhizobium leguminosa- rum were found (i.e., no nodulation occurred) as the Egyptian soil conditions do not support the nodulation of the common bean. Generally, P. vulgaris is considered to be a poor fixer of atmospheric nitrogen compared with CNM CM PNM PM other legume crops (Graham 1981) and generally responds poorly to inoculation with Rhizobium leguminosarum bv. phaseoli strains (Buttery et al. 1987). The response depends on the ability of each strain to adapt to environ- mental stress factors, such as soil pH. Our data shows that AM colonization of common bean plants infected with R. solani resulted in a significant en- hancement in the plant growth parameters and also a signif- icant reduction in both disease severity and disease incidence. 1 day 3 days 7 days 28 days These findings support our molecular findings. Based on Time these results, we conclude that the application of AM fungi played an important role in enhancing plant resistance mo- Fig. 5 Effect of AM colonization on gene expression of the β-1,3- lecularly against R. solani via the activation of a number of endoglucanase gene (a) and DNA concentration (b). Results are pre- sented as the mean of three independent experiments plant defense genes. Concentration (ng/µl) Concentration (ng/µl) Ann Microbiol (2013) 63:1195–1203 1203 Acknowledgments The authors extend their appreciation to the Garmendia I, Aguirreolea J, Goicoechea N (2006) Defense-related Deanship of Scientific Research at King Saud University for funding enzymes in pepper roots during interactions with arbuscular mycor- this work. Our deep gratitude is extended to Dr. Khalid Ghoneem rhizal fungi and/or Verticillium dahliae. BioControl 51:293–310 (Plant Pathology Research Institute, Agricultural Research Center) Gooday GW (1999) Aggressive and defensive roles for chitinases. In: for his sincere help in the fungal identification. Many thanks to all Jolles P, Muzzarelli RAA (eds) Chitin and chitinases. Birkhäuser, those who have helped us in the Plant Molecular Pathology Lab., Basel, pp 157–170 Mubarak City for Scientific Research, especially Ms. Ghada Ali. 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Ann Agric Sci 49:733–745 Trouvelot A, Kough JL, Gianinazzi-Pearson V (1986) Mesure du taux El-Khallal SM (2007) Induction and modulation of resistance in toma- de mycorhization VA d’un système radiculaire recherche de meth- to plants against Fusarium wilt disease by bioagent fungi (arbus- ods d’estimation ayant une signification fonctionnelle. In: cular mycorrhiza) and/or hormonal elicitors (jasmonic acid and Gianinazzi-Pearson V, Gianinazzi S (eds) Physiological and ge- salicylic acid): 2-changes in the antioxidant enzymes, phenolic netical aspects of mycorrhizae. INRA Publ, Paris, pp 217–221 compounds and pathogen related-proteins. Aust J Basic Appl Sci Tu CC, Hsieh TF, Chang YC (1996) Vegetable diseases incited by 1:717–732 Rhizoctonia spp. In: Sneh B, Jabaji-Hare S, Neate S, Dijst G (eds) El-Tarabily KA (2004) Suppression of Rhizoctonia Solani diseases of Rhizoctonia species: taxonomy, molecular biology, ecology, pa- sugar beet by antagonistic and plant growth-promoting yeasts. J thology and disease control. Kluwer, Dordrecht,, pp 369–377 Appl Microbiol 96:69–75 Zeng RS (2006) Disease resistance in plants through mycorrhizal Garcia-Garrido JM, Ocampo JA (2002) Regulation of the plant defence fungi induced allelochemicals. In: Inderjit KG, Mukerji (eds) response in arbuscular mycorrhizal symbiosis. J Exp Bot 53:1377– Allelochemicals: biological control of plant pathogens and dis- 1386 eases. Springer SBM, Dordrecht, pp 181–192 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Molecular defense response of mycorrhizal bean plants infected with Rhizoctonia solani

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Springer Journals
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Copyright © 2013 by Springer-Verlag Berlin Heidelberg and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-012-0578-5
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Abstract

Ann Microbiol (2013) 63:1195–1203 DOI 10.1007/s13213-012-0578-5 ORIGINAL ARTICLE Molecular defense response of mycorrhizal bean plants infected with Rhizoctonia solani Elsayed E. Hafez & Gamal M. Abdel-Fattah & Safwat A. El-Haddad & Younes M. Rashad Received: 14 August 2012 /Accepted: 21 November 2012 /Published online: 19 January 2013 Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract A time course study was conducted to investigate Keywords Arbuscular mycorrhizal fungi . . disease development and molecular defense response in common β-1,3-Endoglucanase Chitinase Differential display bean (Phaseolus vulgaris L.) plants colonized by a mixture of five technique Gene expression and root rot arbuscular mycorrhizal (AM) fungi, namely, Glomus mosseae, G. intraradices, G. clarum, Gigaspora gigantea,and Gigaspora margarita, and post-infected with the soil-borne pathogen Introduction Rhizoctonia solani. Results showed that pre-colonization of bean plants by AM fungi significantly reduced disease severity and Rhizoctonia solani Kühn is a common necrotrophic soil-borne disease incidence. DNA fingerprinting using the differential dis- fungus which can cause seed decay, damping-off, stem canker, play technique revealed a genetic polymorphism (86.8 %) in bean root rot, fruit decay, and foliage diseases in a wide range of plants that resulted from the colonization by AM fungi. Two plant species over a large part of the world (Tu et al. 1996). genetic mechanisms were recorded: (1) switching on of new This unlimited host range, combined with competitive sapro- genes and (2) induction of other active genes, including the phytic ability and lethal pathogenic potential, earns R. solani its defense genes chitinase and β-1,3-glucanase, to a highly status as formidable pathogen. expressed state. During recent years research has focused on identifying potential biocontrol agents to reduce the severity of root-rot disease caused by R. solani. A limited number of fungal E. E. Hafez antagonists against R. solani have been reported, among which City for Scientific Research and Technology Applications, antagonistic and plant growth-promoting yeasts (El-Tarabily Alexandria, Egypt e-mail: elsayed_hafez@yahoo.com 2004), Penicillium spp. (Ciavatta et al. 2006), Trichoderma koningii, T. pseudokoningii, T. viride, T. polysporum, T. aureo- G. M. Abdel-Fattah viride (Shalini and Kotasthane 2007), and Gliocladium roseum Botany Department, College of Science, Mansoura University, (Tarantino et al. 2007) are known to be very efficient. Bacterial Mansoura, Egypt e-mail: Abdelfattaham@yahoo.com antagonism has also been reported against R. solani (Kai et al. 2007). Among the potential biocontrol agents, the arbuscular S. A. El-Haddad mycorrhizal (AM) fungi have received special attention (Aly Mycological Research and Disease Survey Department, Plant and Manal 2009; Abdel-Fattah et al. 2011). Biological control Pathology Institute, Agricultural Research Center, Giza, Egypt using AM fungi is unique, being an eco-friendly and cost- e-mail: safwat@epq.gov.eg effective strategy for disease management that provides greater Y. M. Rashad (*) levels of protection and sustains plant yields, in addition to the Biology Department, Teachers College, King Saud University, positive effects on the plant growth and its nutrition. Riyadh, Saudi Arabia Common bean (Phaseolus vulgaris L.) is susceptible to e-mail: younesrashad@yahoo.com Rhizoctonia root-rot disease in most of the tropical, subtrop- Present Address: ical, and temperate areas of the world where it is grown. G. M. Abdel-Fattah Yield losses of 5–10 % are common, but 60 % yield losses Plant Production Department, College of Food and Agricultural have been reported in Brazil (Tu et al. 1996). Sciences, King Saud University, Riyadh, Saudi Arabia 1196 Ann Microbiol (2013) 63:1195–1203 Mechanisms that can account for the disease control ability Schenck, Gigaspora gigantea (Nicol. & Gerd.) Gerd. of AM fungi may include competition for infection sites and & Trappe, and Gigaspora margarita (Becker & Hall) 6 -1 host photosynthates, root damage compensation, enhance- in suspension at a concentration of 1×10 spores L ment of plant resistance through various physical and physi- (El-Haddad et al. 2004). ological mechanisms, such as increasing the cell-wall thickness of the host or the accumulation of some antimicro- Planting and growth conditions bial substances (Abdel-Fattah et al. 2011), or changes in the composition of the microbial communities in the mycorrhizo- Pots (diameter 25 cm) were each filled with 2.5 kg sterilized sphere (Singh et al. 2000). Several inducible defense-related soil (autoclaving at 121 °C for 2× 1 h). A sandy-loam soil genes, including those encoding isoflavonoid phytoalexins, (sand:silt:clay, 70:20:10 %) was used; soil characteristics such as phenylalanine ammonia lyase, chalcone synthase, and −1 were pH 8, electrical conductivity 270 μScm , total nitro- chalcone isomerase, have been reported to be induced during gen 1.05 %, total organic matter 1.64 %, and phosphorus mycorrhizal establishment (Guillon et al. 2002). The expres- −1 content 4.43 μgg . Five healthy seeds of common bean sion of genes encoding enzymes that synthesize phenolpropa- Phaseolus vulgaris L. (cv. Giza 3) were surface-disinfected noid compounds has also been detected in mycorrhizal roots with 2 % sodium hypochlorite for 2 min, washed with sterile (Garcia-Garrido and Ocampo 2002). Other defense-related distilled water, and sown in each pot. Half of the pots genes shown to be up-regulated in mycorrhizal symbioses received AM inoculum as a suspension at two time points: include genes involved in the metabolism of reactive oxygen in the bean seed bed at the beginning of the experiment and species, chitinase, and β-1,3-glucanase, and genes involved in as a soil drench 14 days after sowing) at a concentration of senescence, including glutathione-S-transferase (Zeng 2006). −1 5mLL water (El-Haddad et al. 2004). Plants were inoc- The role of AM colonization in sustaining bean plants ulated with Rhizobium leguminosarum one time with against R. solani is unclear. Depending on the time after infec- 1 mL of culture suspension at a concentration of 5×10 tion with R. solani and the tissue examined, responses varying −1 CFU mL and watered regularly to near field capacity with from stimulation to suppression to no change in transcript levels tap water. Pots did not receive any fertilizers in this study. All have been detected (Guillon et al. 2002). Therefore, the aim of pots were kept outdoors under natural conditions [day temper- our study was to investigate the molecular aspects of defense ature 25 °C, night temperature 20 °C, 16/8-h (light/dark) responses in mycorrhizal bean plants infected with R. solani. photoperiod]. At 4 weeks after inoculation with AM, the pathogen inoculum was mixed with the upper layer of the pot soil at Materials and methods a rate of 2 % (w/w) potential inoculum. Five pots were treated with tap water to serve as a negative control. Five Causal organism and bean cultivar pots were used as replicates for each treatment. The treat- ments applied in this study can be summarized as follows: Rhizoctonia solani (AG-2-2 IIIB) was isolated originally from untreated control (no mycorrhiza, no pathogen; CNM); my- naturally diseased common bean plants. Fungal identification corrhiza only (CM); pathogen only (no mycorrhiza; PNM); was based on cultural properties and morphological and mi- mycorrhiza + pathogen (PM). All pots were arranged in a croscopical characteristics, as described by Sneh et al. (1991). completely randomized design. Three plants from each The isolate was then assigned an AG designation according to treatment were harvested at 1, 3, 7, and 28 days after hyphal anastomosis with tester isolates from AG 1 to AG 10 pathogen infection for molecular analysis (Guillon et al. using the slide technique of Kronland and Stanghellini (1988). 2002; Mohr et al. 1998). Five plants of each treatment were The inoculum was prepared by growing the pathogen in bottles carefully harvested with their entire roots (2 weeks after containing sterilized sorghum grains for 15 days at 25±2 °C. inoculation with the pathogen), washed under running water The most common bean cultivar in Egypt (Giza 3) was used. to remove soil particles, and evaluated for shoot length, root length, leaf area, and shoot and root dry weights. Dry AM inoculum weights (in grams) were recorded after the samples had been dried at 80 °C for at least 48 h in a hot air oven until a A mixture of Egyptian formulated AM fungi (Multi-VAM) constant weight was reached. Disease severity (DS) and kindly provided by Dr. Safwat El-Haddad (Mycological disease incidence (DI) of the Rhizoctonia root rot were Research and Disease Survey Department, Plant Pathology assessed (2 weeks after inoculation with the pathogen) for Institute, Agricultural Research Center, Giza, Egypt) was each treatment. Disease severity was estimated as the used. This mixture consists of spores (in equal proportions) degree of root damage according to the scale of Carling of Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe, G. et al. (1999). intraradices Schenck & Smith, G. clarum Nicol. & Ann Microbiol (2013) 63:1195–1203 1197 Staining and estimation of mycorrhizal root colonization was visualized and photographed using a gel documen- tation system. Five plants of each treatment were carefully harvested with DNA fingerprints obtained using the differential display their entire roots and washed under running water to remove technique were first analyzed visually and a positive re- soil particles. The roots were separated and fixed in FAA sponse (a score ‘1’) was defined as the presence of a visible (formalin–acetic acid–alcohol) for evaluation of mycorrhi- band of a given size, while a negative response (a score zal root colonization. Roots fixed in FAA were rinsed re- ‘0’) was defined as the absence of any band of the peatedly in tap water, cut into small segments (0.5–1 cm), same size. These scores were then merged in a and stained with 0.05 % trypan blue (Sigma, St. Louis, MO) Microsoft Excel spreadsheet and then inserted in the according to Phillips and Hayman (1970). Fifty randomly SPSS ver. 13.0 computer software (SPSS 2004)for the selected stained root segments of each treatment were construction of the dendrogram using the genetic dis- mounted on slides in lactoglycerol and examined micro- tance method. scopically for estimation of mycorrhizal root colonization according to Trouvelot et al. (1986). Gene expression of two defense-related genes: chitinase and β-1,3-endoglucanase DNA fingerprinting using the differential display technique The expression of two defensin genes [chitinase (Chi) and Total RNA was extracted from treated common bean roots endoglucanase (EGase)] was examined using a gene-specific TM using a GStract RNA Isolation kit II (Maxim Biomedical, RT-PCR (Bishop et al. 2005): Rockville, MD). About 0.5 g of root sample was subjected Chitinase: primer CHI15 (5′-GGYGGYTGGAATG to RNA extraction, and the extracted RNA was dissolved in ATGG-3′) and anti-sense primer CHI25 (5′-GAYTT DEPC-treated water, quantified spectrophotometrically, and AGATTGGGAATAYCC-3′); the amplified fragment is analyzed on 1.2 % agarose gel. 560 bp. Reverse-transcription (RT)-PCR analyses were per- β-1,3-Endoglucanase: primer EGase forward (5′- formed in a total reaction volume of 25 μL. The reaction TCCGGGGTATGTTATGGAAGA-3′) and EGase re- mixture contained 2.5 μL of 5× buffer with MgCl ,2.5 μL verse (5′-GCCATCCACTCTCAGACACA-3′); the am- (2.5 mM) dNTPs, 1 μL (10 pmol) oligo dT primer plified fragment is 681 bp. (Promega, Madison, WI), 2.5 μLRNA,and 0.2 μLM- The PCR for each gene was performed in a total reaction MuLV reverse transcriptase (New England Biolabs, Ipswich, MA). RT-PCR amplification was performed in a volume of 25 μL containing 2.5 μL 5× Colorless GoTaq® Flexi Buffer, 2.5 μL 5× Green GoTaq® Flexi Buffer, with thermal cycler (Eppendorf, Thermo Fisher Scientific, 2.5 μLMgCl ,3 μLdNTPs,2 μL (10 pmol) primer Dublin, Ireland) programmed at 95 °C for 5 min, 42 °C for 1 h, and 72 °C for 10 min; the cDNA was then stored (forward), 2 μL (10 pmol) primer (reverse), 1.5 μL cDNA, -1 and 0.2 μL(5U μL ) GoTaq® Flexi DNA Polymerase at −20 °C (Chen et al. 2005). The differential display PCR reaction was carried out in a (Promega). PCR amplification was performed in a thermal cycler (Eppendorf, Thermo Fisher Scientific) programmed total reaction volume of 25 μLcontaining2.5 μL 5× Colorless GoTaq® Flexi Buffer, 2.5 μL 5× Green GoTaq® Flexi Buffer, for one cycle at 95 °C for 5 min, followed by 34 cycles of 1 min at 95 °C, 1 min at 41 °C for Chi and at 60 °C for 2.5 μL MgCl , 2.5 mM dNTPs, 5 μL (10 pmol) primer, −1 1.5 μLcDNA, and0.2 μL(5 U μL ) GoTaq® Flexi DNA Egase, and 1 min at 72 °C. The reaction was then incubated at 72 °C for 10 min for a final extension. The gel was Polymerase (Promega). PCR amplification was performed in visualized and photographed using a gel documentation a thermal cycler (Eppendorf, Thermo Fisher Scientific) system, and the gene expression was analyzed using PCR programmed for one cycle at 95 °C for 5 min, 40 cycles of Analyzer ver. 1.0 software (Smith et al. 2007). The reaction 1 min at 95 °C, 1 min at 30 °C, and 1 min at 72 °C, followed by one cycle at 72 °C for 10 min. The following primers were was repeated many times, and the concentration was calcu- lated as the mean of the replicates. used: A1A13 (5′-CAGGCCCTTCCAGCACCCAC-3′), Chi15 (5′-GGYGGYTGGAATGARGG-3′), F1 (5′- CCSCSCCGGATCAAYAAGTWYTAYATC-3′), and P2 (5′- Statistical analysis CGCTGTCGCC-3′). Loading dye (2 μL) was added prior to loading of 10 μL per gel slot. Electrophoresis was performed Data were analyzed with the statistical analysis system at 80 V with 0.5× TBE as running buffer in 1.5 % agarose/ (CoStat 2005). All multiple comparisons were first sub- 0.5× TBE gels. To visualize the electrophoresis products, we jected to analysis of variance (ANOVA). Comparisons -1 stained the gel in 0.5 μgmL (w/v) ethidium bromide solu- among means were made using Duncan’s multiple range tion and then destained it in deionized water. The gel test (Duncan 1955). 1198 Ann Microbiol (2013) 63:1195–1203 Results polymorphism (86.8 %) and the other five bands were monomorphic. The molecular weights of the 38 bands Effect of mycorrhizal colonization on plant growth ranged from 100 to 900 bp. Primers Chi15 and A1A13 were and disease incidence the most informative, allowing 100–91.7 % variation be- tween the examined samples, respectively. With primers F1 The growth parameters of mycorrhizal plants infected with and P2, the percentages of the genetic variation obtained R. solani were significantly enhanced when compared to were 88.9 and 62.5 %, respectively. those of the non-mycorrhizal plants infected with R. solani Most of bands obtained in the treated samples were novel (Table 1). However, AM colonization of pathogen-free bands compared with those of the control sample; this was plants significantly increased shoot and root length, shoot especially evident with primers (Chi15 and A1A13) (Fig. 2). and root dry weight, and leaf area when compared with the For primers F1 and P2, the most conspicuous findings was non-mycorrhizal control (CNM) (Table 1). that of the up-regulated bands (high expression of these bands) in all treated samples compared with the control Disease assessment sample (Fig. 2). Mycorrhizal plants infected with R. solani (PM) showed a Cluster analysis of the data significant decrease in both disease severity and disease incidence when compared with the non-mycorrhizal plants The cluster analysis was carried out using the SPSS (PNM) (Table 2). program. The dendrogram illustrated in Fig. 3 shows that the data analysis based on the DNA band pattern sepa- Mycorrhizal root colonization rated the examined samples into two main groups, with each group containing two subgroups and eight samples. The level of mycorrhizal root colonization continued to in- In the first group, subgroup I contained CM 28, PM 28, crease with increasing plant age in all treatments (Table 3). At and PM 7, while subgroup II contained CNM 7, CNM the same time, the level of mycorrhizal root colonization 28, PNM 28, CNM 1, and CNM 3. In the second group, (frequency and intensity of root colonization and frequency subgroup I contained PM 1, CM 7, and CM 1, while of arbuscules) in the PM treatment at 1 or 4 weeks after subgroup II contained PNM 1, PM 3, PNM 3, PNM 7, inoculation with the pathogen was significantly lower than that and CM 3. of the mycorrhyzal control (CM). No mycorrhizal colonization Based on this analysis, in subgroup I of the first group, was observed in the CNM and PNM treatments. Mycorrhizal samples CM 28 and PM 28 were very closely related to each colonizationinthe rootsofbeanplantsisshown in Fig. 1. other, while the third sample (PM 7) was not closely related to the other two samples. Subgroup II of the first group was DNA fingerprinting using the differential display technique divided into two sub-subgroups. The first contained three samples (CNM 7, CNM 28, PNM 28) and the other Total extracted RNA of 16 treated common bean plants was contained two samples (CNM 1 and CNM 3). In the first subjected to analysis using the differential display technique sub-subgroup, the same observation was obtained as in with four different arbitrary primers (Chi15, A1A13, F1, subgroup I; samples CNM 7 and CNM 28 were very closely P2). About 38 bands were obtained using these four differ- related to each other, while sample PNM 28 was poorly ent arbitrary primers (Table 4), of which 33 bands showed related to these two. The other sub-subgroup showed more Table 1 Effect of mycorrhizal colonization on growth parameters of common bean (Phaseolus vulgaris L.) plants infected with Rhizoctonia root-rot disease a 2 Treatment Shoot length (cm) Shoot dry weight (g) Root length (cm) Root dry weight (g) Leaf area (cm ) CNM 32.9 b 0.68 b 21.8 b 0.32 b 46.4 b CM 37.8 a 0.85 a 29.8 a 0.42 a 63.0 a PNM 26.5 d 0.41 c 18.1 c 0.23 d 34.9 d PM 31.6 bc 0.58 b 21.3 b 0.29 c 43.2 bc Data are presented as the mean of five replicates. Values in each column followed by the same letter(s) are not significantly different according to Duncan’s multiple range test (p00.05) CNM, Untreated control (not mycorrhiza, no pathogen); CM, mycorrhiza only; PNM, pathogen only (no mycorrhiza); PM, mycorrhiza + pathogen Ann Microbiol (2013) 63:1195–1203 1199 Table 2 Effect of mycorrhizal colonization on disease incidence and the induced gene (1, 3, 7, and 28 days post-inoculation) was disease severity of common bean Rhizoctonia root-rot disease -1 converted into DNA (gene) concentration (ng μL )using two- dimensional gel documentation software (Fig. 4b). The results Treatment Disease incidence (%) Disease severity (%) obtained on the first day of inoculation showed an increase in CNM 0c 0c Chi gene expression with all treatments (PM, PNM and CM) CM 0c 0c versus the untreated control (CNM). The highest gene expres- -1 PNM 100 a 84.5 a sion was recorded in the PM treatment (450 ng μL ). PM 73.9 b 67.4 b However, gene expression in the PNM treatment was more than that in the CM treatment when compared with the un- Data are presented as the mean of five replicates. Values in each treated control. In increase in gene expression was lower on column followed by the same letter(s) are not significantly different post-inoculation day 3 than on day 1, but the variation in gene according to Duncan’s multiple range test (p00.05) expression between the treatments remained the same as on the Disease severity was estimated according to Carling et al. (1999) first. On post-inoculation day 7 gene expression of all treat- ments decreased compared with that on post-inoculation day 3, difference, where this sub-subgroup contained only two but the same variation pattern remained. The decrease in gene samples which were poorly related to each other. In the expression continued up to post-inoculation day 28 with the second group, subgroup I showed the same behavior as same variation pattern. subgroup I of the first group, where samples PM 1 and CM 7 were closely related to each other more than the third EGase gene one (CM 1). Finally, subgroup II of the second group was divided into two sub-subgroups. One of these contained two Expression of the EGase gene in common bean plants is samples (PNM 1 and PM 3), which were moderately related shown in Fig. 5a. The band intensity for the differential to each other. The other sub-subgroup included three sam- expression of the induced gene was converted into digital -1 ples (PNM 3, PNM 7, CM 3); samples PNM 3 and PNM 7 DNA (gene) concentration (ng μL ) and is illustrated in were very closely related to each other, while sample CM 3 Fig. 5b. The results on the first day showed higher expres- was moderately related to the other two samples. sion levels in the pathogen-incorporated treatments (PNM and PM) than in the pathogen-free ones (CNM and CM). Gene expression of two defense-related genes However, the gene expression in the treatment (PM) was -1 higher than that in PNM) (400 vs. 200 ng μL , respective- Chi gene ly). On post-inoculation day 3, gene expression increased in -1 treatments PM and PNM (420 and 250 ng μL , respective- Expression of the Chi gene in common bean plants was pre- ly), while no variation was recorded in the CNM and CM sented in Fig. 4a. Band intensity for the different expression of treatments when compared with the first day. Gene expres- sion decreased for all treatments on post-inoculation day 7 Table 3 Effect of infection with Rhizoctonia root-rot disease on the in comparison with the third day, but the same variation levels of mycorrhizal colonization in common bean pattern remained. On post-inoculation day 28, gene expres- a b c sion continued to decrease compared with that on the sev- Treatment Weeks after inoculation F (%) M (%) A (%) with R. solani enth day, with gene expression remaining the highest in the PM treatment. CNM 1 0 d 0 e 0 e CM 92.5 b 43.7 b 15.0 bc PNM 0d 0 e 0e Discussion PM 73.8 c 12.4 d 3.3 d CNM 4 0 d 0 e 0 e Results of DNA fingerprinting using the differential display CM 100.0 a 71.5 a 59.3 a technique demonstrated a markedly genetic polymorphism PNM 0d 0 e 0e (86.8 %) in bean plants as a result of AM colonization. Most PM 93.3 b 34.8 c 15.9 bc of the bands obtained in the mycorrhizal samples were novel bands compared with the control sample, especially those Data are presented as the mean of three replicates. Values in each obtained with primers Chi15 and A1A13. In contrast, with column followed by the same letter(s) are not significantly different according to Duncan’s multiple range test (p00.05) primers F1 and P2, the most conspicuous observation was F (%), Frequency of root colonization, that of up-regulated bands (high expression of these bands) M(%), Intensity of cortical colonization in all treated samples compared with the control sample. A (%), Frequency of arbuscules The results obtained here reveal that the colonized cell made 1200 Ann Microbiol (2013) 63:1195–1203 Fig. 1 Arbuscular mycorrhizal (AM) colonization in the roots of common vesicles, d high magnification image of colonized root showing a vesicle, e bean (Phaseolus vulgaris L.) plants. a Non-mycorrhizal root, b heavily low magnification image of colonized root showing many arbuscules, f colonized root, c low magnification image of colonized root showing many high magnification image of colonized root showing arbuscules use of two mechanisms: (1) switching on of new genes; (2) This genetic variation was also supported by the cluster induction of other active genes to a higher expression level. analysis of the data. Table 4 Polymorphism among the bands obtained using the differential display technique No. Primer Sequence (5′–3′) length (bp) Total no. No. of polymorphic Polymorphism (%) of bands bands 1 Chi15 GGYGGYTGGAATG-ARGG 100–500 9 9 100 2 A1A13 CAGGCCCTTCCAG-CACCCAC 100–900 12 11 91.7 3 F1 CCSCSCCGGATCAA-YAAGTWYTAYATC 200–700 9 8 88.9 4 P2 CGCTGTCGCC 300–700 8 5 62.5 Total 38 33 86.8 Ann Microbiol (2013) 63:1195–1203 1201 Fig. 2 DNA fingerprinting using the differential display technique. Four different arbitrary primers were used: Chi15 (a), A1A13 (b), F1 (c), P2 (d). Treatment: CNM Untreated control (not mycorrhiza, no pathogen), CM mycorrhiza only, PNM pathogen only (no mycorrhiza), PM mycorrhiza + pathogen Our results show that infection with the pathogen singly and frequency of arbuscules), the expression of the two led to a higher induction in the expression of the two defense genes in mycorrhizal bean plants infected with the defense genes (Chi and EGase) than that which occurred pathogen was higher than that recorded in plants treated in the case of AM colonization alone (pathogen-free). with either one alone, indicating a synergistic effect when Despite the negative effects of pathogen infection on AM both AM and the pathogen were present. The work of colonization (frequency and intensity of root colonization Lambais and Mehdy (1998) lends support to our results. These authors described Chi and EGase coding mRNA accumulation in arbuscule-containing cells and adjacent CM 28 ones and the repression of EGase mRNA accumulation PM 28 some millimeters distant from the AM fungi colonized zone. PM 7 98 81 CNM 7 In an in situ hybridization study of common bean colonized CNM 28 roots using probes for PAL and Chi, Blee and Anderson PNM 28 (1996) showed that the accumulation of both transcripts CNM 1 CNM 3 occurred only in arbusculated cells. In addition, the accu- PM 1 92 mulation of both PAL and Chi mRNA was greater in cortical CM 7 cells containing young arbuscules than in cells containing CM 1 PNM 1 clumped arbuscules. These results may explain the decrease PM 3 in gene expression of Chi and EGase over time in the PNM 3 mycorrhizal roots in our study. In another study, Pozo et PNM 7 CM3 al. (1999) studied β-1,3-glucanases in tomato roots after AM colonization by Glomus mosseae and G. intraradices Fig. 3 Dendrogram using average linkage (between groups). Number and/or pathogenic infection by Phytophthora parasitica us- on bar Genetic similarity between samples, with genetic similarity increasing with increasing value ing polyacrylamide gel electrophoresis. In control roots, two 1202 Ann Microbiol (2013) 63:1195–1203 acidic EGase isoforms were constitutively expressed, and their activity was higher in that in mycorrhizal roots. Two additional acidic isoforms were detected in extracts from G. mosseae-colonized tomato roots, but not in G. intrara- dices-colonized roots. In addition, when tomato plants were pre-inoculated with G. mosseae and post-infected with P. parasitica, two additional basic isoforms were clearly revealed. The findings of Garmendia et al. (2006) are in agreement with these results. These authors studied the role of AM colonization in the induction of defense-related enzymatic activities in pepper roots before and after infec- tion with Verticillium dahliae. Their results show that the CNM CM PNM PM colonization of pepper roots by Glomus deserticola induced the appearance of new isoforms of acidic chitinases, super- oxide dismutase and, at early stages, peroxidases, but only 350 in mycorrhizal plants, and that the inoculation with V. dahliae slightly increased both phenylalanine ammonia- 200 lyase and peroxidase activities 2 weeks later. In this same context, El-Khallal (2007) also recorded an induction in these enzymes in infected pepper and tomato plants, re- spectively, when the plants were pre-colonized by AM 1 day 3 days 7 days 28 days fungi. These enzymes were found at low levels in healthy Time plants; however, their expression was induced during Fig. 4 Effect of AM colonization on gene expression of the chitinase pathogen attack. In contrast, Guenoune et al. (2001) found gene (a) and DNA concentration (b). Results are presented as the mean that defense responses of alfalfa roots to the pathogenic of three independent experiments fungus Rhizoctonia solani were reduced significantly in roots simultaneously colonized by the AM fungus G. intraradices. The production of chitinases elicits other plant responses, including the synthesis of antifungal phy- toalexins (Gooday 1999). In addition, the production of Chi and EGase are considered important in the biological control of soil-borne pathogens because of their ability to degrade major component of cell walls (chitin and β-1,3- glucan, respectively) (Cota et al. 2007). No effects of inoculation with Rhizobium leguminosa- rum were found (i.e., no nodulation occurred) as the Egyptian soil conditions do not support the nodulation of the common bean. Generally, P. vulgaris is considered to be a poor fixer of atmospheric nitrogen compared with CNM CM PNM PM other legume crops (Graham 1981) and generally responds poorly to inoculation with Rhizobium leguminosarum bv. phaseoli strains (Buttery et al. 1987). The response depends on the ability of each strain to adapt to environ- mental stress factors, such as soil pH. Our data shows that AM colonization of common bean plants infected with R. solani resulted in a significant en- hancement in the plant growth parameters and also a signif- icant reduction in both disease severity and disease incidence. 1 day 3 days 7 days 28 days These findings support our molecular findings. Based on Time these results, we conclude that the application of AM fungi played an important role in enhancing plant resistance mo- Fig. 5 Effect of AM colonization on gene expression of the β-1,3- lecularly against R. solani via the activation of a number of endoglucanase gene (a) and DNA concentration (b). Results are pre- sented as the mean of three independent experiments plant defense genes. Concentration (ng/µl) Concentration (ng/µl) Ann Microbiol (2013) 63:1195–1203 1203 Acknowledgments The authors extend their appreciation to the Garmendia I, Aguirreolea J, Goicoechea N (2006) Defense-related Deanship of Scientific Research at King Saud University for funding enzymes in pepper roots during interactions with arbuscular mycor- this work. Our deep gratitude is extended to Dr. Khalid Ghoneem rhizal fungi and/or Verticillium dahliae. BioControl 51:293–310 (Plant Pathology Research Institute, Agricultural Research Center) Gooday GW (1999) Aggressive and defensive roles for chitinases. In: for his sincere help in the fungal identification. Many thanks to all Jolles P, Muzzarelli RAA (eds) Chitin and chitinases. Birkhäuser, those who have helped us in the Plant Molecular Pathology Lab., Basel, pp 157–170 Mubarak City for Scientific Research, especially Ms. Ghada Ali. 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Published: Jan 19, 2013

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