Crop management effect on arbuscular mycorrhizae and root growth of flax

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1 Crop management effect on arbuscular mycorrhizae and root growth of flax M. A. Monreal, C. A. Grant, R. B. Irvine, R. M. Mohr, D. L. McLaren, and M. Khakbazan Agriculture and Agri-Food Canada, Brandon Research Centre, Box 1000A, R.R. #3, Brandon, Manitoba, Canada R7A 5Y3. Received 18 June 2010, accepted 22 October Monreal, M. A., Grant, C. A., Irvine, R. B., Mohr, R. M., McLaren, D. L. and Khakbazan, M Crop management effect on arbuscular mycorrhizae and root growth of flax. Can. J. Plant Sci. 91: The effects of two tillage systems, two preceding crops and the use of phosphorus (P) fertilizer on arbuscular mycorrhizal fungi (AMF) colonization, root biomass and root size of flax (Linum usitatissimum L.) were evaluated at two sites in Manitoba, Canada. The Brandon Research Centre Farm site had been historically managed under conventional tillage (CT), and the Manitoba Zero Tillage Research Association Farm site under reduced tillage (RT). The preceding crops were a mycorrhizal crop, wheat (Triticum aestivum L.), and a non-mycorrhizal crop, canola (Brassica napus L.), grown under RT or CT using rates of 0, 11 and 22 kg Pha 1 applied as monoammonium phosphate (MAP). Phosphorus was applied to the following flax crop as MAPat rates of 0 or 11 kg Pha 1. Flax root biomass was larger in all site-years when wheat rather than canola was the preceding crop. Also, AMF colonization and root size of flax increased in 3 and 2 site-years, respectively, when grown after wheat. Flax s root biomass decreased under CT and AMF colonization decreased when Pwas added either to the previous crop or to flax only in one site year. Weather conditions may have enhanced the effects of preceding crop, Pfertilizer and tillage. Key words: Mycorrhizae, flax, crop sequence, reduced tillage, phosphorus fertilization Monreal, M. A., Grant, C. A., Irvine, R. B., Mohr, R. M., McLaren, D. L. et Khakbazan, M Incidence des pratiques agricoles sur les mycorhizes à arbuscules et sur la croissance des racines du lin. Can. J. Plant Sci. 91: Les auteurs ont e valué l incidence de deux systèmes de travail du sol, de deux cultures ante rieures et de l application d engrais phosphate (P) sur la colonisation des racines du lin (Linum usitatissimum L.) par les mycorhizes a` arbuscules (MA), ainsi que sur la biomasse et la dimension des racines a` deux endroits, au Manitoba (Canada). Le sol au site BRC-North est travaille de fac on classique (TC) depuis toujours, tandis que celui du site MTRAF fait l objet d un travail re duit (TR). Les cultures ante rieures consistaient en du ble (Triticum aestivum L.), une culture a` mycorhizes, et en du canola (Brassica napus L.), une culture sans mycorhizes. Les deux avaient e té cultive s sur du sol TR ou TC avec application de 0, 11 ou 22 kg de Ppar hectare sous forme de phosphate d ammonium monobasique (PAM). Du PAM a été appliqué à la culture subse quente de lin à raison de 0 ou de 11 kg de Ppar hectare. La biomasse des racines de lin e tait plus importante pour tous les sites-anne es quand le ble et non le canola constituait la culture antérieure. En outre, la colonisation des racines par les MA et la taille des racines ont respectivement augmente a` deux et trois sites-anne es quand le lin était cultivé apre` s le ble. La biomasse des racines du lin diminue apre` s le TC et la colonisation des racines par les MA e tait plus faible quand l engrais Pétait appliqué soit a` la culture ante rieure, soit au lin, mais à un site-anne e seulement. Il se peut que les conditions me te orologiques aient accentue les effets de la culture ante rieure, des engrais Pet du travail du sol. Mots clés: Mycorhizes, lin, succession culturale, travail réduit du sol, engrais phosphate Roots of about 95% of plant species can be colonized by soil fungi and form mutualistic symbiotic associations called arbuscular mycorrhizae (AM) (Smith and Read 1997). Propagules of arbuscular mycorrhizal fungi (AMF) produce hyphae that grow in the soil and penetrate the epidermal cells or root hairs of plant roots. Intraradical hyphae then colonize the cortical cells where bidirectional movement of nutrients can occur (Peterson et al. 2004). The colonizing fungi can benefit by accessing plant photosynthetic products and the host plant might benefit by enhanced availability of soil water and nutrients with extra-radical mycorrhizal hyphae (Smith and Read 1997). The fungal filaments outside the root system can spread and explore soil areas not reachable by plant roots. One centimetre of colonized roots might produce 50 to 150 cm of extraradical hyphae (Harley 1989). Through this mechanism, AMF associations may improve crop yield by increasing the capacity of plants to obtain nutrients that are relatively immobile in the soil such as phosphorus (P) (Rhodes 1980; von Reichenbach and Scho nbek 1995; Al-Karaki and Al-Raddad 1997; Al-Karaki 1998; Jansa et al. 2003b). Numerous agricultural crops can form associations with AMF with the exception of those that belong to the Abbreviations: AM, arbuscular mycorrhizae; AMF, arbuscular mycorrhizal fungi; BRC-North, Brandon Research Centre Farm; CT, conventional tillage; MAP, monoammonium phosphate; MAT, mean air temperatures; MZTRAF, Manitoba Zero Tillage Research Association Farm; RT, reduced tillage Can. J. Plant Sci. (2011) 91: doi: /cjps

2 316 CANADIAN JOURNAL OF PLANT SCIENCE families of the Brassicaceae, Chenopodiaceae, and Caryophyllaceae (Barker at al 1998). Including nonmycorrhizal crops in rotation might affect the concentration and vitality of indigenous AMF species in soil, thereby affecting the growth of AM-dependent crops following in the rotation (Dalpe and Monreal 2004). It has been reported that both the growth of corn (Zea mays L.), including shoot weight, grain yield, and arbuscular mycorrhizal (AM) formation were enhanced when the previous crop formed mycorrhizae (Allen et al. 2001; Arihara and Karasawa 2000; Karasawa et al. 2000). Canola (Brassica napus L.), an important agricultural crops in western Canada, belongs to the family Brassicaceae, and therefore does not form associations with AMF. Plants in the Brassiceae family have been reported to inhibit AMF growth (Glenn et al. 1988; Schreiner and Koide 1993). In a pot study, Kabir et al. (1996) reported that canola did not affect the growth of AMF hyphae when compared with soil receiving corn residues and in control soil without residues. However, when corn or barley were the previous crops, soil AMF hyphal density was more than fivefold larger. Furthermore, in a crop rotation experiment, corn grown after canola showed greater AMF colonization than corn grown after corn (Gavito and Miller 1998). Since the development of AMF is biotrophic (Morton 1990), the absence of mycorrhizae could cause a decrease in soil residual AMF propagules and their vitality for crops seeded afterwards in a rotation. In contrast, wheat, a highly mycorrhizal crop, can be colonized by AMF fungi and may add to the AMF s indigenous vitality, affecting AMF colonization of a crop grown after it (Plenchette et al. 1983). Flax growth has been found to be negatively affected when grown after canola (Gubbels and Kenaschuk 1989), but it was not known whether this was related to AMF colonization. Tillage operations have been shown to reduce AMF spore density in the soil (Kabir et al. 1998) and AMF colonization in some agricultural crops (Jasper et al. 1989; Miller et al. 1995; McGonigle and Miller 1996). Tillage can disrupt the soil network formed by AMF filaments and colonized root systems left by previous AM crops, affecting the potential colonization of subsequent crops. The AMF hyphae can remain viable in frozen soil over winter, connecting the following spring to roots of newly germinated plants and enhancing their ability to acquire soil nutrients and water (McGonigle and Miller 1999). Studies on diversity and structure of AMF communities colonizing corn found that three different tillage practices (ploughing, chiselling and notill) had a significant effect on the AMF community structure affecting sporulation of some species, and Glomus species were more abundant in tilled soils (Jansa et al. 2002, 2003a). However, no-till has been shown to change soil conditions promoting proliferation of soil fauna (House and Stinner 1983), and generating higher densities of soil micro- and macro-arthropods and earthworms (House and Parmelee 1985), thus enhancing the predatory and saprofagous soil arthropods community (House and All 1981; Fox et al. 1999; Hodge 2000), which could affect AMF propagules from year to year. Different tillage practices are used in western Canada, with reduced tillage (RT) being adopted to conserve soil moisture and prevent soil erosion (Lafond et al. 1992). Flax grown under RT has shown positive economic returns (Zentner et al. 2002; Khakbazan et al. 2009), but with low responses to Pfertilization (Lafond et al. 2003; Grant et al. 2009). The growth of crop plants requires Pduring early growth (Grant et al. 2001). However, applications of P fertilizer can affect AMF colonization of crops (Joner 2000; Entz et al. 2004). Also, long-term previous P applications can affect AMF colonization of crops (Kahiluoto et al. 2000; Dekkers and van der Werff 2001). Flax is less efficient in absorbing fertilizer Por soil Pwhen compared with other crops (Kalra and Soper 1968). Thus, Pfertilization of flax crop is not highly effective unless banded Pfertilizers are placed within 2.5 to 5.0 cm of the seed row (Sadler 1980). However, flax responses to applications of Pfertilizer usually are small (Grant and Bailey 1993; Lafond et al. 2003; Grant et al. 2004). Flax may still have a limited response to Pfertilization due to poor proliferation of the flax root system within the Pfertilizer band (Strong and Soper 1974). In a study on annual and residual responses to Papplication to flax as affected by tillage and crop sequence, Grant et al. (2009) found that P fertilization to flax increased flax early-season biomass and seed yield only in 1 out of 5 site-years. Dekkers and van der Werff (2001) reported that after 10 yr without Pfertilization, AMF colonization of winter wheat (Triticum aestivum) and barley (Hordeum vulgare) was greater when previous long-term annual Pfertilization rates were between 0 and 17.5 kg Pha 1 than when P was applied at 52.5 kg ha 1, although crop yields were not affected. Grant et al. (2009) also found that flax yield was not improved by RT compared with conventional tillage (CT). The same study showed that the greatest effect on early-season biomass production, P concentration, Paccumulation and flax seed yield occurred when flax was seeded after wheat rather than canola (Grant et al. 2009). Gavito and Miller (1998) reported that intra-radical AM colonization of corn (Zea mays L.) was delayed in field plots when canola rather than corn was the previous crop, and that notillage, conventional tillage and Pfertilization had little effect on AM colonization of corn. The objective of the present study was to determine if tillage system, preceding crop, and residual Pderived from Pfertilizer applied to the preceding crop or at seeding would influence early season root colonization by arbuscular mycorrhizae, roots biomass and root size of flax.

3 MONREAL ET AL. * MYCORRHIZAL COLONIZATION OF FLAX 317 MATERIALS AND METHODS This study was carried out at two experimental sites (lat ?N, long ?W) near Brandon, Manitoba, Canada. One site was located at the Manitoba Zero Tillage Research Association Farm (MZTRAF), managed under RT since 1993, and the second site was at the Brandon Research Centre Farm (BRC-North) and managed under CT. Soils at the two locations, approximately 4 km apart, were classified as Orthic Black Chernozems and Clay loam (Ehrlich et al. 1958); soil chemical characteristics are summarized in Table 1 (Grant et. al. 2009). Soil Pcontent was determined in the 0- to 15-cm soil depth sampled during the fall and again prior to seeding. Each soil sample was air-dried, ground, and extracted with 0.5 M sodium bicarbonate solution (Carter 1993). Pconcentration in the extracts was measured with an ARL3410 ICPunit (Thermo Fisher Scientific, Inc., Waltham, MA). The average fall soil Pcontent was 11.5 and 8.4 mg kg 1 in 2000 and 2001, respectively (Table 1, Grant et. al. 2009). The data presented in this paper were collected during the flax phase of two crop rotation cycles in 2001 and 2002, and they were collected concurrently and from the same plots as the study reported by Grant et al. (2009). Flax was the crop planted in the second phase of a 2-yr crop rotation. Canola (cv. LG3295), a non-mycorrhizal crop, or wheat (cv. AC Barrie), a mycorrhizal crop, were grown during the first phase. Initially, eight blocks with two tillage systems, reduced tillage (RT) and conventional tillage (CT), were randomly established. The RT consisted of only fertilizer banding and seeding operations and the CT received two tillage passes in the fall and one pass in the spring with a cultivator equipped with tine harrows (Grant et al. 2009). Each tillage block was divided into six sub-plots for the first phase of the study and was randomly seeded to canola or wheat. Each crop seeded during the first phase randomly received Pfertilizer treatments of 0, 11 and 22 kg Pha 1, side-banded 2.0 cm to the side and 3.0 cm below the seed-row as monoammonium phosphate (MAP), completing a total of 48 sub-plots. During the cultivation of flax, sub-plots from the previous crops were divided into sub-sub-plots (2 m5 m), with or without application of side-banded 11 kg Pha 1 (MAP), with a total of 96 plots per site in a randomised complete block with split-split-plot design. Tillage treatments were the main blocks, preceding crop and residual fertilizer combination were the sub-plots, and the flax sidebanded Ptreatments were the sub-sub-plots. The P fertilizer treatments applied to the canola and wheat were considered the residual P treatments for the flax crop. Flax was seeded at a rate of 40 kg ha 1 and all agronomic practices used were as described by Grant et al. (2009). Roots Sampling and Preparation for Analysis At each experimental site, flax roots were sampled by the end of June, approximately 5 wk after seeding. At this time, stand density of flax plants was also measured. Two rows of 1-m row-length each were sampled within each plot. Sampled rows were positioned 1 m into the second row, on opposite sides of the plot. Each plot root-set was sampled with a 30-cm flat-bottomed spade that was pushed into the soil beside the plants and below the root in order to obtain the whole root, which included the stem, tap root, lateral roots, and also the surrounding soil. Sets of whole roots and soil were placed in plastic bags and stored at 58C until further Table 1. Soil characteristics at the Brandon Research Centre (BRC) and at the Manitoba Zero Tillage Research Association Farm (MZTRAF) for soil sampled in the fall of 2000 and 2001 at three soil depths (0 15, 15 30, cm) (Grant et al. 2009) N P K Zn Depth (cm) (mg kg 1 ) Cd (mg kg 1 ) ph EC (ms cm 1 ) BRC MZTRAF BRC MZTRAF

4 318 CANADIAN JOURNAL OF PLANT SCIENCE processing. Next, sampling bags containing whole roots and soil were brought to a root washing area and each set was individually soaked in a solution of Calgon TM (approximately 2.5 g) in a 5-L plastic container. After soaking for at least 15 min, plant stems with root systems were picked from the container and were placed on a sieve with a 20 cm diameter with an 850 mm pore screen. Stems and roots were then carefully washed with running tap water. Debris and other extraneous materials were removed from the root samples using tweezers. After washing, each set of clean stems with roots was randomly separated into three sets of 10 whole roots systems each for future use in different analyses. Two of the three sets were placed in plastic bottles to be preserved in a diluted formalin-acetic-acid alcohol solution (Sass 1958). Preserved stems with roots were later used to determine total lateral root area and root area colonized by AMF. The third set was used to determine root biomass yield. Root Biomass Root-biomass was determined by randomly selecting 10 clean roots from each set of roots sampled in each study plot. Each root set was placed in a 10-mL plastic container and air-dried on a greenhouse bench for 7 d prior to measuring the root biomass weight (g). Plant stand density per plot was used to calculate root biomass yield (kg ha 1 ). Lateral Root Area For each study plot, four roots from the previously preserved whole stem and root system sets were randomly selected and placed on a white plastic sheet engraved with a standard metric grid (mm 2 ). A digital image was captured from each root set with a regular electric digital scanner. These images were safely stored in the computer in jpeg format. Later, images were used to measure total lateral root size using digital image analysis software (Image Pro-Plus TM ) calibrated to measure four root areas in mm 2. An average lateral root area and plant density counts were used to calculate reported root areas (m 2 ha 1 ). Root Colonization by Arbuscular Mycorrhizal Fungi Each set of preserved roots was rinsed with tap water, placed in a glass beaker and covered with a 10% KOH solution. Each beaker was placed on a sand bath (1008C) over a hotplate and the solution was brought to boil and simmered for 2030 min. Afterwards, roots were removed from glass beaker, rinsed with deionised water and acidified with a 1 N HCl solution for 10 min. The roots were transferred to a new beaker containing a 0.01% Trypan blue solution, and placed on a sand bath placed over a hotplate to keep the temperature of the solution between 92 and 1008C for 3 min. The roots were removed from the sand bath, then soaked and rinsed with deionised water until a sufficient amount of the blue dye had been removed to allow for future detection of the mycorrhizae (Phillips and Hayman 1970). The roots were then placed in a sealed 10-mL plastic container, and preserved in 5% acetic acid for future analysis (Brundrett et al. 1984; Vierheilig and Piché 1998; Vierheilig et al. 2005). Root Image Analysis to Determine AMF Colonization To determine root colonization, each set of roots previously preserved and stained was rinsed and cut into fragments approximately 2 cm in length, with a random selection of 30 fragments, then mounted on a glass slide. The glass slip covers were sealed with nail polish to prevent drying of the root sample. A digital camera (Magna-Fire) was attached to a compound microscope set at 5magnification. From each slide, 10 fields of view were randomly selected and an image from each field of view was digitally collected and recorded using bitmap file format. Later, each field of view image was used to assess AMF colonization using an image analysis technique that allows the selection of blue-stained areas within the image using a software tool provided by the software package (Image-Pro Plus TM ). Blue-stained areas selected to represent AMF colonization included mycelium, vesicles and arbuscules. The program recorded the colonized area per root fragment in mm 2. Percent root colonized was calculated by dividing root area colonized (mm 2 ) by total root area observed in each field (mm 2 ) multiplied by 100. Weather Data Collection Rainfall (mm) and mean air temperature (8C) were collected at the Brandon Research Centre Farm (BRC- North) during the 2 yr of this study (Fig. 1). The two study sites were at distance of approximately 3 km and therefore the same data will be used in the discussion. Statistical Analysis Statistical analysis was conducted separately for the two sites and for each year using Proc Mixed of SAS/STAT software (Littell et al. 1996). Differences were considered significant with a P value B0.05. In addition, the correlation procedure was used to evaluate the relationship between percent AMF root colonized, lateral root area, and root biomass (SAS Institute, Inc. 1990). RESULTS AND DISCUSSION Flax Colonization by Arbuscular Mycorrhizal Fungi In 2001, early-season AMF colonization of flax roots was influenced by the preceding crop at the BRC-North site (PB0.0001), but not at the MZTRAF site (Table 2). At the BRC-North, AMF colonization of flax was about 3.5% larger after wheat than when canola was the previous crop (Table 3). Residual wheat roots, a mycorrhizal crop, could have contributed to provide AMF propagules affecting early colonization of flax.

5 MONREAL ET AL. * MYCORRHIZAL COLONIZATION OF FLAX 319 (a) Mean air temperature (C ) Precipitation Mean Air Temperature Precipitation (mm) (b) 40.0 Mean air temperature (C ) May 15-May 29-May 12-Jun 26-Jun 10-Jul 24-Jul 07-Aug 21-Aug May 15-May 29-May 12-Jun 26-Jun 10-Jul 24-Jul 07-Aug 21-Aug The potential of mycorrhizal inocula can be enhanced by a pre-crop improving AMF colonization of the subsequent crop (Barea et al. 1993). AMF hyphae have been found to survive over winter in the absence of roots (Addy et al. 1994). Also, AMF spores have resistant walls and can survive and germinate, sometimes after many years (Peterson et al. 2004). However, extra-radical hyphae have been found to be a principal source of AMF propagules available for annual field crops (Sylvia 1992). Results reported by Grant et al Precipitation Mean Air Temperature Fig. 1. (a) 2001 growing season precipitation (mm) and mean air temperatures (8C) for the Brandon Research Centre site; (b) 2002 growing season precipitation (mm) and mean air temperatures (8C) for the Brandon Research Centre site Precipitation (mm) (2009) on the study conducted concurrently with and on the same sites determined that above-ground flax biomass at 5 wk was consistently higher when flax was seeded after wheat rather than after canola. Although, there might be an advantage to using crop rotation to improve growth of the following crop, research on AMF colonization cautions that the benefits may be limited by the crop type and carbon (C) cost of excessive AM mycorrhization affecting grain yield (Kahiluoto et al. 2001). Table 2. Statistical analysis using Proc Mixed for effects of preceding crop (wheat or canola), phosphorus fertilizer added to previous crop [P (Residual) or added to flax (P (Flax)] and tillage system (conventional and reduced tillage) on arbuscular mycorrhizae colonization of flax (percent AM colonization), on flax dry root weight (kg ha 1 ) and root area (m 2 ha 1 ) at the Brandon Research Centre (BRC-North) and at the Manitoba Zero Tillage Research Association Farm (MZTRAF) sites in 2001 and 2002 Percent AM colonization Dry root weight Root area Source DF BRC-North MZTRAF BRC-North MZTRAF BRC-North MZTRAF 2001 Preceding crop 1 B NS B B NS NS Tillage 1 NS NS B NS NS NS 2002 Source DF PRF P(flax) 1 B.0001 NS NS NS NS NS Preceding crop 1 B.0001 B.0001 B.0001 B.0001 B.0001 B.0001 P(flax)preceding crop 1 B.0001 NS NS NS NS NS P(residual) 2 B.0007 NS NS NS NS NS

6 320 CANADIAN JOURNAL OF PLANT SCIENCE Table 3. Effect of type of preceding crop (canola or wheat), conventional tillage (CT) or reduced tillage (RT), phosphorus application to preceding crop (P previous crop), and P application to flax (P flax) on AM colonization of flax (%) at 5 wk after seeding at the Brandon Research Centre (BRC-North) and at the Manitoba Zero Tillage Research Association Farm (MZTRAF) sites for years 2001 and 2002 BRC-North MZTRAF Previous crop canola Previous crop wheat Previous crop canola Previous crop wheat Pprevious crop PFlax CT RT CT RT CT RT CT RT Mean of P Mean of 25 P Mean across P Mean of P Mean of 25 P Mean across P Tillage treatments did not affect the percent AMF colonization of flax in either study year. Annual soil disturbances produced by traditional tillage management systems have been reported to reduce AMF colonization in conventional tillage when compared with reduced tillage practices (Miller and Jastrow 1992; Miller et al. 1995; Al-Karaki 1998; Miller 2000). Although soil disturbance treatments reduced the density of metabolically active hyphae in a field study, indigenous mycorrhizal hyphae survived winter temperatures (Kabir et al. 1997). At the MZTRAF site, which has been under RT since 1993, early percent AMF colonization of flax in 2001 was not different after either wheat or canola. Furthermore, percent colonization of flax was similar to or smaller than colonization present at the BRC-North site. AMF colonization of flax was expected to be higher at the MZTRAF site as AMF propagules, such as hyphae and spores, would be present in larger amount due to the longterm RT management. Soil microbial diversity and community structure have been found to be influenced by conservation tillage, increasing the catabolic capacity of soil microbial communities (Lupwayi et al. 1998) and microbial composition of soil arthropods (House and Del Rosario Alzugaray 1989). Rainfall and mean air temperature of the growing season period (May through August) were different for the 2 yr of the study. At seeding time, mean air temperature (MAT) was around 118C and several rainfall events of more than 10 mm occurred in 2001 (Fig. 1a), whereas, in 2002, MAT was near 08C and rainfall was scarce (Fig. 1b). These conditions would have favoured the activity of soil arthropods, affecting the abundance of AMF propagules during the early growing season of Unfortunately, the contribution of each type of propagule (spores and mycelium) to AMF percent colonization was not measured in this study. Gavito and Miller (1998), found that tillage did not affect colonization of corn in a field study or in laboratory bioassays. In a study on continuous corn grown under no-till and conventional management, Fox et al. (1999) reported a significant variation in the biotic data that occurred in 2 consecutive years of study (71 and 61%, respectively), which could be explained by variation in the abiotic data, mainly percent soil water (05 cm soil sampling depth). Previous findings indicate that it would be useful to investigate the activity of soil biota in long-term RT systems and their effect on the mycelium and spores resilience of AMF from year to year. Studies on previous crop and tillage interactions on percent AMF colonization of flax have not been reported before. However, the effect of preceding crop on flax mycorrhizal colonization was important (PB0.0001) at both study sites for 2002 (Table 2), with between two- and threefold more colonization when wheat was the previous crop at the BRC- North and the MZTRAF sites, respectively. The 2002 climatic conditions might have affected mycorrhizal colonization of flax to a larger extent, with an enhanced effect on the symbiotic relationship of flax and residual indigenous AMF present after the wheat crop. In the year with low MAT at seeding time and scarce precipitation the effect of previous crop was enhanced (P B0.0001) at the MZTRAF site.

7 MONREAL ET AL. * MYCORRHIZAL COLONIZATION OF FLAX 321 Although applications of Pfertilizer can affect AMF colonization of crops (Joner 2000; Entz et al. 2004), Pfertilizer added to flax or residual Pdid not affect AMF percent colonization of flax at either study site in 2001 or at the MZTRAF site in However, in 2002, Pfertilizer added to flax lowered AMF percent colonization of flax at the BRC-North site (Tables 2 and 3). Also in that site-year, there was a combined effect of previous crop and Papplied to flax at the BRC-North site (PB0.0001) when Pfertilizer was applied to flax at a rate of 11 kg Pha 1 (MAP), where AMF colonization of flax was reduced approximately 5% after wheat and 1% after canola. Also in 2002, residual P(Papplied to the previous crop) reduced early AMF colonization of flax at the BRC-North site (Tables 2 and 3). Although, the Papplied to flax affected AMF colonization at 5 wk after seeding in 2002, overall it did not seem to affect Puptake by flax, as Grant et al. (2009) reported that Pconcentrations in flax tissue at 5 wk increased in 5 of 6 site years of research. However, in spite of the reduction in AMF colonization of flax at the BRC-North site in 2002, its early season biomass and yield increased with P fertilization of flax at BRC-North site. (Grant et al. 2009). In experiments done in Finnish field soils, cumulative low and high Pfertilization decreased AMF colonization of flax, but plant growth and nutrient uptake depended on the soil type and Plevels, where AMF contribution to plant growth and nutrient uptake may be negative if Psupply decreased below a threshold level (Kahiluoto et al. 2000, 2001). Also, the previous authors reported that yields and nutrient uptake were affected differently by AMF colonization of flax, red clover (Trifolium pratense L.), and barley (Hordeum vulgares L.). Flax Root Biomass and Lateral Root Area Previous crop affected early-season root biomass of flax at the two study sites during 2001 and 2002 (Tables 2 and 4). The average root weights of flax were larger after wheat than when flax was grown after canola (Table 4). These results were consistent with larger AMF colonization of flax root grown after wheat than after canola, except at the MZTRAF in 2001, when AMF colonization was not affected by previous crop. These findings support the results reported by Grant et al. (2009) with flax yield higher after wheat than after canola. Research on the northern Great Plains (Mandan, ND) suggests that crop sequence can be used to improve the use of precipitation and improve crop growth, and that seed yield of flax could be increased by threefold when grown after safflower (Carthamus tintorius L.), versus a flax after flax crop rotation (Tanaka et al. 2005). Tillage affected early root biomass of flax at one of four sites, at the BRC-North site only in 2001 (PB0.0001), where root weights of flax were larger after RT (Table 4). Although tillage did not affect early root biomass of flax Table 4. Effect of type of preceding crops (Canola and Wheat), conventional tillage (CT) or reduced tillage (RT), phosphorus applications to preceding crop (P previous crop), and P application to flax (P flax) on root biomass of flax (kg ha 1 ) at the Brandon Research Centre (BRC-North) and at the Manitoba Zero Tillage Research Association Farm (MZTRAF) sites for years 2001 and 2002 BRC-North site MZTRAF site Canola Wheat Canola Wheat Pprevious crop PFlax CT RT CT RT CT RT CT RT Mean of P Mean of 25 P Mean across P Mean of P Mean of 25 P Mean across P

8 322 CANADIAN JOURNAL OF PLANT SCIENCE at the BRC-North site in 2002, or at the MZTRAF site in 2001 or 2002, early root biomass was generally larger when flax was grown under RT (Tables 2 and 4). In 2002, flax s root area colonized by AMF showed a positive correlation with root biomass only at the MTRAF site under RT (r0.427, PB0.0001). Pearson et al. (1991) reported no changes in root growth of wheat using minimum and conventional tillage systems in the first 3 yr of a 4-yr cropping cycle; however, root growth was greater under reduced tillage during the later 2 yr of the cropping cycle, and the changes were attributed to higher rates of water infiltration. Although early lateral root area of flax was not affected by treatments at the two study sites in 2001, root sizes of flax were two- to threefold larger at the MZTRAF site than at the BRC-North site. The main difference between the two sites was the long-term reduced tillage management system. However, in 2002, preceding crop affected the lateral root area of flax at both sites (Table 2). Flax s lateral root area was larger when preceded by wheat than when preceded by canola (Table 5). In 2002, preceding crop affected early percent AMF, root biomass and lateral root area in both study sites; this could have been affected by the growing season climatic conditions. In a study on the AMF formation and growth of corn under various soil moisture conditions, Karasawa et al. (2000) found the influence on AMF formation was pronounced on drier soils when previous crops were mycorrhizal. Furthermore, AMF contributed to improve the tolerance of flax to drought due to the improvement of several physiological functions, including higher nutrient assimilation, lower transpiration, and enhanced root conductance (von Reichenbach and Scho nbek 1995). CONCLUSIONS The early AMF colonization of the flax roots was increased when wheat rather than canola was the preceding crop in 3 of the 4 site-years in this study. Only in 2001 was AMF colonization of flax not affected by preceding crop at the MZTRAF site, which has been managed under reduced tillage since Root biomass of flax was highest when the crop was preceded by wheat in all site years of the present study. Lateral root areas were greater when flax was grown after wheat than after canola in 2 site years. In 2002, early mycorrhizal colonization, root biomass and root size of flax increased when the preceding crop was wheat in both experimental sites, when the crop was grown under lowspring MAT fields and when precipitation was scarce throughout the growing season. When flax received P fertilizer or residual Pfrom previous crops AMF colonization on the roots was lowered at the site that was historically managed under CT. Climatic conditions, especially a cooler spring with less precipitation during the early growth period of flax in 2002, enhanced the effect of the preceding crop on AMF colonization and increased root biomass and lateral root size of flax. Conventional tillage reduced flax root biomass in 2001 at the BRC-North site when the crop was grown Table 5. Effect of type of preceding crop (canola or wheat), conventional tillage (CT) or reduced tillage (RT), P application to preceding crop (P previous crop), and P application to flax (P flax) on lateral root area of flax (m 2 ha 1 ) at 5 wk after seeding at the Brandon Research Centre (BRC-North) and at the Manitoba Zero Tillage Research Association Farm (MZTRAF) sites for years 2001 and 2002 BRC-North MZTRAF Canola Wheat Canola Wheat Pprevious crop PFlax CT RT CT RT CT RT CT RT Mean of P Mean of 11 P Mean across P Mean of P Mean of 11 P Mean across P

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