Validation of Competitive Ability of Diverse Canola Accessions against Annual Ryegrass under Glasshouse and Field Conditions

Weeds are a major constraint in canola (Brassica napus L.) production worldwide, as they cause significant reductions in seed yield and quality. Crop interference is one of the approaches to tackle weed infestation along with other agronomic interventions. In Australia, studies have shown genetic variation in the canola capability to suppress annual ryegrass (Lolium rigidum Gaudin) in the field and under in vitro conditions. Early-season crop biomass accumulation and greater plant height are desired attributes for suppression weeds in canola. However, the canola ideotype for interference traits against this weed has not been studied under glasshouse conditions. In this study, we compared the competitive ability of 26 canola genotypes against annual ryegrass under both glasshouse and field conditions. Five canola genotypes consistently showed the ability to suppress growth of annual ryegrass. Both at glasshouse and field conditions, the shoot biomass, largely con-tributed by leaf biomass, was significantly associated with suppression ability. Our results suggest that a glasshouse-based evaluation approach can be used to determine the suppressive ability of advanced breeding lines for suppression of ryegrass growth. Based on our analysis, we suggest that initial screening of large collections of germplasm can be conducted under glasshouse conditions, with selected genotypes further evaluated in the field.

recent years, canola cultivation has expanded rapidly due to its high grain prices and demand for healthy vegetable oil, stockfeed and biodiesel markets [1]. Weed infestation, however, is a major constraint limiting canola production [2]. In Australia, total yield loss due to weeds in canola and pulses has been estimated at 122,048 tonnes, resulting in a revenue loss of $54 million [3]. Annual ryegrass (Lolium rigidum Gaudin) has been the most widespread weed in winter crops, occurring in 86% of canola crops in south-eastern Australia [2].
The primary method of weed control is the application of herbicides. However, the prolonged and widespread chemical use has been increased especially after the introduction of herbicide-tolerant cultivars to triazine, imidazolinone and glyphosate. This has led to an increase in the evolution of herbicide resistance [4]. Canola seems to be particularly vulnerable for developing to herbicide resistance as there are limited options of commercial herbicide available to control broad-leaf weeds. In recent years, the canola industry is increasingly becoming dependent on herbicide tolerant varieties including genetically modified herbicide tolerant varieties, which are meant to provide control options for the major weeds of that crop, such as annual ryegrass and wild radish (Raphanus raphanistrum). Evaluation of the herbicides against different weed species showed that 8 of the top 15 are likely to be utilised in canola production, including imazamox and imazethapyr for the Clearfield® HR canola, glyphosate for the Roundup Ready® canola, and atrazine and simazine for the triazine tolerant lines are resistant to herbicides [5]. In recent years, some countries are imposing restriction on the usage of certain herbicides such as glyphosate for weed control; this practice necessitates the development of alternative and sustainable options for weed management. In addition to agronomic interventions that can influence weed management including seeding rate, row spacing, row orientation and fertilizer [6] [7], crop interference is worth investigating as a tool for weed management [8]- [14]. Considerable genotypic variation for weed competition exists in crop plants including canola, although some species are considered more competitive than others [15] [25]. The competitive ability of a crop to weeds can be measured either on the basis of the ability of crop to maintain growth and seed yield in the presence of weed, or on the basis of the ability of crop to suppress growth and seed production of weed species [26]. Weed competitiveness in canola has been evaluated mainly under laboratory and field conditions and to a limited extent under glasshouse conditions [9] [27] [28]. Under field conditions, it is difficult to achieve precise and uniform plant densities across a trial site, and this may influence the differential responses obtained [26] [29]. Secondly, field conditions can compromise the outcomes through environmental variance [9] [26] [30]. Lastly, screening large Open Journal of Genetics numbers of genotypes for weed-crop competition under field conditions is labour and space intensive [31].
The objective of this study is to evaluate the suppression ability of different canola genotypes against ryegrass. Obtaining reliable estimates of weed competitive ability and understanding the canola ideotype for interference traits are important for designing sustainable weed control strategies with low herbicide use for improving canola productivity and profitability.

Canola Genotype and Weed Populations
Previously, Asaduzzaman et al. [27] utilised 70 genotypes of canola to investigate genotypic variation for allelopathy and weed competitiveness. In this study, a set of 26 diverse Brassica genotypes (Table 1), including a subset of canola genotypes utilised by Asaduzzaman et al. [8] was characterised for their competitive ability against annual ryegrass cv. Wimmera under glasshouse and field conditions. This rye grass cultivar is well-suited to dry and low fertile soils and extensively used for productive, nutritious pasture crop. Seeds of canola genotypes were accessed from the National Brassica Germplasm Improvement Program (Wagga Wagga, Australia).

Evaluation of Canola-Weed Suppressive Ability under Glasshouse Conditions
The glasshouse experiment was conducted at the Wagga Wagga Agricultural In-

Evaluation of Canola-Weed Suppressive Ability in the Field
The field experiments were conducted at the Wagga Wagga Agricultural Institute, Australia (35˚30'07"S; 147˚21'06"0E) in a duplex Red Kandosol of pH 5.3.
The field had a history of naturally high annual ryegrass population. Herbicides Plots were harvested at maturity with a small-plot header (Kingaroy, Australia) and grain yield was expressed in g plot −1 .

Statistical Analysis
Glasshouse trial: Data were analysed using R software [32] and the ASReml package [33]. Graphics were produced in the lattice package [34].

Genetic Variation for Weedcompetitive Ability under Glasshouse Conditions
The analysis revealed highly significant differences between genotypes and between weed treatments for all traits evaluated, whereas the interaction between the genotype and weed treatments was only significant in stem biomass and plant height ( Table 2). Several genotypes revealed a strong ability to interfere with ryegrass growth under glasshouse conditions. Significant genotypic effects were found on shoot biomass of ryegrass that ranged from 1.05 to 2.28 g plant −1 (Figure 1 Weed treatment influenced shoot biomass of canola genotypes as compared with the weed-free treatment (Figure 1(c)). However, the crop height was less affected by the presence of ryegrass (Figure 1(b)). This explains why crop biomass had higher negative correlation with weed biomass relative to the low correlation between crop height and ryegrass biomass. To identify competitive traits suitable for genetic selection, we sought correlation relationships between ryegrass and canola phenotypes. Leaf and shoot biomass showed a significant negative correlation (r = −0.50 to −0.76) with biomass of annual ryegrass ( Figure 2).
In contrast crop heights and leaf number had positive relationships with ryegrass biomass (Figure 2).

Validation of Genetic Variation for Weed Competitive Ability under Field Conditions
Our glasshouse experiment revealed that vigorous canola genotypes having   higher leaf and shoot biomass control ryegrass better as compared to less vigorous genotypes. In order to validate the competitiveness of vigorous varieties against ryegrass, we conducted a field experiment using 26 canola genotypes.
Results reconfirmed that plant vigour (shoot biomass) is a genetic trait and significantly affects ryegrass (shoot biomass), but the interaction between genotypes and weed biomass was non-significant (Table 3). This indicates that genotype and/or weed treatment is not influenced with growing environment (ryegrass influenced the crop biomass at a constant rate across all genotypes). As observed under glasshouse experiment, there were significant genotypic differences in plant height; however it did not significantly affect ryegrass (shoot biomass). The interaction between genotypes and weed biomass was also significant Open Journal of Genetics Crop biomass (g plot −1 ) n/a n/a <0.001 Crop height 1 (cm plant −1 ) n/a n/a 0.02 Crop height 2 (cm plant −1 ) n/a n/a <0.001 Flowering time n/a n/a 0.159 *n/a, Not applicable, covariate included in the analysis.
( Table 3). The main effect of genotypes on grain yield was highly significant, whereas the effects of weed biomass and flowering time were not significant. The significant genetic effects of crop biomass and plant height may have accounted the variation in yield (Table 3).     have been implicated in competiveness for light [26]. Our study showed a  significant correlation between crop biomass and ryegrass suppression in both glasshouse and field conditions. These results indicate that plant vigour, manifested in higher canola biomass is associated with suppression of annual ryegrass. This is in agreement with previous studies where diverse canola accessions were compared under field conditions and showed that early-season crop biomass accumulation (early vigour) was associated with weed suppression [16] [17] [22]. In the glasshouse experiment, we found that leaf biomass was significantly correlated with weed suppression (r = −0.50), whereas crop stem biomass did not show any correlation (r = −0.05). Number of leaves also was not correlated with weed suppression. These results indicate that larger leaves are likely to provide shade and thus interfere with light inception to ryegrass. Further studies to understand the association between the area and angle of leaves and the suppression of weed growth are required for the germplasm used in our study. This would identify effective phenotypic traits for weed competition in a breeding program. Under field conditions, increase in crop biomass was associated with reductions in ryegrass biomass and with increased crop yields. This may reveal that canola biomass and particularly leaf biomass related to weed suppression and consequently improvement in yield.

Discussion
The glasshouse and field experiments showed a low correlation between crop height and weed suppression but no correlation between crop stem biomass and weed suppression. Such lack of relationship has also been found in screening 111 rice genotypes against Echinochloa crus-galli in the field [37]. However, this re-Open Journal of Genetics sult is not in agreement with previous studies in canola. The glasshouse experiment (Figure 1(b)) indicated that crop height was less affected by the presence of weed compared with those in weed free conditions, whereas crop biomass was highly affected by weed (Figure 1(c)). Similarly, in the field experiment, crop height was not significantly influenced by the weed, whereas the crop biomass was. Most winter varieties found in our study are good weed suppressors in the glasshouse experiment (Figure 1(a)) and only Akela was superior in weed suppression in the field experiment ( Figure 4). A spring genotype, Tarcoola-141 showed better ability in weed suppression under the glasshouse and field conditions with higher yield in the field. Other two spring genotypes, Av-Opal and Av-Garnet and a semi-winter PAK85388-502 demonstrated good weed suppression under both conditions as it was found in other studies [9] [17]. Three spring genotypes including Ag-Spectrum, Lantern and Sturt-TT were found less to be weed suppressive under glasshouse and field conditions and had low grain yield in the presence of weeds.

Conclusion
Our study suggests that: 1) vigorous canola varieties can provide competitiveness to ryegrass under glasshouse and field conditions and 2) glasshouse conditions can be used to evaluate weed suppression ability of a large number of canola accessions while maintaining uniform density of annual ryegrass. We conclude that vigorous genotypes with highest weed suppression can be exploited for weed control in canola. Further research is required to develop structured populations between highest and lowest weed suppression genotypes can be developed to understand genetics underlying this valuable trait. Molecular markers can be developed for marker assisted selection leading to an acceleration of improved varietal development pipeline.