Echinochloa crus-galli and Echinochloa colona mostly grown as weeds in different economic crops and cause losses in their yield. Flooding and Crop rotation ca not suppress these weeds. Herbicides are widely applied to control barnyard grasses. Bisbyric-sodium was applied at FRD (Field Recommended Dose) on both species, the results of preliminary experiment indicated that E. crus-galli was susceptible to bispyribac-sodium but E. colona was resistance. The Confirmation experiment for E. colona resistance consist of two parts, Part of experiment contained both E. colona and rice and other part contained E. colona alone. Although the herbicide effect on the biomass of plants which caused reduction in fresh and dry weight in presence and absence of rice 7 days after treatment, the plant was able to grow and can recover after 20 days in absence of rice. The photosynthetic pigments were affected by herbicide, which decreased by increasing the herbicide concentration. While anthocyanins content were increased to protect the leaves from the stress of photoinhibitory light fluxes by absorbing the excess photons. The soluble and insoluble sugar content was increased by increasing the concentration of herbicide. The transcript level of ALS in E. colona was modified by different concentrations of herbicide. The present study indicated that ALS expression level was responsive to herbicide treatment. This suggests that ALS is a target site of bispyric-sodium. But 50% FRD can be considered as sub-lethal dose which upregulated the expression of ALS by 4 folds. The regrowth of plants at 20 days after treatment could be due to a time-dependent process with an initial weak inhibition followed by a slow transition into a final steady-state where the inhibition is more potent.
The genus Echinochloa includes serious weeds in agriculture being the third and fourth most important weeds in the world (Holm et al., 1977). Echinochloa colona, a vigorous C4 annual species, is one of the world’s most serious grass weeds in rice (Holm et al., 1991; Rao et al., 2007). More than 60 countries have reported it as a weed problem in 35 crops, including rice (Oyza Sativa), maize (Zea mays L.), sorghum (Sorghum bicolor L.), sugarcane (Saccharum officinarum L.), cotton (Gossypium hirsutum L.), and peanuts (Arachis hypogea L.) (Holm et al., 1991). This weed has been reported to occur in 24 countries in dry-seeded rice, in 12 countries in wet-seeded rice, and also in transplanted rice (Rao et al., 2007). E. colona is an example of crop mimicry because it closely resembles rice at the seedling stage and is sometimes unintentionally transplanted into fields together with the crop. Echinochloa crus-galli (L.) Beauv is a C4 weed that grows essentially in paddy fields, well known weed of this genus and is distributed worldwide. Yield losses caused by Echinochloa sp. invasion in rice can be very severe and variable in relation to the cultivar and the duration of competition.
The importance of Echinochloa species in rice is increasing and this is due to the development of resistance to herbicides in several populations. Within the major herbicides applied in rice, resistances have been reported to bispyribac-sodium, cyhalofop-butyl, molinate, propanil, and quinclorac (Fischer et al., 2000; Busi et al., 2002). Several management practices, including intensive chemical control, are used to reduce E. crus-galli infestation in rice production systems in many countries (Gibson et al., 2003; Hoagland et al., 2004). Intensive herbicide used on E. colona and E. crus-galli has already led to the evolution of herbicide resistance to propanil, quinclorac, azimsulfuron, and fenoxaprop-p-ethyl (Rao et al., 2007).
Bispyribac-sodium, sodium 2, 6-bis [(4, 6-dimethoxy-2-pyrimidinyl) oxy] benzoate , is an herbicide used for rice farming . It is indicated for the post-emergence control of cyperaceous grasses and some dicotyledonous grasses and is sold commercially as a suspension concentrate under the name of Nominee. The herbicides bispyribac-sodium, penoxsulam and imazamox are acetolactate synthase (ALS; EC 2.2.1.6)-inhibiting herbicides and have a propensity rapidly to select for resistant weed accessions (Tranel and Wright, 2002). This is confirmed by the fact that 126 weed species have already developed resistant accessions to ALS-inhibiting herbicides (Tranel et al., 2012). In most cases, resistance is due to point mutations in one of the following codons: Ala122, Pro197, Ala205, Asp376, Arg377, Trp574, Ser653 and Gly654, numbered on the basis of the sequence of Arabidopsis thaliana (L.) Heyhn (Tranel et al., 2012). In most cases, resistance to ALS-inhibiting herbicides is caused by an altered ALS enzyme (Tranel and Wright, 2002).
Acetolactate synthase (ALS, EC 2.2.1.6; also referred to as acetohydroxyacid synthase) is the first common enzyme in the biosynthetic pathways leading to the branched-chain amino acids (isoleucine, leucine, and valine). ALS does not exist in mammals, and thus ALS-inhibiting herbicides are thought to be less toxic to mammals. Resistance to ALS-inhibiting herbicides in plants has in most cases been conferred by either single or double amino acid substitutions at a particular position in ALS (Kawai et al., 2007; Okuzaki et al., 2007). ALS-inhibiting herbicides have become widely used in world agriculture. Consequently, there has been evolution of many ALS herbicide–resistant weed populations, with resistant biotypes of 110 weed species worldwide (Heap, 2011).
The knowledge of the response of Echinochloa populations to different herbicides may have several implications. This information, when coupled with the knowledge of the Echinochloa population structure at field or regional scale, is fundamental for the elaboration of appropriate weed management strategies, including the development of models capable to predict the overall weed control level achievable with a certain herbicide. Theoretically, herbicide sensitivity can also be evaluated as an additional trait for the identification of the different species of the genus Echinochloa. This approach has been rarely adopted, even though herbicide sensitivity has been used often to compare the fitness of different populations (Lopez-Martinez et al., 1999). Investigations on differences between Echinochloa populations in terms of response to herbicides can also give potential information for conducting ecological surveys and for studying shifts of the relative abundance of the different species or populations.
Preliminary experiment: investigation the response of E. crus-galli and E. colona to Bispyribac-sodium (Nomineetm 0.015%) herbicide.
Experimental design
The seeds of the two species E. crus-galli and E. colona were sown in a green house in June 2012. Greenhouse conditions were: 1550 µmole m-2 s-1 maximum light intensity, 28/19ºC day/night temperature, 14 h photoperiod, 70% relative humidity, 4.97 m s-1 wind speed. After one month, 10 seedlings of each species were transplanted into blocks 50×50 cm. The experimental design was a randomized complete block design with 3 replicates, 12 blocks represent all treatments 20 plants in each block (10 E. crus-galli and 10 E. colona). After 10 days of transplanting, the plants were treated with 50%, 100% and 200% field recommended dose (FRD) of nominee. The herbicide applications were made using sprayer. Nitrogen fertilizer was applied as urea (50 gm / Sq.m.) one week before herbicide application, the plants was fertilized according to regional practices. The herbicide treatment was carried out as recommended by the manufacturer.
Harvesting of the plants
Seven days after treatment with herbicide, the plants were collected, placed in paper envelops and oven-dried at 65 Co for 48 hours.
Biomass yield
Determination of fresh and dry weights and water contents
After recording the fresh weights of shoots, they were dried in oven at 80°C for 2 days and the dry weights were then recorded. The water contents were calculated on fresh weight basis. Three plant replicates were used for each treatment.
Confirmation experiment: The resistance of E. colona to Nominee.
Seeds of both E. colona and rice were planted in the green house in July 2012. Greenhouse conditions were: 1550 µmole m-2 s-1 maximum light intensity, 28/19ºC day/night temperature, 14 h photoperiod, 70% relative humidity, 4.97 m s-1 wind speed. Part of experiment contained both E. colona and rice and other part contained E. colona alone. The experimental design was a randomized complete block design with 3 replicates, the block area 100 cm×100cm. After 25 days, the plants were treated with 50%, 100%, 200% and 300% FRD of nominee. The herbicide applications were made using sprayer. Nitrogen fertilizer was applied as urea one week before herbicide application. The herbicide was applied as recommended by the manufacturer
Harvesting of the plant material
After Seven and 20days of treatment with herbicide, plant leaves (the second leaf from the top) from 3 replicates of each treatment were collected at 10 AM. Frozen immediately in liquid nitrogen and stored at -800C until used for subsequent biochemical and molecular analyses.
Biomass yield
Determination of fresh and dry weights and water contents
The fresh weight, dry weight and water content were determined as described in preliminary.
Biochemical Analysis
Estimation of photosynthetic pigments: according to the method described by Well burn and Lichtenthaler (1984).
Determination of Anthocyanins: According to the method adopted by Hoagland (1980).
Determination of protein content: The protein content of fresh plant material was measured according to the method of Bradford (1976).
Molecular Analysis
Quantification of gene expression
Quantification of gene expression by semi-quantitative RT–PCR
Total RNA was extracted from about 50 mg frozen leaves using TRI-reagent (Sigma, UK) according to the manufacture’s protocol. To prevent DNA contamination, the extracted RNA was treated with DNA-free kit (Ambion, UK) for 30 min at 37ºC. Poly A tail mRNA was then isolated by reacting 10 ll of RNA with 2 µl of oligo dT(18) and 3 µl RNase and DNase free H2O for 5 min at 70ºC and the reaction was terminated on ice for 2 min. The reverse transcription was conducted by using MMLV-reverse transcription kit according to the supplier’s recommendations (Promega, UK). Primers for gene were designed to recognise conserved regions resulting from the alignment of the characterized gene in E. colona. The primers used for amplifying ALS and 18S rRNA are listed in Table 1. The PCR conditions were as follows: initial denaturation at 94ºC for 3 min, followed by 35 and 45 cycles of denaturation at 94ºC for 30 s, annealing at 50ºC for 30 s and extension at 72ºC for 50 s, the number of PCR cycles was optimized to show the maximal differences among samples within the linear phase of amplification. PCR products were resolved by electrophoresis on 1% agarose gels, stained with ethidium bromide in 1X TAE (Tris–acetic acid-EDTA) using Bio- Rad equipment and visualized and documented using Trans illuminator UViTec. The band volumes were measured by using Lab Image V 2.7.2 software. The measurement was normalized for equal 18S rRNA bands.
Statistical analysis
Each measurement was repeated until consistent results were obtained with at least three independent measurements. In order to compare between samples one way ANOVA-LSD was performed using SPSS 18.0. and CoStat 6.311. (Carver and Nash, 2011).
Results:
First Experiment: Effect of Herbicide on plant growth ( E. Crus-galli and E . colona)
The negative effect of herbicide on the fresh and dry weights of plants increased by increasing the concentration compared to the control. The Fresh weight was decreased by 75.7%, 93.87% and 96.5%, respectively at50%, 100% and 200% in E.crus-galli and also in E.colona it decreased by 75%, 84% and 84.9%, respectively. The Dry weight also decreased by 78%, 86.3% and 90%, respectively at 50%, 100% and 200% in E.crus-galli and in E.colona it decreased by 75.5%, 71% and 70.6%, respectively at (Fig.1 and Fig.2).
Second experiment: Resistance of E. colona to Nominee
The results of plant grown with rice after 20 days of herbicide application are not presented because the plant cannot be tolerate.
Biomass yield (Fresh weight, Dry weight and Water content)
The fresh and the dry weight was decreased by increasing the concentration of the applied herbicide compared to the control. The fresh weight at 300% FRD was decreased by 62.4%, 92% and 98%, respectively in presence of rice 7 days after treatment, absence of rice 7 days after treatment and absence of rice 20 days after treatment (Fig.3A & B) (Fig.4a). The dry weight at 300% FRD was decreased by 81.9% and 95%, respectively in absence of rice 7 days after treatment and absence of rice 20 days after treatment, but in presence of rice 7 days after treatment DW was decreased at 50% then increased at 100% then decreased by increasing the concentration (Fig.4b). Water content was varied at different herbicide concentrations in presence of rice 7 days after treatment where it increased at 50% compared to the control and then decreased at 100% then increased by increasing the concentration but remained lower than the control. In absence of rice 7 and 20 days after treatment WC was decreased compared to the control by increasing the concentration of herbicide (Fig.4c).
Physiological parameters
Photosynthetic pigments
Chlorophyll a, b and carotenoids content were affected with herbicide concentration, which decreased by increasing the concentration in chlorophyll a and carotenoid but it was varied in chlorophyll b. Chlorophyll a was decreased by 56.5%, 43.8% and 36.6% at 200% FRD, also carotenoids were decreased by 41.6%, 41.3% and 39.4% at 200% FRD compared to the control, respectively in presence of rice 7 days after treatment, absence of rice 7 days after treatment and absence of rice 20 days after treatment. chlorophyll b was increased at 50% compared to the control then decreased by increasing the concentration of herbicide in both presence of rice 7 days after treatment and absence of rice 7 days after treatment but in absence of rice 20 days after treatment it decreased by increasing the concentration (Table.2) .
Anthocyanins
The anthocyanins content were increased in comparing to the control by increasing the herbicide concentration, it increased by 69.7%, 2 folds and 3.9 folds, respectively in presence of rice 7 days after treatment, absence of rice 7 days after treatment and absence of rice 20 days after treatment (Fig.5).
Soluble and insoluble Sugars
Both soluble and insoluble sugars content were affected according to the herbicide concentration. Soluble sugars were increased by 5.6 folds and 3 folds at 200% FRD, also insoluble sugars were increased by 21.6% and 72.2% at 200% FRD, respectively in presence of rice 7 days after treatment and in absence of rice 7 days after treatment. In absence of rice 20 days after treatment, both soluble and insoluble sugars were decreased by increasing the concentration of applied herbicide (Table.2).
3.3.2.1.7 Protein Content
Protein content was affected with herbicide concentration. Protein was increased by 63.4% and 2.14 folds at 200% FRD, respectively in presence of rice 7 days after treatment and in absence of rice 20 days after treatment but in absence of rice 7 days after treatment it significantly decreased by 18.4% at 300% FRD (Table.2).
3.3.2.2 Expression of Acetolactate Synthase (ALS) gene in response to Nominee treatments.
The transcript level of ALS in E. colona was modified by different concentrations of nominee. In presence of rice 7 days after treatment, the transcript level of ALS was not detected in treatment 50% but was detected at 100% and 200% which decreased compared to the control by 6.4% and 80.4%, respectively, then was not detected at 300%. In absence of rice 7 days after treatment, it increased at 50% by 4 folds, then decreased at 100% by 93.7%, and was not detected in 200% and 300%. In absence of rice 20 days after treatment, it increased at 50% and 100% by 34.88% and 4.42 folds, respectively, then decreased at 200% by 79.67% (Fig.6).
Discussion
Herbicides have been used to enhance the productivity of crops by killing the weeds which compete with the growth of cultivated plants. In the present study, Nomineetm (bispyribac-sodium) was applied as herbicide and plant responses was studied on E. crus-galli and E. colona. A preliminary experiment indicated that the growth of E. crus-galli in terms of fresh and dry weight was reduced by 93.87% and 86.3%, respectively at 100% FRD. While E. colona growth in terms of fresh and dry weight was decreased at 100% FRD by 84% and 71%, respectively, but nonetheless, the plants were still growing and young leaves were present (Fig. 1). Different responses to bispyribac-sodium were reported by Riar et al., (2012), who found that three Echinochloa crus-galli populations from rice fields in Arkansas (AR1 and AR2) and Mississippi (MS1) showed different responses, reduction in dry weight of AR2 was 99% (susceptible population), in resistant populations, the dry weight reduction was from 54% to 62% and from71% to 73% in AR1 and MS1 populations, respectively. Kaloumenos et al., (2013) also indicated that bispyribac-sodium applied at the recommended rate reduced the fresh weight of nine susceptible E. oryzicola accessions by 83–100%. The present results suggest that E. crus-galli was susceptible to bispyribac-sodium but E. colona showed some resistance.
Investigation of the resistance of E. colona to Nominee
The dose response studies on plants treated with bispyribac-sodium confirmed that E. colona was resistant. Although the herbicide effect on the biomass of plants which caused reduction in fresh and dry weight in presence and absence of rice 7 days after treatment, the plant was able to grow and can recover after 20 days in absence of rice.
The fresh and dry weight of E. colona, was decreased by increasing herbicide concentration from 50% to 300% FRD (Fig. 3&4). Kaloumenos et al., (2013) reported that bispyribac-sodium applied at the recommended rate reduced the fresh weight of six E. oryzicola accessions by 63–77%. These accessions were labelled ‘less Susceptible’. and applied at 4 times the recommended rate reduced the fresh weight of two E. oryzicola accessions only by 16–33% and 12–31% respectively and these accessions were labeled ‘Resistant’. Based on the present results, E. colona can be considered resistant when treated in rice fields where the fresh weight decreased by18-62%. . Presence of rice with E. colona could remove part of herbicide from the plant due to shading effect so the complete dose did not reach to the plant. But when grown alone, E. colona can be considered susceptible (fresh weight decreased by >80%) because the plants exposed to all herbicide concentrations. Studies on reduced doses of herbicide have been conjunction with various cultural practices, such as seeding rate and crop density (Shibayama, 2001).
The photosynthetic pigments were affected by herbicide, where the chlorophyll a, chlorophyll b and carotenoide content was decreased by increasing the herbicide concentration from 1000 to 300% FRD (Table. 2).
Reduction in chlorophyll content may be due to its incorporation into the cell membrane function through physiological processes, such as depolarization of membrane potential (Wright, 1994). Reduction also might be due to the enhanced activity of chlorophyll degrading enzyme chlorophyllase and/or disruption of the fine structure of chloroplast and instability of chloroplast or pigment-protein complex, which leads to oxidation of chlorophyll and decreased its concentration (Hamza et al., 2012).
The anthocyanins content were increased by increasing the herbicide concentration from 100 to 300% FRD (Table. 2). E. colona have anthocyanine pigment in leaves through their life span, or else they are induced and retained only after the plant has experienced stress (Chalker-Scott, 1999). The function of anthocyanins in leaves is the photoprotection of chloroplasts; under saturated light, anthocyanins potentially mitigate photoinhibitory and photo-oxidative damage by absorbing a proportion of the photons surplus to the requirements of the light reactions of photosynthesis (Esteban et al., 2008; Zeliou et al., 2009). This may be the case in the present study where, herbicide affects photosynthetic pigmentation which decreases. So anthocyanine pigments increased to protect the leaves from the stress of photoinhibitory light fluxes by absorbing the excess photons.
The soluble and insoluble sugar content was increased by increasing the concentration from 50 to 300% FRD in presence of rice, 7days and in absence of rice, 7 days after treatment. But they decreased in absence of rice 20 days after treatment at 50% and 100% FRD (Table. 2). Carbohydrate accumulation in leaves has been repeatedly reported after treatment with ALS inhibitors (Wittenbach and Abell, 1999). Zabalza et al., (2004) reported that decrease in sink strength seems to cause the carbohydrate accumulation in leaves. Plants with lowered sink strength (tuber excised) accumulated carbohydrate in the leaves and displayed a considerably reduced maximum photosynthetic rate (Basu et al., 1999).This agrees with the present results where accumulation of carbohydrates in leaves attached coincided with a decrease in plant growth rate.
The protein content was increased by increasing the concentration from 50 to 200% FRD, in presence of rice, 7 days and in absence of rice, 20 days after treatment. But in absence of rice, 7 days after treatment it decreased by 18.4% at 300% FRD. Several biochemical and physiological effects have been described as a secondary consequence of the primary action of ALS inhibitors like decrease in protein levels (Sidari et al., 1998). It could propose that an increase in the protein content was detected because it measured in young leaves that may have been produced after application of herbicide. This view agrees with Rhodes et al., (1987) who suggested that decline in protein content was caused by an increase in the protein turnover. This turnover increased the degradation rate of the already existing proteins, providing amino acids for de novo protein synthesis since the synthesis rate itself is not inhibited. Increase in protein content in the present result might be detected due to decrease in growth rate and proteins become concentrated in less biomass or the remobilizing proteins from old to young leaves as a component of senescence resulting from herbicide treatment.
Expression of Acetolactate Synthase (ALS)
The transcript level of ALS in E. colona was modified by different concentrations of bispyric-sodium (nominee). In presence of rice, 7 days after treatment the transcript level of ALS was decreased by increasing herbicide concentration. In absence of rice, 7 days after treatment the transcript level of ALS increased at 50%, then decreased at 100%, and was not detected in 200% and 300% FRD. In absence of rice, 20 days after treatment the transcript level of ALS increased by increasing the concentration at 50% and 100% and then decreased at 200% FRD. The present study indicated that ALS expression level was responsive to herbicide treatment. This suggests that ALS is a target site of bispyric-sodium. But 50% FRD can be considered as sub-lethal dose which upregulated the expression of ALS by 4 folds. The present results indicate that growing the plants with rice reduces the effect of herbicide on the plants perhaps because leaves of rice can shield the weed leaves.
Often, evolved ALS herbicide resistance in plants is target-site ALS based, endowed by one or more specific resistance-endowing point mutations in the ALS gene. Generally, ALS resistance mutations at Pro-197 confer SU and TP resistance, mutations at Ala-122, Ala-205, Ser-653 or Gly-654 confer IMI resistance, and mutations at Asp-376, Arg-377 or Trp-574 confer broad spectrum resistance across ALS herbicides (Yu et al., 2012).So in the present results the resistance of E. colona to bispyric-sodium might be due to mutation in one of these points on ALS gene. Kaloumenos et al., 2013 reported that the sequence alignment comparison between the susceptible E. crus-galli and resistant E. oryzicola and putative resistant E. oryzicola accessions revealed a heterozygous mutation in resistant and putative resistant accessions at the second nucleotide of codon 574. The presence of Trp574Leu mutation in the 12 analysed E. oryzicola ALS sequences from resistante E. oryzicola supports the evidence that cross resistance to ALS-inhibiting herbicides was due to mutant ALS alleles encoding an amino-acid replacement at codon 574. So the resistance of E. colona to bispyric-sodium could be due to presence of Trp574Leu mutation.
The regrowth of plants at 20 days after treatment could be due to a time-dependent process with an initial weak inhibition followed by a slow transition into a final steady-state where the inhibition is more potent (Chang and Duggleby 1997). An explanation for time-dependent irreversible inactivation of AHAS by herbicides is the oxygenase side reaction of the enzyme (Schloss, 1994). The transcription level of ALS decreased after 7 days of treatment but after 20 days of treatment an increase in transcription level of ALS was detected. Similar results were reported by Li et al., 2013 who found that the expression levels for E. crus-galli glutamate receptor mRNA (EcGLR1) increased 4–9 times in the resistant plants compared to that in the susceptible plants after exposure to quinclorac.