Nod factor induces soybean resistance to powdery mildew

Plant Physiology and Biochemistry 43 (2005) 1022–1030 www.elsevier.com/locate/plaphy Original article Nod factor induces soybean resistance to powdery mildew Haifa M. Duzan, Fazli Mabood, Xiaomin Zhou, Alfred Souleimanov, Donald L. Smith * Department of Plant Science, Macdonald Campus of McGill University, 21, 111 Lakeshore, Sainte-Anne-de-Bellevue, Que., Canada H9X 3V9 Received 24 January 2005; received in revised form 15 June 2005; accepted 5 August 2005 Available online 28 September 2005 Abstract Plants possess highly sensitive perception systems by which microbial signal molecules are recognized. In the Bradyrhizobium-soybean (Glycine max (L.) Merr.) symbiosis, recognition is initiated through exchange of signal molecules, generally flavonoids from soybean and lipo-chitooligosaccharides (Nod factors) from the microsymbiont. Application of the Nod factor Nod Bj-V (C18:1, MeFuc) induced soybean resistance to powdery mildew caused by Microsphaera diffusa. Addition of Nod factor (concentrations ranging from 10−6 to 10−10 M) to soybean root systems led to reductions in disease incidence. The lowest disease incidence was caused by Nod factor treatment at 10−6 M. The effect of Nod factor application on fungal growth and development was measured at 4, 12, 48, and 96 h after inoculation. Colony diameter and number of germ tubes per conidium were decreased by 10−6 M Nod factor. Phenylalanine ammonia lyase (PAL, EC.4.3.1.1.) is the first enzyme of the phenyl propanoid pathway, and is commonly activated as part of plant responses to disease. Treatment of soybean seedlings with Nod factor, through stem wounds, induced PAL activity; the most rapid increase followed treatment with 10−6 M Nod factor. These data show that soybean plants are able to detect root applied LCO and respond by increased disease resistance. © 2005 Elsevier SAS. All rights reserved. Keywords: Disease resistance; Nod factor; Phenylalanine ammonia lyase; Soybean 1. Introduction Soybean (Glycine max [L.] Merr.) is the world’s most widely grown grain legume [52]. It is affected by a number of pathogens, including powdery mildew, caused by the obligate foliar parasitic fungus Microsphaera diffusa Cke. and Pk [30]. Powdery mildew can disrupt leaf structure, leading to declines in soybean photosynthetic activity as great as 50%, and yield losses of up to 35% [12,30,35]. The earliest event in the establishment of the rhizobia– legume association is a highly specific exchange of signal compounds. Plant roots exude signal molecules, mainly flavonoids, which induce bacterial nod gene expression, resulting in synthesis of bacteria-to-plant signal molecules, Nod factors [26]. The structure of the Nod factor produced is a component of specificity in the rhizobia–legume nitrogen fixing symbiosis [41]. In general, Nod factors are composed of Abbreviations: ANOVA, analysis of variance; LCO, lipo chitooligosaccharides; PAL, phenylalanine ammonia lyase; RZT, root zone temperature. * Corresponding author. Tel.: +1 514 398 7866; fax: +1 514 398 7897. E-mail address: Donald.Smith@McGill.Ca (D.L. Smith). 0981-9428/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2005.08.004 a tri- to penta-chitin backbone possessing an N-acyl group at the non-reducing end and a variety of substitutions along the chitin backbone [41]. The main Nod factor produced by the soybean microsymbiont, Bradyrhizobium japonicum, is Nod Bj-V (C18:1, MeFuc) [37,44]. Application of appropriate Nod factor to soybean roots evokes a number of physiological responses: plasma membrane depolarization [16], calcium spiking [20], root hair deformation [14,24,36], induction of incipient nodule meristems [48], enhancement of early growth stages of legume and non-legume plants [45], and complete, although empty of rhizobial bacteroids, nodule structures on G. soja roots [48]. It is interesting that the signaling occurring at the beginning of the N2 fixation symbiosis involves exchange of flavonoids and chitin based compounds. Plants have a well characterized ability to detect chitin fragments, elicitors of plant defense reaction and constituents of fungal cell walls, and to produce phytoalexins, often flavonoids, in response [15]. It may be that, during the course of evolution, a hostile signaling system, established to detect and respond to the presence of pathogenic fungi, has been converted to a friendly signaling system, used in the establishment of an extremely impor- H.M. Duzan et al. / Plant Physiology and Biochemistry 43 (2005) 1022–1030 tant symbiosis. It may also be that vestiges of the original protective function remain in the symbiotic signaling system. Sub-optimal root zone temperatures (RZTs) inhibit soybean growth, nodulation and nitrogen fixation [56], and affect the N fixing symbiosis by suppressing signaling. Thus, it is possible that RZT could affect the ability of root applied Nod factors to induce disease resistance in soybean. In addition, the phenylpropanoid pathway, which is involved in plant disease resistance, can be induced by abiotic stress factors, such as low temperatures [9], so that lower RZTs could induce disease resistance themselves, and direct Nod factor effects could be additive with lower RZT effects. The potential role of rhizobia to protect the macrosymbiont against pathogenic organisms was reviewed by Dakora [6]. Tu [49] reported decreases in root rot (Phytophthora megasperma) development on soybean when B. japonicum was applied to the soil of pot-grown plants. Buonassissi et al. [4] reported a reduction in root rot caused by Fusarium solani f.sp. phaseoli on snap bean in the presence of appropriate rhizobia. Under field conditions, soil inoculation with rhizobia resulted in protection of crops including soybean against Rhizoctonia solani, Macrophomina phaseolina, and Fusraium spp. [18]. Tu [50] suggested that rhizobia directly interfere with early stages of pathogenic infection. Recent evidence suggests roles for rhizobial Nod factor that go beyond the nodulation process. Nod factors are known to affect phytoalexin concentrations in plant tissues [6]. Plant isoflavonoid biosynthesis was stimulated by the presence of rhizobial cells [7]. Nod factor also induced expression of genes encoding enzymes of the isoflavonoid biosynthetic pathway in Medicago roots, including a pathogen related protein [39]. Phenylalanine ammonia-lyase (PAL) initiates the first reaction in the phenylpropanoid pathway [10] which produces intermediates for the synthesis of secondary metabolites such as the antimicrobial phytoalexins [33]. PAL activity can be induced during plant–pathogen interactions as well as by nonpathogenic agents, such as environmental conditions [9]. Infection of soybean roots with B. japonicum induces PAL genes [19]. PAL activity can also be stimulated by elicitors, including chitin and chitosan. Recently, the stimulation of PAL activity in soybean by chitin and chitosan oligomers was observed in soybean, suggesting the induction of this defenserelated metabolic pathway in soybean plants [25]. The present study investigates the capacity of Nod Bj-V (C18:1, MeFuc) to induce PAL activity and powdery mildew resistance in soybean plants. 2. Results 2.1. Effects of Nod factor on disease progression Nod factor slowed disease development at all concentrations at the first sampling (1 week after inoculation). At 17 °C 1023 RZT, a reduction in disease incidence was detected for all Nod factor concentrations, with the least development of disease symptoms occurring at the highest Nod factor concentration (10−6 M, Fig. 1). Disease intensity in the control treatment was approximately threefold higher than for the 10−6 M Nod factor treatment. A similar pattern was observed at RZT 25 °C; at higher Nod factor concentrations the incidence of disease was 25% lower for the 10−6 M Nod factor treatment than the control (Fig. 1a). Between the first and second samplings (weeks 1 and 2 after inoculation) disease incidence increased for all treatments, however, the 10−6 M Nod factor treatment continued to have the lowest level of powdery mildew infection at both RZTs (Fig. 1b). By the third observation (third week) disease intensity had increased markedly. The overall progression of disease was slower for the lower than for the higher RZT and at the higher RZT infection levels were high and not different among Nod factor concentrations. At 17 °C RZT plants treated with 10−6 M Nod factor had a lower incidence of disease than the control plants (Fig. 1c). 2.2. Effect of RZTs on disease development Disease incidence was always higher at 25 °C RZT than at 17 °C RZT. There was an interaction between Nod factor and RZT at the first sampling (1 week after inoculation). At the second and the third samplings there were no differences among treatments at 25 °C RZT, but differences persisted at 17 °C RZT (Fig. 1a). 2.3. Effect of Nod factor on fungal growth at 17 °C RZT There was no evidence that Nod factor treatment affected conidial germination or appressorium formation by M. diffusa on soybean leaves at 4 and 12 h after inoculation. However, a reduction was observed in colony diameter and number of germ tubes per colony due to treatment with 10−6 M Nod factor 48 h after inoculation. At 96 h after inoculation, colony diameter was markedly reduced by all Nod factor treatments, as compared to the control treatment, and this effect was dose-dependant, the greatest reduction in colony size occurring at 10−6 M, the highest Nod factor concentration tested (Table 1). 2.4. Effect of Nod factor on fungal growth at 25 °C RZT Nod factor did not affect fungal conidial germination on soybean leaves 4 and 12 h after inoculation, while appressorium formation was reduced to some extent by 12 h. By 48 h after inoculation reduced colony size and number of germ tubes per colony were observed at the highest Nod factor concentration (10−6 M), compared to the control (Table 2). By 96 h after inoculation, over lapping of fungal colonies was observed and it was not possible to take data of the colony size. 1024 H.M. Duzan et al. / Plant Physiology and Biochemistry 43 (2005) 1022–1030 Fig. 1. The effect of Nod factor concentrations on disease progression on soybean leaves. A–C represent data at weeks 1–3, respectively. Bars associated with the same lower case letter are not significantly different, by an ANOVA (P = 0.05) protected LSD0.05 test. 2.5. PAL response to Nod factor treatment PAL specific activity was induced in soybean leaves by all Nod factor treatments (Fig. 2). At 24 h plants treated with 10−6 M Nod factor exhibited a maximum level of PAL specific activity (23.2 U; U = 1 unit = 1 nM (trans-cinnamic acid) mg per protein min−1) followed by 10−8 M (19.2 U) and 10−10 M (18.9 U), all of which were greater than the control plants (16.9 U). After 48 h PAL activity declined for the 10−6 M treatment (20.6 U), although it remained higher than the control, while levels continued to increase for 10−8, 10−10 M treatments (20.2, and 19.2 U, respectively), although the latter was not greater than the control (18.4 U) at this time (Fig. 2). 3. Discussion Our data are generally in agreement with Savoure’ et al. [39], where Nod factor-mediated a defense reaction in Medicago cell cultures and roots. Alkalinization of soybean suspension cultures by treatment with Nod Bj-V (C18:1, MeFuc), the same Nod factor tested in the current study, was felt to suggest elicitation of a defense-related response [8]. However, Felle et al. [20] reported that an increased cytosolic cal- H.M. Duzan et al. / Plant Physiology and Biochemistry 43 (2005) 1022–1030 1025 Table 1 Effect of Nod factor treatment on the growth and development of powdery mildew on soybean leaves at 17 °C RZT Treatment *(M) Hour after treatment 10−6 10−8 10−10 Control 10−6 10−8 10−10 Control 10−6 10−8 10−10 Control 10−6 10−8 10−10 Control 4 4 4 4 12 12 12 12 48 48 48 48 96 96 96 96 Germination percentage (%) 5.2 7.7 5.6 6.7 11.7 10.6 14.0 14.6 – – – – – – – – Appressorium formation (%) ** – – – – 70.5 72.2 66.6 68.8 – – – – – – – – Colony diameter (µm) – – – – – – – – 146.0 b 192.2 a 190.5 a 202.5 a 670.8 c 744.2 c 852.5 b 931.6 a Number of germ tubes per colony – – – – – – – – 2.6 c 3.1 ab 3.0 b 3.3 a – – – – Values in the same column followed by the same letter are not significantly different by an ANOVA-protected LSD0.05 test. * M = molar. ** = not applicable. Table 2 Effect of Nod factor treatment on the growth and development of powdery mildew on soybean leaves at 25 °C RZT Treatment *(M) Hour after treatment 10−6 10−8 10−10 Control 10−6 10−8 10−10 Control 10−6 10−8 10−10 Control 4 4 4 4 12 12 12 12 48 48 48 48 Germination percentage (%) 18.6 22.2 20.9 20.5 25.5 24.3 29.5 29.5 – – – – Appressorium formation (%) ** – – – – 80.9 b 84.5 b 88.8 ab 92.5 a – – – – Colony diameter (µm) – – – – – – – – 382.4 b 404.5 b 418.2 ab 453.1 a Number of germ tubes per colony – – – – – – – – 3.7 b 3.8 ab 3.9 a 4.0 a Values in the same column followed by the same letter are not significantly different by an ANOVA-protected LSD0.05 test. * M = molar. ** = not applicable. cium concentration due to Nod factor treatment of alfalfa root hairs did not exceed the (hypothetical) threshold required for signal transduction associated with activation of defenserelated reactions. In the present study, the lowest incidence of disease occurred when plants were treated with 10−6 M Nod factor. This concentration evokes defense related responses in M. sativa roots, by inducing genes related to synthesis of isoflavonoids and pathogen related proteins [39], and non-defense responses in the form of root hair deformation [13,36]. Nod factor treatment suppresses endogenous salicylic acid levels in soybean [28], suggesting that host defense responses might be compromised during the early stages of symbiosis. This may explain how rhizobia are able to successfully infect legume roots. However, suppression of SA related defense responses is often associated with concomitant activation of jasmonate induced disease resistance [11]. Methyl jasmonatemediated induction of powdery mildew resistance has been demonstrated in barley leaves, and this was associated with marked increases in PAL activity [51]. Thus, Nod factor treat- ments may be activating one set of disease responses and inhibiting another. We found an effect of Nod factor treatment on later stages of fungal growth: colony size and number of germ tubes per conidium, while germination and appressorium formation were not affected at either RZT. Powdery mildew germination and appressorium formation are generally not affected by elicitor treatments [22,42]. Germination and appressorium formation do not necessarily indicate successful infection, however, establishment of biotrophy by powdery mildew can be observed by 4 h after inoculation, indicating the formation of functional haustoria, which support further secondary fungal growth [43]. At 17 °C RZT and 96 h after inoculation, colony diameter was smaller for all Nod factor treatments than the control, indicating indirect effects on haustorium function. At 25 °C RZT, appressorium formation, colony size, and number of colonies per conidium were affected at 12 and 48 h after Nod factor treatment. This may have affected infection establishment at later stages. 1026 H.M. Duzan et al. / Plant Physiology and Biochemistry 43 (2005) 1022–1030 Fig. 2. PAL specific activity as function of time in soybean leaves treated with Nod factor. Each value is plotted as the mean ± S.E. (N = 6). Application of Nod factor to cut soybean seedling stems increased PAL activity, with the most rapid increase occurring at the highest concentration of Nod factor (10−6 M). The lowest level of Nod factor applied (10−10 M) caused the slowest increase in PAL activity. It may be that the levels of PAL activity at the lowest Nod factor concentration rose too slowly and the disease became established before the plant defenses were in place. One must be cautious about making direct comparisons between cut tissue and root applications of Nod factor. It has been reported that treatment of cell cultures with 10−6 M Nod factor increases production of medicarpin (a phytoalexin with antimicrobial activities) by M. sativa [39]. Accumulation of flavonoids (genistein, coumestrol, and daidzein) was reported in soybean root exudates treated with 10 nM of Nod Bj-V (C18:1, MeFuc, OH) and various types of NodNGR over a range of 10−8 to 10−10 M [40], in keeping with our findings. Several studies report increases in calcium flux associated with Nod factor applications [41], a response commonly observed in plants treated with defense elicitors. The concentrations of both cytosolic calcium and the phytoalexin glyceollin increased in chitin fragment challenged soybean [32]. A similar pattern of increase in calcium concentration was reported for alfalfa treated with Nod factor [17]. Nod factor and chitin oligomers induced cytosolic calcium [Ca 2+] increases in transgenic soybean cells, suggesting calcium as a second messenger in signal transduction of Nod factor [34]. Nod Bj-V (C18:1, MeFuc) enhanced overall calcium up take by soybean (Supanjani et al. unpublished). The apparent increases in Nod factor-related calcium flux and uptake serve two possible functions in the plants: 1. enhancement of cell integrity [29], fortifying the cell wall against pathogenic attacks [23]; 2. calcium flux in stimulation of plant-defense machinery through calmodulin signaling. The current data do not allow us to test these possibilities. In the present study, we have shown that Nod factor induces soybean resistance to powdery mildew, and that greater concentrations induce more resistance, at least over the range tested. Because of the chemical similarity between Nod factor and chitin oligosaccharides, known elicitors of plant defense reactions, it is possible that Nod factor induction of plant defense responses is related to Nod factor structure [1], particularly the chitin backbone [8]. Foliar spraying and root treatment with chitin oligosaccharide induced resistance of wheat to powdery mildew [53]. Moreover, Nod factor and chitooligosaccharide applications to Medicago cell suspension cultures elicited transcription of genes encoding enzymes of the phenylpropanoid pathway [39]. On the other hand, Minami et al. [31] reported that, like Nod factor, chitin oligomer induced the expression of an early nodulin, ENOD40, in soybean. In addition, it is known that Nod factors imitate chitin oligosaccharides in their induction of chitinase activity in both host and non-host plants [46]. Nod factors also act as elicitors and provoke alkalinization and oxidative burst reactions in non-legume plants, similar to the defense response provoked by chitin fragments [1]. The presence of chitin as a structural unit of Nod factor suggests possible degradation by root hydrolytic enzymes, such as chitinases. However, Staehelin et al. [46,47] suggested that certain Nod factor substitutions protect the compounds from inactivation. Moreover, perception of substituted and nonsubstituted Nod factors by the host plant has been reported, suggesting “the existence of two signal perception systems, one transmitting the host-specific signal, the other representing an ancient reception system for a generic Nod factor structure” [21]. Structural similarities, and binding interactions between Nod factor [Nod Bj-V (C18:1, MeFuc)] and chitin oligosaccharide suggest that soybean perceives both molecules by the same binding site so that Nod factor might be anticipated to induce plant defense responses [8]. In addi- H.M. Duzan et al. / Plant Physiology and Biochemistry 43 (2005) 1022–1030 1027 Fig. 3. Effect of Nod factor on disease incidence at 17 °C RZT. (A) Control without Nod factor treatment. (B) Nod factor treated soybean. Arrows show typical powdery mildew symptoms, irregular white patches on soybean leaves. tion, we have observed PAL induction by Nod factor, which has also been reported for chitin oligomers [25]. Sub-optimal RZTs negatively affect the growth rate of soybean [55,56] so that plants growing under favorable RZTs exhibit faster growth. It is possible that this is, indirectly, the reason for the differential rates of disease progression at the two RZTs. Disease development was slower at the lower RZT. This is interesting as the disease developed on the leaves, which were at the same temperature for both RZTs, implying that the difference between the two RZTs was due to materials translocated from the roots to the shoots. It is known that the phenylpropanoid pathway can be induced by abiotic stress factors, such as low temperature, and that levels of PAL induction can vary with the stage of plant development, genotype, and environmental conditions [9]. Thus, it is possible that the low RZT and Nod factor treatment might have an additive effect on plant defense reactions, through PAL induction. Our disease data suggest that PAL induction was greater or somehow more effective, at the lower RZT. It was not possible to show a correlation between PAL induction and disease incidence for two reasons: 1. the PAL assay was conducted under optimal temperature conditions, 2. Nod factor was applied to cut plants for PAL assay and to intact roots in the disease development work. It is worth to note that two types of infection, symbiotic [55,56] and pathogenic, can be slowed by low RZT, while N2 fixation occurs in the roots and the powdery mildew development is on the leaves, suggesting the presence of common factor in the plant effecting both types of infection, even if the infection is caused by a symbiont in one case and a pathogen in the other. In conclusion, these data show that Nod factor produced by B. japonicum is able to substantially increase soybean resistance to powdery mildew. Given that PAL activation is involved in the soybean response to Nod factor and that PAL activation plays an import role in resistance to a wide range of diseases it seems likely that Nod factor treatment will confer disease resistance to a wide range of pathogens (Fig. 3). 4. Methods 4.1. Plant materials Plants were produced following, in general, the methods of Zhang and Smith [54]. Briefly, soybean cv. OAC Bayfield seeds were surface sterilized in 2% sodium hypochlorite for ~ 2 min and washed several times with distilled sterilized water. The seeds were planted in trays containing sterilized vermiculite. After about 4 days, three germinated seeds showing vigorous seedling growth were selected and transferred together into each growth pouch; the pouches were then placed in one of four tanks with two tanks set at 17 ± 2 °C RZT, and two at 25 ± 2 °C RZT. RZTs were controlled by circulating cooled water around the growth pouches. After several days two of the three seeds in each pouch were removed; the ones left were selected for uniformity of growth. 4.2. Bacterial culture B. japonicum 532C was obtained from Liphatech Inc. (Milwaukee, WI, USA). This strain was selected as it has been widely used in commercial B. japonicum inoculants in Canada. The culture was grown at 28 °C in 250 ml yeast extract mannitol (YEM) broth, with shaking at 150 rpm, for 4–6 days, and thereafter subcultured into 2 l (2.5 ml per 200 ml of YEM medium). After 7 days of subculture (OD620 0.4– 0.6), bacterial cultures were induced through the addition of 5 µM genistein and incubated for an additional 48–96 h, followed by Nod factor extraction. In addition, a non-induced bacterial culture was prepared, to act as a control. This was 1028 H.M. Duzan et al. / Plant Physiology and Biochemistry 43 (2005) 1022–1030 subject to the same Nod factor isolation procedure as the induced cultures. Bacterial OD was adjusted to an A620 of 0.08 (approximately 108 cells ml−1) with sterilized water, adjusted to temperatures appropriate to the treatment RZTs, and 1 ml was applied, with a sterilized syringe, onto each plant root system. 4.3. Nod factor extraction and purification The 2 l culture was extracted with 40% HPLC-grade 1-butanol by shaking the mixture for 5–10 min and then allowing the two phases to separate for 24 h. The organic phase (butanol layer) was collected and evaporated at 80 °C in a Yamato RE500 Rotary evaporator (Yamato Scientific American Inc., New York, USA). The resulting material (volume of 2–3 ml) was dissolved in 4 ml of 18% of acetonitrile and stored in the dark in glass tubes at 4 °C for 24 h. Samples were then centrifuged for 10 min at 12,000 × g, and the supernatant was collected for HPLC analysis. Two hundred microliters of the Nod factor extract were injected into a Waters HPLC system (Waters Associates Inc., Milford, MA) consisting of a model 712 WISP HPLC, fitted with two model 510 pumps and a model 441 UV detector operated at 214 nm. Separation was carried out with a Vydac C18 reversed-phase column (5 µm, 46 × 250 mm, Vydac, USA). To elute Nod factor from the column, a program of acetonitrile and water gradients was used: 18% acetonitrile (10 min), a linear gradient from 18 to 60% acetonitrile (20 min), a linear gradient from 60 to 100% acetonitrile (5 min). A chromatographic peak was identified as Nod Bj-V (C18:1, MeFuc) by comparing its retention time with a standard of this Nod factor (a gift from Professor G. Stacey, Center for Legumes Research, University of Tennessee, Knoxville, USA). The identity of this peak has also been confirmed by mass spectrometry. The resulting Nod factor was freezedried and redissolved in distilled sterilized water. ity, was maintained throughout the experiment. Nod factor was added to each growth pouch with a sterile syringe. Inoculum from naturally infected soybean was maintained by infecting soybean leaves that were grown in a separate room, under the same conditions, in the greenhouse. Given the type of symptoms caused by powdery mildew infection and the presence of such symptoms, and microscopic observations, the anamorph stage of the fungus was confirmed on soybean leaves and, there was no other fungal growth or secondary infection seen on the leaves. Because Nod factor stimulate transcription of genes encoding enzymes of plant defenserelated responses over at least a 48 h period Savoure’ et al. [39] powdery mildew fungus was applied to soybean leaves 48 h after Nod factor addition to soybean root systems. Soybean leaves heavily infested with powdery mildew were used to inoculate the test plants when the first trifoliate leaf was fully developed [27] by tapping the infected leaves over the leaves of the test plants, thus depositing spores on the surface of the leaves of the test plants. The inoculated plants were monitored for initial signs of powdery mildew and once initial disease symptoms were evident, data on disease incidence were taken weekly for 3 weeks. By 3 weeks after inoculation powdery mildew infection reached 100% on at least some plants in most treatments at 25 °C RZT. The degree of infection was scored from 0 to 5 according to percent of leaf tissue showing powdery mildew symptoms, with 0 = none, 1 = trace to 10%, 2 = 10.1–25%, 3 = 25.1–50%, 4 = 50.1– 75%, 5 = over 75% [42]. Disease intensity was calculated for each replicate of each treatment, using the following equation: Disease intensity = Sum of rating (0–5)/(Maximum possible score × Total number of leaves examined) × 100. The entire experiment was conducted twice, with similar results in both instances. Data were combined and subjected to statistical analysis. 4.5. Experiment 2 4.4. Experiment 1 In this experiment, four levels of Nod Bj-V (C18:1, MeFuc) were tested. Seedlings were maintained in the growth pouches and watered with sterilized H2O for the first week, after which plants were watered with nitrogen-free Hoaglands solution [55,56]. Because low RZT is known to inhibit stages of the infection process during establishment of the soybean– bradyrhizobia symbiosis [54], and work by this group (unpublished data) has shown that low RZTs inhibit the ability of soybean roots to perceive LCOs, two RZTs [17 °C (critical temperature for soybean nodulation and nitrogen fixation) and 25 °C [optimum temperature for soybean nodulation and N2 fixation [55]] were included in this experiment. The experiment was organized following a completely randomized splitplot design. Temperature was the main plot factor. The subplot factor was Nod factor concentration (10−6, 10−8, 10−10, 0.0 M) with six replicates per treatment. A photoperiod of 16/8 h day/night cycle, at 25 ± 2 °C and 75% relative humid- In this experiment, the effect of Nod factor treatment on fungal growth and development was studied. As in experiment one, after Nod factor treatment and fungal inoculation, three plants from each treatment (three replicates) were collected at each sampling, at each of the two RZTs (17 and 25 °C). Samples were collected from one tank of each RZT. Therefore; there were two experiments, one at each RZT, and the structure of each experiment followed a completely randomized design. The data were collected from the third nodal leaves of each plant at intervals of 4, 12, 48, and 96 h after fungal inoculation. Data were taken on germination at 4 h, germination and appressorium formation at 12 h, number of germ tubes per conidium and colony size, measured as the greatest colony diameter, at 48 h, and colony size at 96 h after fungal inoculation. At least, 100 conidium per replicate were observed in order to calculate frequency of conidial germination and appressoria formation at 4 and 12 h after fungal inoculation. Collected samples were fixed following the H.M. Duzan et al. / Plant Physiology and Biochemistry 43 (2005) 1022–1030 method of Carver and Adaigbe [5]. Briefly, samples were placed carefully into Petri plates containing ethanol/acetic acid 3:1 until completely cleared of chlorophyll, then placed on other filter paper in a Petri plate containing lactoglycerol (glycerol/lactic acid/water; 1:1:1; v:v). Slides were prepared with methylene blue (0.01%) for microscopic observations. Data were subjected to angular transformation [22] before statistical analysis. The experiment was conducted twice with similar results at the both instances. 4.6. Enzyme assay (PAL) 4.6.1. Plant materials Assays from PAL activity followed the method of Khan et al. [25], for both treatment of plant materials and application of treatments. Briefly, soybean cv. OAC Bayfield seeds were surface sterilized using 2% sodium hypochlorite for approximately 2 min, rinsed thoroughly with distilled sterilized water, and then planted in sterilized perlite in a greenhouse under a 16/8 h day/night cycle at 25 ± 2 °C, and 75% relative humidity. 4.6.2. Nod factor treatment Plants at the first trifoliate stage were selected for growth uniformity. Plant stems were excised using a sharp scalpel, cutting each at the base of the stem and placing each into a 4 ml glass test tube containing 0.5 ml of Nod factor solution. When the solution was completely taken up, seedlings were immediately transferred into glass tubes containing distilled water. Plants were kept under constant light (85 µmol m−2 s−1). The leaves of three plants, representing each of the three blocks of each treatment, were collected at regular intervals (4, 12, 24 and 48 h) after Nod factor treatment, weighed, and stored immediately at –80 °C. 4.6.3. Enzyme extraction Leaf samples (300 mg fresh weight) were ground with a cold mortar and pestle containing 4 ml of buffer (50 mM Tris pH 8.5, 14.4 mM 2-mercaptoethanol, 5% w/v insoluble polyvinylpolypyrorolidone). Samples were kept on ice at all times. Extracts were centrifuged at 6000 × g for 10 min at 4 °C and the supernatant was collected and centrifuged at 10,000 × g for an additional 10 min at 4 °C. 4.6.4. PAL enzyme assay PAL activity was assayed as described by BeaudoinEagan and Thorpe [2]; 100 µl of enzyme preparation was mixed with 500 µmol of Tris–HCl buffer (pH 8.5), and 6 µmol of L-phenylalanine in a final volume of 1 ml of reaction mixture and incubated at 40 °C for 60 min. A blank with no L-phenylalanine was also prepared. The reaction was stopped by the addition 50 µl of 5 N HCl. The product (transcinnamic acid) was detected and quantified by measuring absorbance at 290 nm against a blank. Enzyme activity was expressed in nM (trans-cinnamic acid) mg per protein min−1. The total protein concentration in enzyme extracts was deter- 1029 mined by the Bradford [3] method. The experiment was conducted twice, and because similar results were observed, data from the two experiments were pooled before calculating standard error values. 4.7. Statistical analysis Data on disease progression and fungal growth and development were analyzed through analysis of variance (ANOVA) using the Statistical Analysis System [38]. Treatment effects were considered significant when detected at P ≤ 0.05. When this occurred a least significant difference (LSD) test was conducted to test for differences among means (P < 0.05). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] R. Baier, K. Schiene, B. Kohring, E. Flaschel, K. Niehaus, Alfalfa and tobacco cells react differently to chitin oligosaccharides and Sinorhizobium meliloti nodulation factors, Planta 210 (1999) 157– 164. L.D. Beaudoin-Eagan, T.A. 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