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Economics of Insecticides Usage among Cowpea Farmers in Kaduna State, Nigeria

This study carried out an economics of insecticides usage among cowpea farmers in Kaduna State. Specifically, the study estimated insecticides marginal productivity; the degree of response of demand for insecticides to changes in its prices and the return to cowpea production due to insecticides usage. A multi-stage sampling technique was used to select 150 cowpea farmers who used insecticides in controlling pest in cowpea production in the study area. Information collected includes those of inputs quantities and prices as well as quantity of cowpea output and its farm prices. The logistic specification of the damage control model and its corresponding demand function were used to estimate insecticides marginal productivity and the degree of demand’s response to changes in insecticides prices respectively. A budgetary analytical model was used to estimate the return to cowpea production. The study showed that insecticide marginal value product was N310.06 and the ratio of MVP to insecticide price was 0.48. This is an indication that insecticides were not efficiently utilized. The demand elasticities for the various insecticides were greater than unity indicating that demand for insecticides used in cowpea production in the area studied was own price elastic. The study also found that a return of N787.52 per hectare was obtained due to insecticide usage.

INTRODUCTION
In West Africa and many parts of the world, Cowpea (Vigna unguiculata (L) Walp) is an important grain legume (Singh et al., 2002). The crop is very unique as an important source of food, income and fodder. It has high potential to increase income of both farmers and traders, being widely consumed and traded outside the producing localities (Owoade et al., 2004). Nigeria, being the largest producer of cowpea in the world accounts for about two million metric tonnes which is about 50% of the world production output (Singh et al., 2002). However, the continued cultivation of cowpea as a profitable agribusiness enterprise is being threatened by a number of problems which include those of pests and diseases. Singh and Allen (1980), and TJRI (2009) identified various kinds of pests and diseases that have led to a considerable decrease in the output of cowpea. Effective control of these pests and diseases require the use of chemical pesticides which level of use has been found to depend on its price, risk, credit availability and cropping pattern (Sukume, 1999).
Insecticides are agro-chemicals (pesticides) that are used for crop protection. They are substances intended to prevent, destroy, repel or control any animal pest or diseases caused by micro organisms as well as weeds (Dugje et al., 2009). Tijani and Oshotimehin, (2007) posited that pesticides are protective resources which are unique and differ from other productive resources. This is because they do not affect productivity directly but are applied to eliminate those factors that directly reduce productivity. For a good result in cowpea production, pest control is a major task. Even though efforts are being intensified to reduce the amount of chemical insecticides used in the control of pests through the introduction of integrated pest management, insecticides use in cowpea production has been reported to be skyrocketing dramatically (IITA, 2003). This goes in line with the assertion of Davies (1992) that "pesticide treatments are likely to remain the most important component of crop protection programs for the foreseeable future in agriculture even in the face of new methods of pest management exploiting genetic resistance in plants and biological control". It is therefore obvious that the use of pesticides in cowpea production is inevitable if success must be recorded among small scale farmers who control the bulk of domestic output.
A report given by NFRA (2008) revealed that the total land area cultivated by small holder farmers and their output of cowpea in Kaduna State, Nigeria has dropped drastically in recent times. The low yield obtained in most cowpea producing areas of West Africa is largely due to field insect pests which feed on reproductive plant parts causing most economic damage thereby necessitating appropriate control measure (Karungi et al., 2000). Effective control of pest infestation on cowpea can only be achieved through the use of convectional insecticides. The efficacy of pesticides (which include insecticides) depends mainly on their level of use which in turn depends on their market prices. Prices are particularly important since most of the pesticides are imported into the country (Tijani and Oshotimehin, 2007). With the low exchange rate of the Nigerian currency against major currencies of the world, the prices of the pesticides are becoming comparatively high. It is expected therefore that farmers would respond to changes in prices by adjusting the level of use of the resource alongside changes in its market prices. Lagoke (1982) has attributed the low output per hectare of cowpea to the attack of pest and diseases.
The broad objective of this study is to examine the economics of insecticides use among cowpea farmers in Kaduna state. The specific objectives are to: (i) estimate the production function and marginal value products from the usage of insecticides in controlling pests in cowpea production, (ii) estimate the demand functions and price elasticity of demand for the different insecticides used for cowpea production and (iii) estimate the financial returns (profitability) from insecticide usage in cowpea production.
According to Chambers and Litchtenberg (1994), existing empirical information on pesticide productivity and pesticide demand is at best inadequate. Information about the degree of responsiveness to changes in the market price of pesticides by farmers is an important one, which will help the policy makers and researchers in their policy recommendations. Also, earlier studies in cowpea production in the area have not taken into cognizance the uniqueness of insecticides as a protective production resource differing from other productive resource. This study is justified on the above premises.

Conceptual Frame Work
All marginal product estimates of pesticides before 1986 use the standard Cobb-Douglass production function. In a 1986 analysis, Lichtenberg and Zilberman argued that the single equation Cobb-Douglass model may not be appropriate because it leads to overestimation of the marginal product of pesticides and to address this problem, they introduced a different model of the pesticide yield relationship. This approach proposes a damage abatement model. Essentially, the approach considers the effects of pests on yield separately from the effect of pesticides on pests (Anna et al., 2007). One key feature of the damage control model is the distinction between inputs in standard factors of production and damage control agents. This distinction is important because control agents like pesticides and biological control do not enhance productivity directly as standard inputs do but contribute indirectly to the actual output by preventing output losses. Lichtenberg and Zilberman (1986) noted that damage abatement function may not only include damage control agents, but also other variables which affect the damage abatement effort. Thus, exogenous variables such as weather conditions and the state of nature interacting with pest prevalence could be included in the function but are ignored since they are considered to be uncontrollable.
From the characterization of actual output as a combination of potential output and the quantity lost that production (Q) can be characterized as a function of productive inputs, Z, and damage abatement G(X) The abatement function G(X) can be defined as the proportion of the destructive capacity of the damaging agent eliminated by the application of a level of control agent X. This definition suggests that the abatement function will possess the properties of a probability distribution. It will be defined on the (0,1) interval with G=1 denoting complete eradication of the destructive capacity and G=0 denoting zero elimination i.e., maximum destructive capacity; it will be monotonically increasing and it will approach a value of unity as damage control agent X increases. That is GሺXሻ → 1 as X → ∞. The derivative of G with respect to X,G ଡ଼ ሺXሻ = gX, represents the marginal damage control effectiveness or marginal productivity; simply the density of GሺXሻ.
The production function F (•) is assumed to posses the standard properties of production functions notably concavity in (Z, G). When the destructive capacity of damaging agent is completely eliminated, losses will be zero and actual output will equal potential output, that is potential output can be expressed as F(Z, 1). When G=0, F (Z, 0) denotes the output obtainable under maximum destructive capacity that is minimum actual output.
Assuming that the output elasticity of abatement, G, and all other inputs, Z are constant and that the elasticity of substitution differs only negligibly from one over the relevant range, then the Cobb-Douglass specification; (2) represents the underline production function and the random error associated with it. This as argued by Lichtenberg and Zilberman gives a better estimate of abatement agent rather than using X instead of abatement function GሺXሻ in Cobb-Douglass Q = α + Zβ + γX + V Where β and γ are the parameter estimates of the productive inputs and protective inputs respectively, Z are the productive inputs and X the damage control agent.

Hypothesis
The null hypothesis for this study is: H 0 : the ratio of revenue to pesticide price does not affect pesticide use level.

MEHODOLOGY
The study was conducted in Kaduna state, Nigeria. The state is in the Northern part of Nigeria and is located between latitudes 10 0 20 1 N to 10 0 33 1 N and longitudes 7 0 45 1 to 7 0 75 1 E (Wikipedia March, 2008). It shares common borders with Abuja in the south-east, and six other states namely; Katsina, Kano, Zamfara in the North, Nassarawa, and Plateau in the North-East and Niger in the North-West. The hottest months are March-April while the coldest are December-January. Rainfall is heaviest in the south, and decreases northwards with an annual mean rainfall varying from 942mm to 1000mm. The rainfall lasts from May till October (NAERLS, 2002). The people of the state are mainly engaged in agricultural production activities with the main crops being Maize, Sorghum, Millet, Rice, Cowpea, Ground-nut, Yam and Sugar cane. The National population commission puts the projected population of the state for 2010 at 6,066,562 peoples (NPC, 2006). Kaduna state has a land area of about 46,053Km 2 .
Primary data were used for this study. The data were collected by interview method with the aid of a well structured questionnaire administered to cowpea farmers using multistage and random sampling procedures. In the first stage, three Local Government Areas (Giwa, Kudan and Sabon Gari) were purposively selected from the 23 LGA in the state based on their level of involvement in cowpea production in the state. In the second stage, three villages were randomly selected from each of the selected LGA. These include: Zabi, Hanwa and Ugwan Nashuka in Sabongari LGA; Giwa, Yakawada and Doka in Giwa LGA and Jaja, Hanyin Makada and Honkuyi in Kudan LGA. In the final stage, 150 cowpea farmers were randomly selected and interviewed for the study.

The Damage Control Model
A damage control production function by Litchenberg and Zilberman (1986) begins with a multi-factor production function mathematically expressed as Q = fሺZ ଵ , Z ଶ , Z ଷ , Z ସ , Z ହ … Z ୬ ሻ + U………………………………………… (3) Where Q equals the quantity of output in Kgha -1 , Z 1 -Z n = input used in production purposes. They argued that damage control agents such as insecticides have no direct relationship with output as specified by the equation above. Their argument is based on the work of Feder, (1979) who stated that the output realized from a production process in which damage agents are present is assumed to be given by Where Y is equal to output realized, Π equals to potential output and D equals to quantity lost due to pest attack. The quantity D depends on the pressure of pests. A rational farmer applies pesticides so that D can be reduced as much as possible while Y is increased. The increased in Y therefore depends on the amount of pest the insecticide is able to abate. This established the indirect relationship between the output and the insecticide as other productive inputs do.
If the insecticide used is denoted by X, ψ(X) is the percentage of insect killed and an additive error ε is included. Then yield can be written as It is on the basis of equation (5) that Lichtenberg and Zilberman proposed their damage control model (LZ model). The relationship between output and pest level is given by: Where Q= output, Z= productive inputs used and B = the pest pressure. Also, B=B(X,B 0 )………………………………………………………………………… (7) X being the level of protective input used and B 0 the uncontrolled pest level. Lichtenberg and Zilberman (1986) therefore proposed a production function which recognized the abatement function of the damage control agents like pesticides among other variable inputs. The model is stated as: Q = αZβሾGሺXሻሿ ஓ … … … … … … … … … … ………………...………………… (8) Where Q = output realized from cowpea production (Kg), α= constant of the model, Z = productive inputs used, X= damage control agent (insecticides in litres) and β and γ are positive parameters estimated. G(•) is the proportion of the capacity of the damaging agent abated by the use of X; 0<G(X)<1. Explicitly, LZ model is given by: Where: Z 1 = farm size in hectare, Z 2 = fertilizer (SSP) in Kgha -1 , Z 3 = quantity of labour (hired + family) used in mandays, Z 4 = quantity of cowpea seed used in Kgha -1 , X= quantity of insecticides used measured in litres/ha and μ = white noise error term. Equation (9) was estimated.
Different forms of G(•) exist as alternative relationship between damaging capacity and the level of control agent. Assuming a logistic damage control function and the neo-classical profit maximization objective on the part of the farmers, Lichtenberg and Zilberman derived the following specification for the abatement function The logistic production function specification of the LZ model obtained by substituting the value of G(X) in equation 10 into equation 9 is therefore The corresponding demand function is This can be further expressed as X = a + a ଵ ln ቂ  Table 1 shows the result from the estimated coefficients of the Logistic production function in equation (9). The result shows that insecticide usage (variable X) in cowpea production was statistically significant at 5% level and has positive effect on the output. Thus insecticide has a direct positive relationship with output and the damage agent. The higher the pest pressure, the higher the level of insecticides a farmer is expected to use.  Table 2 shows that the marginal value product of insecticide was N310.06 while the ratio of MVP to factor price of insecticides was 0.48. This is an indication that insecticides were inefficiently used by the farmers. A ratio of less than unity signifies an overuse of the resource or high factor cost in relation to the product market price. This result agrees with the findings of Mshelia and Kwanga, (2006) but inconsistent with the findings of Alimi (1999) which had earlier indicated that insecticides were not overused. This inconsistency might be possible if insecticides prices are high in the study area since the two studies cited were carried out at different times and in different ecological zones of Nigeria.

Demand Functions for Insecticides
All the respondents used insecticides to control insect pests on their farms. However, about 48% of the respondents stated that they have grown cowpea in the past without using insecticides and discovered a significant difference in the marketable quantity of cowpea realized. Majority of the respondents,(99%) obtained their insecticides from the open markets. Four different types of insecticides were used (Table 3). The results of the demand equation (equation 3) are presented on table 3. The R 2 -adjusted ranged from 41.4% for Cymbush to 88% for Shapper plus. Thus, the revenue, insecticides cost ratio explained 41% of the demand for cymbush while it accounts for 88% of the demand for shapper plus. The hypothesis that the ratio of revenue to insecticide cost does not affect insecticides was rejected at 5% level of probability because in all the cases coefficients were statistically different from zero at 5% level of probability. It shows therefore that the quantities of all the insecticides used by the respondents were influenced by their prices. This agrees with the findings of Tijani and Oshotimehin (2007).

Price Elasticity of Demand for Insecticides
The insecticide demand elasticities were estimated and are as shown on table 4. Each of the insecticides has absolute values of elasticity greater than unity and is therefore elastic. This suggests that a 1% increase in the price of cymbush (holding product and other input prices constant) would lead to 1.63% decrease in its use. Furthermore, a 1% increase in the prices Karrate/cypermethrine, shapper plus and uppercott would lead to a decrease in their use by 4.27, 3.0 and 3.41 percent respectively. The policy implication from this result is that increase in prices of insecticides would substantially reduce insecticides usage by farmers in the study area. On the other hand, the provision of subsidy would significantly enhance insecticide usage by the farmers in cowpea production.  Table 5 shows the net returns from insecticides usage derived from equation (16). The average is the percentage increase in marketable yield of cowpea as a result of insecticides usage to control pests was about 260%. It means that insect pests of cowpea of different categories were effectively controlled through the use of insecticides. The mean cost of pest control of cowpea farm was found to be N 787.52 per hectare. However the cost ranges from N354.38 to N5, 709.50 per hectare. The average area of land sprayed among the cowpea farmers was 1.95ha. The average net return from cowpea farms was N 48,308.8 per hectare. However, the net returns ranged from 25,933 to 417,812. The net return analysis result shows that cowpea production using insecticide was profitable.

CONCLUSION AND RECOMMENDATION
This study has shown that insecticides use has a positive and significant effect on cowpea grain output. In addition, land, labour, and seed have positive and significant effect on cowpea yield while fertilizer has a negative and also significant effect on yield. However, the price elasticity of demand is elastic suggesting that the price need not increase astronomically, or else, the farmers will use less of them. It was therefore recommended that federal government input policy should be such as to minimize sudden increase in prices to ensure farmers continual patronage.


extfieldsofstudy: Biology Medicine

provenance: AgriculturalAndFoodSciences,1970-2015-Agricultural And Food Sciences-2011.jsonl.gz.gz

text:
Preliminary Phytochemical Screening and In Vitro Anti-Helicobacter pylori Activity of Extracts of the Stem Bark of Bridelia micrantha (Hochst., Baill., Euphorbiaceae)

Helicobacter pylori is a major risk factor for gastritis, ulcers and gastric cancer. This study was aimed to determine the antimicrobial activity of the stem bark of Bridelia. micrantha on H. pylori isolated in South Africa. Extracts and clarithromycin were tested against 31 clinical strains, including a standard strain (NCTC 11638) of H. pylori, by measuring the diameters of the corresponding inhibition zones, followed by determination of the Minimum Inhibitory Concentration (MIC) (using metronidazole, and amoxicillin as control antibiotics) and the rate of kill. Preliminary phytochemical screening was also done. Inhibition zone diameters which ranged from 0–23 mm were observed for all five of the extracts and 0–35 mm for clarithromycin. Marked susceptibility of strains (100%) was noted for the acetone extract (P < 0.05), followed by ethyl acetate extract (93.5%). The MIC50 values ranged from 0.0048 to 0.156 mg/mL for the ethyl acetate extract and 0.0048 to 0.313 mg/mL for the acetone extract. The MIC90 values ranged from 0.0048 to 2.5 mg/mL for the ethyl acetate extract and 0.078 to >0.625 mg/mL for the acetone extract, respectively. Insignificant statistical difference in potency was observed when comparing the crude ethyl acetate extract to metronidazole and amoxicillin (P > 0.05). Complete killing of strain PE430C by the ethyl acetate extract was observed at 0.1 mg/mL (2 × MIC) and 0.2 mg/mL (4 × MIC) at 66 and 72 h. For strain PE369C, 100% killing was observed at 0.1 mg/mL (2 × MIC) in 66 and 72 h. The ethyl acetate extract could thus be a potential source of lead molecules for the design of new anti-Helicobacter pylori therapies as this study further confirmed the presence of phytochemicals including alkaloids, flavonoids, steroids, tannins and saponins.

Introduction
Helicobacter pylori is one of the most common chronic bacterial pathogens of humans. It colonizes the gastric epithelial surface and withstands the stomach's hostile environment by microaerophilic growth capacity and the production of numerous virulence factors which may lead to chronic gastritis, peptic ulceration and gastric cancer in later life [1,2]. Infections in the developing countries have been reported to be higher than in developed countries. In Africa, 70 to 80% are infected with the organism and 61 to 100% harbour the organism in sub-Saharan Africa [3][4][5].
Treatment could be achieved by a combination of therapeutic agents such as antibiotics, bismuth subsalicylate, proton pump inhibitors and H2-blockers. However, treatment failure rates remain as high as 5 to 20%, along with frequent relapses of gastric ulcers and the cure achieved is incomplete and undesirable side effects are known to occur [6]. The organism has developed resistance against most antibiotics, especially metronidazole, which therefore limits their use in the treatment of infections and this problem is being encountered more in Africa [4,7]. Other factors including poor patient compliance, the significant cost of combination therapy, and the non-availability of medications in rural areas, especially in Africa, reveals the need to develop alternative approaches to suppress/cure the infection.
Plant materials have been reported to be present in or have provided the models for about 50% of Western drugs, with herbal remedies demonstrating encouraging results in the cure of many diseases [8]. Medicinal plants such as Thymus kotschyanus, Ageratum conyzoides (Linn), Scleria striatinux (De Wild) and Lycopodium cernua (Linn) Pic. Serm have shown potential anti-Helicobacter pylori activities [8,9].
Bridelia micrantha Hochst., Baill. (Euphorbiaceae), also known as Coast gold leaf in English (Munzere in Venda) is a semi-deciduous to deciduous tree, up to 20 m tall, with a dense rounded crown and tall, bare stem, widespread in South Africa. The stem bark has been traditionally employed in South Africa to treat several clinical conditions, including intestinal parasites, gastritis, salmonellosis and gastro-enteritis, stomach problems, human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS), infertility, neurosis and psychosis [10,11]. This plant has been reported to possess compounds like friedelin, alpha-amyrin, gallic acid, luteoforol and also being active against E. coli, K. pneumoniae, S. flexneri, P. aeruginosa and other organisms [12].
To the best of our knowledge, this plant has not been evaluated for its antimicrobial activity on clinical isolates of H. pylori isolated in South Africa; an organism with profound heterogeneity compounded by an emerging trend of resistance to the current treatment regimen employed in South Africa [7]; hence the search of potential sources of easily available starting materials for the synthesis of new drugs against the pathogen.

Anti-Helicobacter pylori Activities
It was observed in our results that the zones of inhibition ranged from 12 to 20 mm for the ethyl acetate extract; 16 to 23 mm for the acetone extract; 0 to 15 mm for the water extract and 0 to 35 mm for clarithromycin (Tables 1 and 2).   PE11A  16  22  0  0  8  10  PE26A  17  22  8  8  10  18  PE93A  18  20  0  0  0  28  PE93C  18  20  0  8  9  8  PE102C  16  20  0  0  11  16  PE115A  17  20  0  0  11  One hundred percent susceptibility was noted for the acetone extract. Susceptibility to the ethyl acetate, methanol and water extracts were 93.5%, 3.2%, and 12.9%, respectively. For the control antibiotic, clarithromycin it was 58.1% ( Figure 1). The mean difference of the acetone extract of B. micrantha was statistically significant (P < 0.05) compared to the other extracts and clarithromycin at 95% Confidence Interval (Table 2).  For the MIC determination, an inhibition zone of ≥14 mm was chosen as representative of bacterial susceptibility to the extracts (acetone and ethyl acetate) and antibiotic. Ethyl acetate (20%) and DMSO (10%) used as negative control, showed no activity.
The high prevalence of H. pylori infection as well as the increasing trend of antibiotic resistant strains to the current treatment regimen in the Eastern Cape Province of South Africa has been reported, but few studies have examined the activities of medicinal plants against the pathogen in Africa in general and South Africa in particular [7,8,13]. We are not aware of any study which evaluated the anti-H. pylori activity of B. micrantha in spite of its profound antimicrobial potential against several microorganisms, including Campylobacter spp., a close relative to H. pylori [10,12,14].
The present study revealed that the ethyl acetate extract demonstrated inhibition zones against this pathogen that ranged from 12 mm to 20 mm and for acetone, 16 mm to 23 mm ( Table 2). This is in line with similar zone diameters of inhibition for ethyl acetate and other solvent extracts of some medicinal plants from Cameroon against H. pylori [8]. However, Adeleye et al. [15] reported a diameter of 0 mm zone of inhibition for the water extract of the leaves of this plant. In the present study, we documented a zone diameter of 0 mm to 15 mm. This may be due to the fact that the different parts of the plant may contain different phytochemicals.
The acetone (100%) and ethyl acetate (93.5%) extracts were observed to be the most active against all the H. pylori strains tested, compared with the other solvents used in our study ( Figure 1). This is similar to the results of other investigators [16,17], who equally reported marked activity of ethyl acetate extracts compared to other solvents. Djipa et al. [18] and Asha et al. [19] reported potent antimicrobial activity of an acetone extract against pathogenic bacteria compared to other solvents used. This may be due to the fact that the active compounds against H. pylori strains in the plant were less polar, semi-polar solvents dissolve semi-polar compounds best and different solvents extract different compounds [8].

MIC determination at MIC50 and MIC90
The MIC 50 values for B. mic. EA ranged from 0.0048 to 0.156 mg/mL; 0.0048 to 0.313 mg/mL for B. mic A, and 0.0048-0.156 for metronidazole and amoxicillin respectively. The MIC 90 for B. mic. EA ranged from 0.0048 to 2.5 mg/mL; 0.078 to >0.625 mg/mL for B. mic A; 0.0098 to >5 mg/mL for metronidazole and 0.078 to >2.5 mg/mL for amoxicillin (Table 3).  Table 3. Cont.
One hundred percent of the strains were suppressed by B. mic. EA, followed by amoxicillin, 12 (38.7%) and metronidazole, 11 (35.5%) at a concentration of 0.0048 mg/mL (at MIC 50 ). At 0.078 mg/mL, 11 (35.5%) strains were inhibited by B. mic A followed by amoxicillin, 6 (19.4%) and metronidazole, 5 (16.1%) (Figure 2). The activity of B. mic. EA was not statistically significant (P > 0.05) to the two antibiotics tested. However, a statistically significant difference in potency was observed for B. mic. A compared to the two antibiotics (P < 0.05), but not between metronidazole and amoxicillin (P > 0.05).
The average MIC 50 values of the extract in this study, which ranged from 0.0048 to 0.156 mg/mL, was 0.052 mg/mL (Table 3). Samie et al. [12], found that B. micrantha was mostly active against S. flexneri (1.5 mg/mL) and most of the other organisms tested with a MIC value that ranged between 3 to >12 mg/mL. This was less active compared to our current result which may be attributed to the different organisms used, solvent used in extraction, season in which the plants were collected as well as storage conditions amongst others, as all these factors have been reported to affect the antimicrobial activity of plants [20,21].  For all the isolates tested, the MIC values of the antibiotic ranged from 0.0048 mg/mL (4.8 µg/mL) to 0.156 mg/mL for amoxicillin and metronidazole, respectively, which is similar to a recent study in the same locality conducted by Tanih et al. [7] who reported 2.5 µg/mL-5.0 µg/mL for amoxicillin. Metronidazole and amoxicillin which served as the positive control however, had no statistically significant difference (P > 0.05) observed at 95% Confidence Interval in activity compared to B. micrantha ethyl acetate extract.

Rate of Kill
Time course of the extract at different concentrations was examined. B. mic. EA completely inhibited the growth of H. pylori strain PE430C at 0.025 mg/mL, 0.05 mg/mL, 0.1 mg/mL and 0.2 mg/mL in 12 h and at 0.1 mg/mL and 0.2 mg/mL in 18 and 24 h of incubation, respectively. Growth and inhibition were later observed from 30 to 60 h before 100% killing at 0.1 mg/mL and 0.2 mg/mL after 66 and 72 h. Like strain PE430C, strain PE369C was totally inhibited at all the concentrations in 12 h. Inhibition was also observed at 0.1 mg/mL and 0.2 mg/mL in 54 and 66 h. One hundred percent killing was however observed at 0.1 mg/mL (2 × MIC) in 66 and 72 h (Figures 3a and 3b).
Apart from five strains with MIC90 > 0.5 mg/mL, others ranged from 0.0048 to >0.156 (Table 3). This is in line with the highest killing effect observed at a concentration of 0.2 mg/mL (4 × MIC) (Figure 3a and 3b), which corroborates the study of Ali et al. [22]; and more potent compared to the observation reported in other studies [23,24]. The organism was completely killed when the exposure time was increased to 66 and 72 h. From the results, the rate of kill exhibited by the extract against the test strains appeared to be both concentration and time dependent, which is in agreement with previous observations [22,24].

Phytochemical Compounds
Qualitative phytochemical analysis revealed the presence of flavonoids, steroids, tannins, alkaloids, and saponins. It was observed from the reactions (colours, heamolysis, turbidity, layers, emulsification and precipitation) that there may be much more of the steroids, tannins, flavonoids, and saponins present in the ethyl acetate extract of B. mic. EA compared to alkaloids (Table 4). This plant part (stem bark) was observed in this study to possess the common compounds present in many active plants, which have been reported by many investigators to be high in antimicrobial properties [25]. These biologically active phytochemical compounds contribute to the antimicrobial activities of many plants. For example, the importance of tannins for the treatment of inflamed or ulcerated tissues has been reported together with remarkable activity in cancer prevention and anticancer conditions symptomatic of H. pylori infection [24]. Flavonoids in the human diet reduce the risk of various cancers [26], while saponins are known to produce an inhibitory effect on inflammation [11]. Alkaloids have been reported to have analgesic, anti-spasmodic and bactericidal effects [25]. Ali et al. [22] had also documented compounds from Eugenia caryophillis and Cinnamomum verum, including eugenol and cinnamaldehyde, respectively, which at a concentration of 2 μg/mL completely inhibited all their H. pylori strains. It may therefore be the effects of these compounds that are responsible for the activities observed in the ethyl acetate extract of B. micrantha tested in the present study.

Bacterial Strains
Thirty-one strains of H. pylori, in addition to a control strain NCTC 11638, were subjected to antimicrobial assays in this study. Strains were isolated from patients presenting with gastric related morbidities at the Livingston Hospital, Port Elizabeth for endoscopy and confirmed following our previously reported scheme [4]. Informed consent was obtained from the patients and ethical approval (Protocol number EcDoH-Res 0002) from the Eastern Cape Department of Health, and the institutional review board of the University of Fort Hare (GMRDC).

Preparation of Plant Extracts
Bridelia micrantha (stem bark) was selected based on ethnobotanical information. The plant was collected and identified in Limpopo Province, South Africa in collaboration with a botanist at the University of Venda where voucher specimens (number BP03) have been deposited. The extraction method described by Ndip et al. [8] was employed. Briefly, the plant was harvested and processed. Technical grade ethyl acetate, acetone, ethanol, methanol (100%) and water were employed for extraction. The samples were dried in an oven (30-40 °C) and milled before extraction. Dried plant material (2 kg) was macerated in the solvent and the slurry was put in a shaker incubator (Edison, N.J. U.S.A.) regulated at room temperature (RT) for 48 h then centrifuged (Model TJ-6 Beckman, U.S.A.) at 3,000 rpm for 5 min. The combined extracts (exhaustive extraction) were concentrated in a rotavapor (Buchi R461, Switzerland) and a 6.2 g sample of each plant extract was used for the preliminary bioassay. Stock solutions were prepared in 20% ethyl acetate for B. mic. EA extract and 10% dimethyl sulphoxide (DMSO) for other extracts, which we established to be non inhibitory to H. pylori.

Screening of Crude Extracts for Anti-H. pylori Activity
The agar-well diffusion method was used in accordance with the method previously described with some modifications [27]. H. pylori inoculum was prepared from subcultures of bacteria in sterile distilled water and the turbidity adjusted to 1.5 × 10 8 CFU/mL (corresponding to 0.5 McFarland standards). It was evenly inoculated on Brain Heart Infusion (BHI) agar (Oxoid, U.K.) supplemented with 7% horse blood (Oxoid) and Skirrow's supplement (Oxoid) and allowed to dry for 3 to 5 min. Wells were punched in the plates using a sterile stainless 6 mm cork borer. The wells were filled with 30 µL of the extract (50 mg/mL). DMSO (10%) and ethyl acetate (20%) were used as negative controls and 0.05 µg/mL clarithromycin as a positive control. The tests were repeated in duplicate and incubated microaerophilically at 37 °C for 72 h (Anaerocult Basingstoke, U.K.). H. pylori control strain NCTC 11638 inoculated plate was included in all the experiments.

Determination of Minimum Inhibitory Concentration (MIC 50 and MIC 90 )
The MIC was carried out in accordance with the method of Banfi et al. [28] with modifications. Extracts that gave a zone of inhibition ≥14 mm were chosen for MIC determination by the microdilution test method in 96-well plates. Two-fold dilutions of the most potent extracts (ethyl acetate and acetone) and antibiotics, metronidazole and amoxicillin were prepared in the test wells in complete BHI broth supplemented with 7% horse serum and Skirrow's supplement; the final extracts and antibiotics concentrations ranged from 0.0048-10 mg/mL respectively and the tests were carried out in duplicate. Each strain applied into the wells was serially diluted to correspond to 0.5 McFarland standards and incubated under microaerophilic condition at 37 °C for 3 days. After incubation, resazurin solution was added per well and further incubated at 37 °C for 1 h. It was read with a microtiter plate reader adjusted to 620 nm (Model 680 Bio-Rad, Japan) and the MIC 50 and MIC 90 were determined.

Determination of the Rate of Kill
The time kill assay of bacterial isolates by B. mic. EA (which gave the best potent activity) was carried out as described by Ali et al. [22] with little modification. The test organism was standardized to 10 8 CFU/mL and an aliquot from each strain suspension (0.5 mL) was added to BHI broth (4.5 mL) supplemented with 7% horse serum and Skirrow's supplement and the solution of the extracts (½ × MIC, MIC, 2 × MIC and 4 × MIC) added. These were incubated at 37 °C in a microaerophilic cabinet shaking at ~120 rpm over a period of 72 h at 6 h interval (0, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72 h). The experiment was performed in duplicate. An exact volume (0.5 mL) of each suspension was withdrawn at time intervals and transferred to BHI broth recovery medium (4.5 mL) containing 3% "Tween 80". The suspension was then serially diluted and plated out for viable counts. The control plates contained the bacterial cells without the extract.

Phytochemical Analysis
Adopting the methods of Adegboye et al. [25], the qualitative phytochemical analysis of the ethyl acetate extract of B. micrantha was carried out to test for alkaloids, flavonoids, tannins, steroids and saponins.

Statistical Analysis
Analysis was performed using the SPSS Version 17.0 (Illinois USA, 2009). The one way ANOVA test was used to determine if there was any statistically significant difference in the diameter of zones of inhibition of the plant extracts and clarithromycin; the MIC of the most active extracts (ethyl acetate and acetone) and the control antibiotics (metronidazole and amoxicillin). P-values <0.05 were considered significant.

Conclusions
This study demonstrated the in vitro inhibitory and bactericidal activity of the crude extracts of B. micrantha bark. The plant may provide novel or lead compounds, which could become template for the synthesis of new anti-H. pylori drugs. Isolation and characterization of the bioactive compounds would be our major focus in future studies.


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Competition between Avena sterilis ssp. sterilis and wheat in South Western Spain

Avena sterilis spp. sterilis has different growth and demographic characteristics than other weedy taxa of Avena . Preliminary studies have shown that wheat yield losses caused by this species were larger than those produced by the spp. ludoviciana . These losses were estimated experimentally in 19 wheat fields distributed throughout western Andalusia. The relationship between A. sterilis spp. sterilis density per square meter and the wheat grain weight was estimated using several simple models. The results obtained confirm that this weed species was tremendously competitive even at low densities, obtaining wheat yield losses up to 70 kg ha –1 panicle –1 m –2 . The potential crop yield of the field was considered as an important factor to estimate the yield losses. A relationship between the expected weed-free yield of wheat and the yield loss per panicle of A. sterilis spp. sterilis was used to determine the economic threshold. This threshold ranged from 4 to 81 panicles m –2 depending on the potential yield of each field. Farmers with high potential yields (> 6,000 kg ha –1 ) should treat their fields if A. sterilis density is above 10 panicles m –2 . Farmers with low potential yields (< 3,000 kg ha –1 ) should not applied any herbicide control to their fields whatever the infestation density. And if the potential yields are between 3,000 and 6,000 kg ha –1 they should estimate the economic threshold value from the exponential equation described in this work.

Introduction
The genus Avena L. includes several species which are important crop and pasture weeds throughout the world, causing major crop losses. In Spain, these species include Avena fatua L., Avena sterilis ssp. ludoviciana (Durieu) Nyman and Avena sterilis ssp. sterilis L. (Syn. Avena macrocarpa Moench) in cereal crops and Avena barbata Pott ex. Link in pastures and roadsides (Carretero, 2004). Plants of the four taxa are very robust, reaching up to 150 cm tall, and they are highly competitive (Fernández-Quintanilla et al., 1997). Avena fatua is mainly found in the North, with cooler weather. Although A. sterilis ssp. ludoviciana is found all over Spain, it is most abundant in the Central and North. Apparently, it is better adapted to harsh winters (Fernández-Quintanilla et al., 1997), being very rare in the South (Romero-Zarco, 1987). A. sterilis ssp. sterilis, apparently more thermophilic, infest cereal fields in the south and west of Spain (Garcia-Baudín and Salto, 1978;Fernández-Quintanilla et al., 1997).
In western Andalucia, A. sterilis ssp. sterilis has been reported as the most important grass weed in cereals, with 58.8% of the cereal fields being infested by this species (Saavedra et al., 1989), although infestations were widespread, densities were very low in most sites. However in this area it is rare to find subspecies' ludoviciana. In contrast, in eastern Andalusia, where the two Avena sterilis subspecies coexisted, A. ludoviciana was more frequent and presented higher plant densities (Saavedra et al., 1989). However, both subspecies are clearly distinguishable by the sizes of their vegetative and reproductive traits. Although numerous studies have been conducted on the competition and population dynamics of A. fatua and A. sterilis ssp. ludoviciana (Scursoni and Satorre, 2005;González-Díaz et al., 2007;Dadnia et al., 2009), only a few studies have been published on A. sterilis ssp. sterilis (Madeira et al., 1984;González-Ponce, 1988;Saavedra et al., 1990;Palma et al., 1990Palma et al., , 1991González-Ponce and Santin, 2001). According with these studies, the competitive ability and the population growth rates of A. sterilis are higher than for A. ludoviciana. This fact is perceived by farmers in western Andalucia that generally apply more selective herbicides to control this weed than in the rest of Spain.
Competition between Avena spp. and cereal crops is a complex process that involves many different factors. Numerous authors have shown the large effect of Avena spp. density on crop yield (Torner et al., 1991;Murphy et al., 2002;Scursoni and Satorre, 2005). Crop density is another important factor: weed competition increases when crop densities are low, increasing yield losses, favoring the production of Avena seeds and increasing medium-long term infestations (Wilson et al., 1990;Torner et al., 1991). Weed emergence time also influences the competition process: Avena plants emerged earlier are more competitive than late emerging plants (Martin and Field, 1988). The crop species (and variety) is other factor: barley is generally considered more competitive than wheat (Torner et al., 1985;Cousens et al., 1991;Satorre and Snaydon, 1992) and among the wheat varieties, tall varieties are more competitive than semi-dwarf varieties (González-Ponce and Santin, 2001). In relation to soil fertility, Ruiz et al. (2008) found differences in biomass production of A. sterilis ssp. ludoviciana depending on soil texture, organic matter and nitrogen content. Climatic factors also have an important effect. Torner et al. (1991) estimated different crop losses in barley attributable to differences in the amount and distribution of rainfall. González-Ponce and Santin (2001) studying competition between wheat varieties and A. sterilis spp. sterilis found that nitrogen fertilization benefited the growth and seed production in both species, being this response greater in wet years than in dry years.
Previous competition studies conducted with A. sterilis spp. sterilis and wheat have yielded inconclusive results (Madeira et al., 1984;Saavedra et al., 1990). Apparently, various factors may be involved in the interference process between these two plants, resulting in variable effects. This study was conducted to obtain a reliable estimation of crop losses attributable to this species in the wheat fields of Western Andalusia through a simple model, trying to identify the possible factors that have most influence on this competition process.

Material and methods
The studies were conducted in three season 1986-87, 1987-88 and 1988-89 in nineteen dry-land winter wheat (Triticum aestivum) fields distributed throughout Western Andalusia (Southern Spain), using natural infestations of A. sterilis ssp. sterilis ("sterile oat") ( Fig. 1). The cropping system in all fields consisted of a wheat-sunflower rotation with not too intensive tillage (one or two disk plough passes followed by one or two harrow passes) which have not changed in recent decades. In each field, in an area between 2 and 5 ha, plots from 200 to 1,000 m 2 were chosen carefully, with homogeneous soil and slope, avoiding valleys and hills. These plots had A. sterilis ssp. sterilis patches exclusively. Throughout the crop cycle it was checked that there were not changes or inconsistencies neither in the plots nor in the crop due to agronomic factors or diseases. Winter wheat growing practices were standard for that area. Sowing took place between November 15 th and December 10 th , at doses of 150-180 kg ha -1 in rows 17 cm apart. Main cultivars used were Cajeme, Yecora and Anza, all with similar growth patterns. Soil was fertilized at about 50 to 70 kg N 2 ha -1 and 100 kg P 2 O 5 ha -1 at the sowing time and similar amount of N 2 in the tillering growth stage. Phenoxy-type or sulfonylureas postemergence herbicides were applied in middle February-early March for the control of broadleaf weeds. Harvesting time was early June. Rainfall during the growing season (15/Nov-10/June) varied among years and locations, ranging from 300 to 560 mm (Fig. 2). Soils varied between clay to clay loam texture, with pH between 7.5 and 8.2.

Wheat and weed samplings
The first year (1987), two fields (Cañete and Carlota) were sampled, with 6 plots on each field. In each plot, 20 samples of 1 m 2 were taken, with densities 864 J. Barroso et al. / Span J Agric Res (2011) 9(3), 862-872    Three crop variables (number of stems with ears, aerial dry weight and grain weight) and five weed variables (number of plants, number of panicles, aerial dry weight, number of spikelets per panicle and total number of spikelets) were analyzed. The second year (1988), seven fields (Écija, Osuna, Fuente Palmera, Bujalance, Castro, Alcolea and Sta. Cruz) were sampled. The first field had 5 plots and the rest of them had 2 or 3 plots on each field. In each plot, the samples were taken as in the previous year. In each sample, wheat yield, number of A. sterilis ssp. sterilis plants and panicles and number of spikelets per panicle were determined. In Écija we also determined the biomass and number of tillers of wheat and the biomass of the weed. The third year (1989), ten fields (Marchena, Santaella, Trebujena, Utrera, El Arahal, Bujalance, Espejo, Fernán Núñez, San Sebastián de los Ballesteros and Arjona) ( Fig. 1) were sampled with a single plot per f ield. In each plot, between 15 and 18 pairs of samples of 1 m 2 were taken. Each pair consisted in one sample took in an area with 0 density of A. sterilis spp. sterilis plants and the other sample took in a near area with low density of the weed species (between 1 and 37 panicles m -2 ). In each sample, wheat yield and numbers of A. sterilis spp. sterilis panicles, spikelets and number of spikelets per panicle were determined. The different methodology in the third year of study was based on the previous year results to try to get more information in weed competition in the range of low densities. All samples in the three seasons were obtained a few days before harvest.

Data analysis
The relationship between the different variables measured for A. sterilis spp. sterilis and wheat the first year (in all fields) and in the second year (only in the Ecija field) was studied through a correlation analysis (Pearson correlation). In the three years of study, regression techniques were used to relate A. sterilis spp. sterilis panicle density and wheat yield. Three empirical models where fitted to the data to explain and quantify the competitive relationship.
The equations used were: where Y is the yield obtained in the presence of A. sterilis spp. sterilis (kg ha -1 ), Y o is the weed-free yield (kg ha -1 ), X is A. sterilis spp. sterilis density (panicles m -2 ), b is the yield loss per weed density unit, c estimates the rate of reduction in crop yield as A. sterilis spp. sterilis infestation increases, i is the yield loss per weed density unit as weed density approaches zero, and a is the yield loss as weed density approaches infinity. The models were fitted to data, which were not transformed, using the statistic program R v. 2.11 (R Core Development Team, 2009, http://www.Rproject.org). The AICc (Akaike's Information Criterion corrected) was used as a measure of the fit of the models. The relationship between the parameters Y o and b of the linear model equation were studied through regression techniques with the same R program mentioned above, adjusting an exponential equation.
The economic threshold (ET), which represents the weed density at which the cost of treatment equals the economic benefit obtained from that treatment, was calculated according to the equation proposed by and Fernández-Quintanilla and García-Torres (1991): ET = C * 10,000 / (b * Y o * P * K) [4] where C is the cost of control (€ ha -1 ), b and Y o were defined previously, P is the price of the crop (€ kg -1 ) and K is the efficiency of control (%). To calculate the ET a constant wheat price (€ 0.232 kg -1 ) (MARM, 2009), an average herbicide treatment cost of € 45 ha -1 , and a percentage of efficiency (98%) was assumed. The C value estimated came from the average of the two most widely post emergence herbicides used for Avena spp. in the area [0.6% iodosulfuron-methylsodium and 3% mesosulfuron methyl (Atlantis ® WR, Bayer) and 24% clodinafop-proparguil (Topik ® , Syngenta)] plus the application cost (€ 6.61 ha -1 ) (Barroso et al., 2004). Label rates for these herbicides are: Atlantis, 300-500 g ha -1 and Topik, 200-250 g ha -1 , and their prizes in Spain per kg of product are € 135 and € 220 respectively. Finally, the relationship between the ET and the potential yield was established through regression techniques, adjusting an exponential equation.

Results
The first two years of the study A. sterilis spp. sterilis densities were moderate, with not many patches with densities above 50 panicles m -2 and few samples ex-ceeding 100 panicles m -2 . The average density per field ranged between 15 and 38 panicles m -2 and the average spikelet production per panicle ranged between 8 and 31 (Table 1). Cañete was the field with lowest values in all the A. sterilis spp. sterilis variables (81.3 g m -2 of biomass, 1.4 panicles per plant and 8 spikelets per panicle), indicating that this weed was less competitive in this site. The third year of study A. sterilis spp. sterilis densities were relatively low in all sites, the mean values per field ranged between 7.35 and 10.81 panicles m -2 . The mean number of spikelets per panicle ranged between 9.36 and 24.42 (Table 2). In 1987 and 1989, the fields with the highest density of panicles per square meter also had the lowest number of spikelets per panicle.
Average weed-free wheat yields ranged from 1,565 kg ha -1 (field I of year 3) to 7,700 kg ha -1 (Field Sta.Cruz, plot 2 of year 1988). The largest yields were obtained in the second year, with the highest rainfall (Fig. 2).
For A. sterilis spp. sterilis, very high positive correlations were obtained among numbers of plants, numbers of panicles, numbers of spikelets and aerial biomass (Table 3). However, the correlation between number of plants and number of panicles per plant was negative and significant for the three locations (Table 3). In other words, when weed density increased the intraspecific competition resulted in a lower panicle production per plant. However, there was no correlation between the density of infestation and panicle size (measured as the number of spikelets per panicle), indicating that intraspecific competition did not affect significantly this variable in this range of data (Table 3).
For wheat, the three measured variables (number of stems with spikes, aerial dry weight and weight of grain) were positively and strongly correlated (data not shown).
The Pearson correlation between crop and weed variables showed that wheat was strongly affected by the presence of A. sterilis spp. sterilis. All the wheat variables were negatively and significantly correlated with all the measured variables of A. sterilis spp. sterilis (Table 4). Wheat yield and panicle density of A. sterilis spp. sterilis were the two variables with the highest correlation values.
In 1987 and 1988, the relationship between wheat yield and A. sterilis spp. sterilis panicle density was well described by the three models tested. However, in 866 J. Barroso et al. / Span J Agric Res (2011) 9(3), 862-872 1989, none of the models fitted well to the data due to the narrow range of densities measured (0 to 37 panicles m -2 ) and to the enormous variability found in the data. Generally, the linear and exponential models gave very similar fitting. The hyperbolic model was difficult to converge in some plots due to the yield in weed free points sometimes was lower than in points with infestation because panicle density was from moderately to low in most of the plots. In addition, this model gave worse fitting or higher AICc than the other two (Table 5). We considered that the linear model explained reasonably well the process of competition and simplified the calculation of ET (Table 6). When we compared the linear models for each individual plot we found that the parameter b (yield loss per panicle) ranged between 7.5 and 52.1 kg ha -1 in 1987 and between 6.6 and 70 kg ha -1 in 1988 (Table 6). In 1989, with low infestation levels and taking paired samples, the losses per panicle ranged between 0.96 and 27.6 kg ha -1 . This wide variation among plots was related with the expected weed-free yield. In Sta. Cruz, in 1988, the yield losses per panicle were much higher than in all the other fields and this corresponded with much higher expected weed-free yield ( Table 6). The same trend was observed with the data from 1989 (data not shown). Using the data from the three years, the relationship between the potential weed-free yield and the yield loss per panicle could be fitted to an exponential equation (Fig. 3).
Competition between sterile oat and wheat 867   The ET values, as a function of Y o and b varied between 3.57 and 80.92 panicles m -2 ( Table 6). The relationship between this high range of ET values and Y o , showed that when the potential yields were lower than 3,000 kg ha -1 a herbicide control of this weed species is not profitable independently of its density. When the potential yields were higher than 6,000 ka ha -1 the ET were lower than 10 panicles m -2 . When the potential yields were between 6,000 and 3,000 kg ha -1 the ET values followed and exponential increase (Fig. 4).

Discussion
This work highlights the high competitiveness of A. sterilis spp. sterilis growing in wheat f ields in 868 J. Barroso et al. / Span J Agric Res (2011) 9(3), 862-872    Table 6. Southern Spain, supporting previous results from Saavedra et al. (1990). The only exception was found in one field (Cañete) and it was attributed to the late emergence of the weed. This effect has already being described by Willenborg et al. (2005) for Avena fatua. This lower competitiveness (lower yield losses) was accompanied with a lower number of spikelets per panicle and fewer seeds produced. Consequently, a good seedbed preparation and/or a delayed sowing will contribute to destroy early emerging plants (the most competitive ones) and may be particularly useful when infestation levels are under the ET level. Due to the relatively low population densities of A. sterilis spp. sterilis present in most fields, intraespecific competition was expected to be low. However, although weed density influenced the number of panicles per plant it did not influenced the number of spikelets per panicle. The low correlation coefficient found between the number of plants and the number of panicles per plant was probably due to the enormous variability of the data (among plots and among fields). The experimental design used in this study has the advantage of covering a wide geographic area and a wide range of environmental conditions, being representative of the agricultural reality of the region. However, it has the disadvantage of a high variability in the data (Fernández-Quintanilla and González-Andújar, 1988).
Although there are various regression models available to describe the response of crop yield to increasing weed densities, we decided to use the linear model due to its simplicity. Previously, Streigbig et al. (1989) and Zanin et al. (1993) had used the hyperbola rectangular model whereas Torner et al. (1991) used the exponential model. Other models more complex, involving several environmental factors (such as rainfall) or agronomic factors (such as the use of herbicides and fertilizers, the variety or the seeding rate) (Wagner et al., 2007), were not taken into account because of the lack of information on all these factors. Furthermore, given the complexity of factors that can influence in the competition process, our purpose was to use simple models that can be easily understood and applied by farmers. We found the linear model to describe the data similar to the exponential model and slightly better than the rectangular hyperbola model. However, this similarity of fitting observed could vary if the range of infestation densities had been higher in favor of non-linear models.
Several authors working on Avena spp. in cereal crops have estimated ETs in terms of simple variables such as plant density, crop value or herbicide efficiency (Madeira et al., 1984;Zanin et al., 1993;Weaver and Ivany, 1998;Mennan et al., 2003;or Scursoni and Satorre, 2005) but few have highlighted the influence of the expected weed-free yield in the establishment of the ET (O'Donovan et al., 2005) and none had quantified it. Instead of establishing a comparison with the values given by these authors (determined by the wild oat species and highly fluctuating economic factors over the years and countries), we would like to emphasize Competition between sterile oat and wheat 869 spp. sterilis plant has 2 to 3 panicles per plant at those densities). Famers with low potential yields (< 3,000 kg ha -1 ) should not applied any herbicide control to their f ields whatever the infestation density. If the potential yields are between 3,000 and 6,000 kg ha -1 they should estimate the ET value from the exponential equation described in this work. So far, it is not possible to determine the long-term ET in a wheat-sunflower rotation. Although demographic variables of Avena sterilis spp. sterilis are known for an annual cycle of 870 J. Barroso et al. / Span J Agric Res (2011) 9(3), 862-872 wheat (Palma et al., 1990), the population annual growth rates in wheat-sunflower rotation have not yet been investigated. Future research in this direction would improve the knowledge of the population dynamics of Avena sterilis spp. sterilis in this cropping system and would help to find effective measures for management of this species in order to reduce the high competitive ability showed in this study.


s2fieldsofstudy:
Agricultural And Food Sciences
Biology

text:
Antibodies Induced by Lipoarabinomannan in Bovines: Characterization and Effects on the Interaction between Mycobacterium Avium Subsp. Paratuberculosis and Macrophages In Vitro

Lipoarabinomannan (LAM) is a major glycolipidic antigen on the mycobacterial envelope. The aim of this study was to characterize the humoral immune response induced by immunization with a LAM extract in bovines and to evaluate the role of the generated antibodies in the in vitro infection of macrophages with Mycobacterium avium subsp. paratuberculosis (MAP). Sera from fourteen calves immunized with LAM extract or PBS emulsified in Freund's Incomplete Adjuvant and from five paratuberculosis-infected bovines were studied. LAM-immunized calves developed specific antibodies with IgG1 as the predominant isotype. Serum immunoglobulins were isolated and their effect was examined in MAP ingestion and viability assays using a bovine macrophage cell line. Our results show that the antibodies generated by LAM immunization significantly increase MAP ingestion and reduce its intracellular viability, suggesting an active role in this model.

Introduction
Paratuberculosis is a chronic granulomatous enteric disease affecting ruminants. The causative agent, Mycobacterium avium subsp. paratuberculosis (MAP), enters orally, crosses the intestinal barrier, and is phagocytized by macrophages within the lamina propria. These cells serve as the intracellular site in which MAP survives and multiplies [1,2]. Several studies have been carried out to evaluate the MAPmacrophage interaction, due to its importance in paratuberculosis pathogenesis [3]. It has been proved that various receptors are involved in endocytosis of mycobacteria [4,5] and that different routes of entry can alter the intracellular fate of pathogens. For example, ligation to receptors for the Fc portion of the immunoglobulins (FcR) is generally accompanied by activation of the respiratory burst [6], and maturation of phagolysosomes [7], whereas uptake mediated by complement receptors occurs in the absence of proinflammatory signals [8].
Generally, the humoral immune response against mycobacterial infections has been considered nonprotective. However, evidence for an active role of B cells and antibodies in some intracellular infections has been accumulated during the last years [9][10][11][12][13][14][15]. As regards paratuberculosis, it is accepted that the humoral immune response appears late in the infection and probably associated with the progression of disease from a subclinical to a clinical stage [16]. However, few works have suggested that antibodies could enhance some immune mechanism against MAP. A recent report has evaluated the effect of immune serum on the MAP macrophage interaction suggesting an active role of antibodies [17]. In addition, our group has previously reported 2 Veterinary Medicine International that purified specific antibodies against MAP could enhance the MAP-macrophage interaction in vitro and improve the activation of the nuclear factor NF-κB in infected cells [18].
Lipoarabinomannan (LAM) is the main glycolipidic antigen on the mycobacteria envelope and has a molecular weight of approximately 40 kDa. Its structure presents similarities among pathogenic mycobacteria and differences in relation to LAM of nonpathogenic members of the genus [19,20]. The role of LAM in mycobacterial pathogenesis has been studied by different research groups [21,22]. Antibodies against LAM have been shown to be beneficial in passive protection experiments in murine tuberculosis models [10]. As regards paratuberculosis in bovines, the serological response against this compound has been extensively studied in order to improve diagnosis. However, little is known about the role of LAM-specific antibodies in this infection and, to our knowledge, there are no published reports on this topic.
The aim of this work was to characterize the humoral immune response induced by immunization with a LAM extract in bovines and to evaluate the role of the generated antibodies in the in vitro infection of macrophages with MAP.

Materials and Methods
2.1. LAM Extract. Mycobacterium avium subsp. avium (MAA) was grown to log phase in Dorset-Herley medium, heat-inactivated and kindly provided by Dr. A. Bernardelli (Servicio Nacional de Sanidad Animal, Argentina). The bacterial pellet was centrifuged and resuspended in PBS (NaH 2 PO 4 3 mM, Na 2 HPO 4 7.5 mM, NaCl 145 mM, pH 7.2-7.4) for further sonication. LAM was extracted from 5.2 g of total bacteria according to the method previously described elsewhere [23] and adapted to our laboratory conditions [24]. Carbohydrate concentration was determined by the phenol-sulphuric acid method [25] using glucose as standard. Protein concentration was determined by the Bradford method [26] using bovine serum albumin as standard. From these data, the percentage of protein removal achieved was estimated as total protein amount in the LAM extract × 100/initial total protein amount. The LAM extract was characterized by SDS-PAGE, stained with Bio-Rad Silver Stain (Bio-Rad Laboratories Inc., Hercules, CA, USA) modified for carbohydrate detection [27]. Electrophoresis was performed in a Mini-Protean II electrophoresis cell (Bio-Rad) on 12% polyacrylamide gels, following the manufacturer's instructions. Samples containing 5 μg carbohydrate/lane were twofold diluted in sample buffer and heattreated (85 • C, 5 min) before running (2 h at 96 mV). An ELISA was carried out using anti-LAM of Mycobacterium tuberculosis monoclonal antibody (mab CS-35) and purified M. tuberculosis LAM as pattern (both reagents were kindly provided by Dr. J. Belisle, Colorado State University, Fort Collins, CO, USA). Flat-bottomed 96-well polystyrene plates (Greiner Microlon, Greiner Bio-One North America Inc., Monroe, NC, USA) were coated with LAM extract or LAM pattern at 25 μg carbohydrate/well. An HRP-conjugated antimouse IgG (KPL, Kirkegaard & Perry Laboratories Inc., Gaithsburg, MD, USA) was used at a dilution of 1 : 500. Plates were developed using ortho-phenylendiamine dihydrochloride (OPD, Sigma-Aldrich Corp., St. Louis, MO, USA) in citrate-phosphate buffer (Sigma-Aldrich). The reaction was stopped after 10 min by the addition of 50 μL/well of 1 M sulphuric acid, and plates were read in an OpsysMR spectrophotometer (Dynex Technologies, Chantilly, VA, USA). Results are expressed in ELISA Units (EUs), estimated as the mean optical density value obtained at 490 nm (OD) for each sample × 100/OD for the negative control. In this case, an irrelevant mab was tested as negative control.

Animals and Samples.
A total of fourteen five-month-old Holstein calves from tuberculosis-and paratuberculosis-free herds from the Pampas region of Argentina were kept under field conditions during all the experimental period. Calves were randomly assigned into the LAM group (n = 9), which subcutaneously received 2 mg of LAM extract dissolved in 1 mL of PBS and emulsified in 1 mL of Freund's Incomplete Adjuvant (FIA, Sigma-Aldrich), or the normal control group (NC group, n = 5), which were mock-immunized with 1 mL of PBS emulsified in 1 mL of FIA. The first immunization was received on day 0 and the booster 35 days later. Blood samples were taken on days 0 and 65. This experiment was performed under the approval and supervision of the Institutional Committee for the care and use of experimental animals of Facultad de Ciencias Veterinarias of Universidad de Buenos Aires, Argentina.
Serum samples from five naturally infected bovines with clinical signs of paratuberculosis were included in the current study as the infected control group (IC group). The diagnosis was confirmed by fecal culture and amplification of the IS900 fragment from isolated colonies by PCR [28].  Rad) was carried out in a Trans-Blot SD cell (Bio-Rad) following the manufacturer's instructions. Membranes were incubated in blocking buffer and then with bovine sera diluted 1 : 250. Subsequently, HRP-conjugated goat antibovine IgG (KPL) was added in a 1 : 1000 dilution. The reaction was developed using 0.5 mg/mL DAB (HRP Color Development Reagent 3,3 -diaminobenzidine, Bio-Rad) and 1 μL/mL H 2 O 2 100 vol. in TBS buffer (20 mM Tris, 500 mM NaCl, pH 7.5) for 10 min. All incubations were performed at 37 • C for 1 h, and each step was followed by three washes in rinsing buffer. In order to confirm specificity against nonprotein molecules, a proteolytic treatment of LAM extract with 0.2 mg/mL proteinase K (Amresco Inc., Solon, OH, USA) was performed before running the SDS-PAGE, as described by Reichel et al. [29]. All sera were analyzed by immunoblot against digested and undigested LAM extract. As a digestion control, 1 mg/mL ovalbumin (OVA, Sigma-Aldrich) solution was incubated with proteinase K in the same conditions as the LAM extract. Digested and undigested OVA were evaluated with a hyperimmune bovine serum against OVA.

Precipitation and Purification of Antibodies.
Serum antibodies of two animals from LAM-immunized, infected, and normal control groups were isolated and purified for further use in functional assays. Sera were heat-inactivated at 56 • C for 30 min. Ammonium sulfate precipitation (44% saturation) was performed to obtain precipitated immunoglobulins (Igs). Then, purification by protein G affinity chromatography (protein G-agarose, Exalpha Biologicals, Shirley, MA, USA) was carried out, and purified Igs were obtained. In order to check the purity of the Igs fractions, SDS-PAGE and electrophoresis were conducted. The concentration of Igs was estimated by the Bradford method, considering the percentage of gamma-globulins detected by densitometric analysis of electrophoresis. The Igs fractions were filtered by 0.22 μm and stored at -70 • C until use.

Ingestion Assay.
Bomac cells (1 × 10 6 viable cells/mL) were seeded onto 20 mm × 20 mm sterile coverslips, allowed to adhere for 2 h and incubated overnight in RPMI medium. Bacteria were opsonized with precipitated Igs (100 μg/mL) from LAM-immunized, infected or normal control bovines, at 37 • C for 1 h in a shaker. Immediately prior to inoculation of monolayers, the bacterial suspension was disaggregated as described above. MAP-macrophages interaction was allowed for 45 min. Cells were washed with cold PBS, fixed with 0.37% formaldehyde, and stained with Ziehl-Nielsen. A minimum of 100 cells/coverslip were counted in immersion fields (1000x) by light microscopy. The percentage of phagocytic cells (%PhC) and the mean number of internalized MAP detected in each cell (iMAP) were recorded. The phagocytic index (PI) was calculated as % PhC × iMAP [32].

Intracellular Viability Assay.
To evaluate the viability of ingested MAP, Bomac cells (1 × 10 6 viable cells/mL) were seeded onto 24-well tissue culture plates and incubated at 37 • C overnight in 5% CO 2 . Bacteria were opsonized and disaggregated as described above. Then, inoculated into Bomac cultures, in duplicate. After 2 h, monolayers were washed with PBS and one of each duplicate well was lysed with 0.2% sodium dodecylsulfate for initial CFU counting. The other duplicate well was incubated with RPMI medium containing 0.1 mg/mL gentamicin for 2 h in order to avoid contamination [33], then replacing it with antibiotic-free medium. Infected macrophages were cultured for 72 h and then lysed for final CFU counting. Lysates were serially plated on 7H9 agar and cultured at 37 • C for 5 weeks until the CFU were counted. Results are expressed as percent change in viability (final CFU/initial CFU × 100). Additionally, an FcRblocking assay was conducted as described by Manca et al. [34]. Bomac cells were preincubated for 1 h with 100 μg/mL of protein G-purified Igs from a normal control bovine that had been heat-aggregated. 2.6. Statistical Analysis. Data were analyzed for statistical significance using STATISTIX 8.0 software. ANOVA followed by Tukey's test was used except for isotype analysis. In that case, Kruskal-Wallis test followed by pairwise comparisons was run. The level of significance was set at P value <.05.

Characterization of LAM Extract.
We obtained an extract containing 105 mg of total carbohydrate. Protein presence was largely reduced (46.8 mg of protein initially versus 0.4 mg of residual protein in the LAM extract), thus showing a percentage of protein removal of 99.2%. SDS-PAGE results demonstrated that the LAM extract was mostly composed of a carbohydrate mixture, as revealed by the presence of many bands when modified silver stain was performed. Indeed, the predominant component migrated similar to M. tuberculosis LAM (Figure 2(a), lane 1 and 2). As expected, sera from the MAP-infected control group recognized our LAM extract of MAA (Figure 2(a), lane 3). In the mab CS35-ELISA, the result obtained for our LAM extract was 493.5±35.1 EU, whereas for purified tuberculosis LAM it was 657.8 ± 10.6 EU. These results show cross reactions between LAM of pathogenic mycobacteria.

Humoral Immune Response against LAM Extract.
The reactivity of sera against LAM extract was assessed and the level of specific antibodies and the isotypic profile were determined. All the calves in the LAM group generated a specific humoral immune response, with antibody levels at 1 : 100 serum dilution between 180 and 790 EU. Comparable levels were detected in the infected control group (range between 180 and 723 EU). As regards the isotype analysis, the presence of specific IgM, IgG1, IgG2, and IgG3 was evaluated in sera from LAM-immunized, infected, and normal control bovines. We could not detect specific IgM (LAM group 106.8 ± 5.5 EU, IC group 97.9 ± 15.8 EU, NC group 101.6 ± 8.1 EU). The results obtained for specific IgG1, IgG2, and IgG3 are shown in Figure 1. The humoral immune response of LAM-immunized bovines was mostly predominated by IgG1 with minor presence of IgG2 and IgG3, although statistically significant differences were found for the three of them when comparing with the normal control group (P < .05). In the infected control group, a similar trend was detected, with IgG1 as the only isotype with levels significantly higher than in the normal control group.
Sera were also tested against the LAM extract by immunoblot assay (Figure 2(b)). For all LAM-immunized calves, only one band of molecular weight between 25 and 50 kDa was detected. This band remained at equal strength after proteolytic digestion of the extract, demonstrating the nonprotein nature of the antigen involved (Figure 2(b), D versus UD lanes).

Functional Effects of Antibodies.
The precipitated and purified Igs obtained (Figure 3(b), boxes) were used in functional assays.
Effect on MAP Ingestion. As shown in Figure 3(a), opsonization of MAP with specific antibodies increased the ingestion of bacteria. The antibodies from both LAM-immunized and infected control bovines showed a phagocytic index significantly higher (P < .01) than the one obtained when opsonizing MAP with normal control antibodies (phagocytic index of 59.4, 61.5, and 33.2, resp.).
Effect on MAP Intracellular Viability. When assays were conducted by opsonizing MAP with precipitated antibodies, LAM group Igs significantly reduced (P < .05) the percentage of MAP viability, as compared with that obtained for normal and infected control Igs opsonization (Figure 3(b)(1)).
When MAP was opsonized with purified Igs, comparable and more repeatable results were obtained for LAMimmunized and normal control groups (P < .01). For the infected control group, the result was similar to that of the treatment with LAM group Igs. Preincubation of macrophages with aggregated Igs (FcR blockade) resulted in a significant increase of MAP viability as compared with the effect of antibodies from LAM group without previous incubation with aggregated Igs (P < .05) (Figure 3(b)(2)). This result suggests that the effect observed for the opsonization with LAM group Igs could be FcR-mediated.

Discussion
In the present work, we examined the immune response induced in cattle by immunization with a LAM extract and the effect of the generated antibodies in the MAPmacrophage interaction.
We used MAA for LAM extraction instead of MAP due to the antigenic homology between both [29] and the 6 Veterinary Medicine International faster growth in culture of the former. Besides, our ELISA and immunoblot results support cross reactions among mycobacterial glycolipids previously described [19,20,29] and show that the method of LAM extraction applied preserved its antigenicity. To our knowledge, this is the first report of bovine immunization using a mycobacterial LAM extract. With the immunization protocol used, we were able to induce high levels of specific antibodies with increases of all IgG isotypes and the predominance of IgG1 in most of the studied calves. IgG1 was also detected as the predominant isotype in the infected control group. Similar responsiveness against LAM was previously described in bovines with clinical paratuberculosis [35]. Taking into account that this isotype represents the most relevant immunoglobulin in mammary gland secretions of bovines [36], the presence of local specific IgG1 might be relevant for the passive protection of newborn calves.
The influence of opsonization with antibodies on MAP phagocytosis and intracellular viability has been previously evaluated [17,18,33,37,38]. However, published reports have been generally based on assays where total serum was examined as a source of antibodies. While many other serum components are known to possess opsonic activity and could affect intracellular viability of bacteria, we purified Igs for functional evaluation. The purification methodology applied was successful. However, a heavy band was observed in SDS-PAGE, probably corresponding to the presence of incompletely reduced Igs molecules, as suggested by the reactivity with an antibovine IgG antibody (data not shown). We found that the phagocytic level of Bomac cells increased almost twofold when MAP was opsonized with antibodies from either LAM-immunized or infected bovines. MAP-phagocytosis enhancement by hyperimmune sera has been previously reported in Bomac cells [18] and bovine blood monocyte-derived macrophages (BMDMs) [17,37]. The phagocytic levels detected herein were comparable to those published by Woo et al. for Bomac cells [38], although our indexes were lower than those described for BMDM when ingesting MAP [17,37]. Therein, the kinetics of MAP uptake was evaluated and a significant increase in the ingestion level was detected after 60 to 120 min of incubation [17,37]. Taking these observations into account, it is possible that the lower phagocytic index detected herein could be related to the shorter MAP-macrophages incubation time or to the use of Bomac cell line instead of BMDM.
Controversial results in viability assays opsonizing MAP with whole sera from healthy and infected bovines were published [17,37,38]. In our model, opsonization with precipitated Igs from normal and infected bovines shows a beneficial effect on MAP viability. However, we found different results when precipitated and purified Igs from infected bovines were used for opsonization. The presence of other opsonins in precipitated Ig fractions, such as collectins, C-Reactive protein, or fibronectin, could explain the difference found [39][40][41]. Noteworthy, our results show that antibodies induced by LAM immunization could significantly reduce the intracellular viability of MAP.
These results raise questions about the biological relevance of LAM antibodies in paratuberculosis. We here found that purified antibodies present in sera from bovines immunized with LAM of MAA could reduce MAP intracellular viability as well as antibodies from MAP clinically infected cattle. However, in the natural infection, the antibodies appear late and probably associated with the progression of disease. It remains to be established whether specific antibodies present at the moment of the infection could modify the course of paratuberculosis.
Our findings provide new data about basic aspects of the role of antibodies in MAP-macrophage infection in vitro. More studies, using other MAP strains, especially field isolates, and macrophages derived directly from bovines, must be conducted in order to better approach to the role of antibodies in the natural MAP-macrophage interaction that takes place in the host.