**Clinical, Serological, Hormonal, Bacteriological and Molecular Detection of Brucellosis in Aborted Cows and Buffalos**

Jabbar A.A. AL-Sa'aidi, Mohsen A. Al-Rodh, Ali Anok Najum

Dept. Physiol. & Pharmacol., College of Vet. Med., Al-Qadisiya Univ., Iraq Dept. Vet. Med., College of Vet. Med., Al-Qadisiya Univ., Iraq Dept. Microbiol., College of Science, Al-Muthana Univ., Iraq

#### **Abstract**

Abortion is the most obvious manifestation of *Brucella* infection. In this study, 59 aborted buffalos and 91 aborted cows were included. Diagnosis of *Brucella* infection in these abortions was based on clinical, serological, bacteriological, hormonal, and molecular assays. Serological studies included the use of RB and ELISA tests as screening tests for infection. Argumentative differences between RB and ELISA results have been shown. *Brucella* isolated and identified from aborted fetuses, vaginal discharge and milk samples were 7 isolates from aborted cows and 3 from aborted buffalos. *Brucella* isolates revealed amplification of a 223-bp fragment with B4 and B5 primers. Hormonal assessment in both, brucella infected cows and buffalos, registered significant decrease in progesterone and P/E ratio in comparison with that aborted due to other causes. Immunohistochemical study revealed down expression level of 3BHSD enzyme in placentas of *Brucella* positive animals. ELISA technique was the valuable serological test to confirm the diagnosis of brucellosis. In conclusion, both RB and ELISA are necessary to be performed together as screening tests in diagnosis of brucellosis, whereas serum hormonal, placental immunohistochemical, and molecular (PCR) assessments have an efficient diagnostic values which can be included for confirmation of brucellosis.

**Keywords**: *B. abortus*, *B. melitensis*, Brucellosis, ELISA, RB, PCR.

## **1. Introduction**

Brucellosis, is a major infectious disease afflicting humans and a wide range of domesticated animals and wildlife. It is known to be a worldwide problem and one of the most important among zoonoses in the Mediterranean region, India, and Central and South America (1). Brucellosis results significant human morbidity (2). Reports from the areas where *Brucella melitensis* infection is endemic, suggest that there is an increased rate of abortion in asymptomatic pregnant women (3). Outbreaks of bovine brucellosis are associated with abortion during the last trimester of gestation, and produces weak newborn calves, and infertility in cows and bulls (4). The diagnostic method known to produce the best results in terms of specificity is the isolation of *Brucella* organisms from the suspected animal. However, this method has a limited sensitivity, is expensive and cumbersome and has the added difficulty of being unpractical to apply at a large scale in control

© 2012 AL-Sa'aidi et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

campaigns. Accordingly, the indirect diagnosis of disease based on serological tests is of choice in the eradication programmes. The standard Rose Bengal (RB) is the main serological test used to detect antibodies against *B. abortus* and *B. melitensis* infections. This test has been used for several decades, proving to be successful for eradicating bovine brucellosis in some countries. Nevertheless, there is evidence that RB test is significantly less effective for the diagnosis of brucellosis in sheep and goats than in cattle (5). During recent years, different ELISAs have been developed using more or less purified S-LPS as the antigen and have been reported to be at least as sensitive and specific for the diagnosis of brucellosis in ruminants (6, 7, 8). The aims of the present work were: (i) to compare the diagnostic performance of serological: standard RB and ELISA, hormonal; in the blood and placenta, and molecular; PCR studies in cattle with known clinical and bacteriological status, (ii) to determine the diagnostic performance of these tests in aborted cattle positive in the classical RB test, but in which *B. abortus* and *B. melitensis* could not be isolated.

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**Identification and isolation of** *Brucella***:** *Brucella* growth was confirmed by bacteriological and

B-The PCR amplification process: Enzymatic amplification of DNA was carried out in a final volume of 25 µl according to the recommendations of manufacture. The following reaction mixes

GoTaq® Green Master Mix, 2X 12.5μl 1X

upstream primer, 10μM 0.25–2.5μl 0.1–1.0μM downstream primer, 10μM 0.25–2.5μl 0.1–1.0μM DNA template 1–5μl <250ng

Nuclease-Free Water to 25μl N.A.

PCR consisted of a preheating at 95ºC for 5 min. After this initial denaturation step, the mixture

**Loop's steps Temperature Time Number of cycle**

denaturation 94 ºC 1 min 40

Final extension 72 ºC 7 min 1

Hold 4 ºC Indefinite 1

C-Detection of PCR products by agarose electrophoresis: A horizontal slab gel electrophoresis apparatus was used. Ten µl of amplified products were mixed with 3 µl of loading buffer, analysed by electrophoresis in 2% agarose gel, and stained with 0.5 µg /ml ethidium bromide at 100 V for 1hr. in 1 X TBE buffer. Then visualized under UV light using ultraviolet transelumenater. DNA ladder (100-1000) was used and the gel was photographed when necessary by digital camera. A sample was considered positive for *Brucella* spp. when a specific fragment of 223 bp was

**Statistical Analysis:** All data were analyzed using the statistical package for social science (SPSS) for Windows program on the computer. Chi-square was used to compare between the frequencies. Student *t* test was used to compare between means of groups. The significance was accepted

annealing 55 ºC 1 min extension 72 ºC 1 min

**Component Volume Final Conc.**

A-DNA purification: was performed according to the manufacturer instructions.

biochemical tests as suggested by Alton *et al*., (12). **Molecular Identification by PCR-based assay:**

were prepared on ice. For a 25µl reaction volume:

was subjected to 40 amplification cycles as follow:

detected in the gel (10).

as P value < 0.05 and <0.01.

**3. Results**

Clinical observation:

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### **2. Materials and Methods**

**Materials**: Bovine brucellosis kit was provided by EUROPEAN VETRINARY LABORATORY; EVL, Netherland. Gram's stain solution and modified Ziehl-Neelsen stain solution was prepared and used as described by Alton *et al .,*(9). Rose Bengal antigen was provided by Omega company, UK. Monospecific antiserum; antibrucella abortus and antibrucella melitensis were supplied by Difico, USA. It was provided as a gift from FAO. Antibiotic supplements were provided by Himedia, India. Fetal calf serum was provided by Difico company, USA. Materials used in PCR; Wizard Genomic DNA purification kit GoTaq® Green Master Mix were provided by Promega company, USA. Primers used for diagnosis; the system used was B4/B5 (Baily) primers system; advised by Baily *et al*.,(10), and provided by Alpha DNA Company, as described below:


#### **Methods**:

**Samples collection.**

**1. Clinical observations:** was performed on aborted cows and fetuses throughout the last few days of gestation period. At parturition, retained placenta, body temperature and bleeding status has been registered.

**2. Blood samples**: 150 blood samples (91 from cows and 59 from buffalos) were obtained. serum samples have been obtained for serological assessments using RB and ELISA tests.

**3. Aborted fetuses**: Specimens from 15 aborted fetuses, at the first and last stage of pregnancy (9 from aborted cows and 6 from aborted buffalos) were cultured in duplicated *Brucella* agar plates as described by Alton *et al* (9).

**Serological assessments**: were preformed according to OIE, (11).

**Identification and isolation of** *Brucella***:** *Brucella* growth was confirmed by bacteriological and biochemical tests as suggested by Alton *et al*., (12).

#### **Molecular Identification by PCR-based assay:**

A-DNA purification: was performed according to the manufacturer instructions.

B-The PCR amplification process: Enzymatic amplification of DNA was carried out in a final volume of 25 µl according to the recommendations of manufacture. The following reaction mixes were prepared on ice. For a 25µl reaction volume:


PCR consisted of a preheating at 95ºC for 5 min. After this initial denaturation step, the mixture was subjected to 40 amplification cycles as follow:


C-Detection of PCR products by agarose electrophoresis: A horizontal slab gel electrophoresis apparatus was used. Ten µl of amplified products were mixed with 3 µl of loading buffer, analysed by electrophoresis in 2% agarose gel, and stained with 0.5 µg /ml ethidium bromide at 100 V for 1hr. in 1 X TBE buffer. Then visualized under UV light using ultraviolet transelumenater. DNA ladder (100-1000) was used and the gel was photographed when necessary by digital camera. A sample was considered positive for *Brucella* spp. when a specific fragment of 223 bp was detected in the gel (10).

**Statistical Analysis:** All data were analyzed using the statistical package for social science (SPSS) for Windows program on the computer. Chi-square was used to compare between the frequencies. Student *t* test was used to compare between means of groups. The significance was accepted as P value < 0.05 and <0.01.

### **3. Results**

Clinical observation:

All positive cases for *Brucella* infection were found in late stage of pregnancy (5-9 month), some cases accompanied with abortion with retention of placenta, aborted buffalos were in herds not sporadically like cows (table 1). Pneumonia and pericarditis were the main complications shown in aborted fetuses positive for brucellosis. Placentas were, edematous and opaque with bleeding in some of cases**.**

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**period**

**NO R.B ELISA Bacterial isolation**

**+ - % + - % + - %**

**Table 1.** History, clinical signs, pregnancy period, Samples and isolation, RB and ELISA results of aborted

Cow 91 26 65 28.5 21 70 23 7 79 13.18 Buffalo 59 7 52 11.86 17 42 28.8 3 55 6.77

*Brucella* organisms first recognized in smears obtained from fetal stomach stained with modified Ziehl Nielsen stain, which appeared red clumps against blue background. *Brucella* culture recognized on the basis of colonial morphology which appeared round translucent pale honey. Routine bacteriological examination has been carried out for identify the genus *Brucella* before they submitted for *Brucella* typing tests any isolate that was differ in even one test was excluded from further consideration as member of the genus *Brucella*. So the obtained isolates were Gramnegative, coccobacilli, arranged singly, in short chain pairs, with small groups, negative for haemolysis on blood agar, and it neither grow nor perform lactose fermentation on MacConkey agar, positive for nitrate reduction oxidase, catalase and urease, negative for MR-VP, gelatinase, Citrate utilization and indol production. Out of 91 aborted cows, 7 (13.18%) were positive by culture, whereas out of 59 aborted buffalos, 3 (6.77%) were positive for culture (table 1 and 2). Out of 15 aborted fetuses from cows and buffalos 4 from aborted cow's fetuses and 2 from buffalo's aborted fetus were positive by culture. Out of 12 uterine fluid swabs 2 from aborted cows and 1 from aborted buffalo were positive by culture. Out of 122 milk samples positive 1 from aborted cows, was positive by culture. Uterine swabs and milk samples where firstly cultured on the selective media, so *Brucella* colonies were recognized first by colonial morphology then subcultured to obtain pure culture before submitted to the bacteriological and biochemical test. According to the results of Pearson Chi- Square tests, isolates from cows specimens were significantly higher

**Table 2.** Results of RB test, ELISA and bacterial isolation for aborted cows and buffalos.

**No. History & clinical signs Pregnancy** 

15 Aborted before 40 d with retained

16 Aborted before 60 d. (Abortion also occurred in all herds before 5 months at late pregnancy)

cattle and their fetuses.

placenta

**\*Only one** case had ELISA titer 10.09 (suspected grey zone)

(P<0.05) than that isolated from buffalos (figure 2).

**Bacterial isolation & identification:**

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**Samples & isolation of agent**

6 Months Milk (-) ++ 1.026

9 Months Milk (-) \_ 1.016

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**R.B ELISA**

#### **Serological assessments:**

ELISA, RB tests and clinical signs results are shown in tables (1 and 2) that RB test results were positive in 33 cases (22%) including 26 cases (28.5%) in cows and 7 cases (11.86%) in buffalo. ELISA results were positive in 38 cases (25.3%), including 21 cases (23%) in cows and 17 cases (28.8%) in buffalo. ELISA was positive in aborted cattle which had previous history of abortion, whereas RB test was positive in 2 cows that was negative for ELISA & bacterial isolation, also RB test was negative in 10 case of buffalo that aborted before several months, which was positive with ELISA test. According to the results of Pearson Chi-Square test, the difference in RB positive cases between groups was significant (P<0.05), whereas difference in RB negative cases between cows and buffalos was not significance (P>0.05). According to ELISA technique, statistical analysis showed insignificant difference (P>0. 05) between groups.



**Table 1.** History, clinical signs, pregnancy period, Samples and isolation, RB and ELISA results of aborted cattle and their fetuses.


**\*Only one** case had ELISA titer 10.09 (suspected grey zone)

**Table 2.** Results of RB test, ELISA and bacterial isolation for aborted cows and buffalos.

#### **Bacterial isolation & identification:**

*Brucella* organisms first recognized in smears obtained from fetal stomach stained with modified Ziehl Nielsen stain, which appeared red clumps against blue background. *Brucella* culture recognized on the basis of colonial morphology which appeared round translucent pale honey. Routine bacteriological examination has been carried out for identify the genus *Brucella* before they submitted for *Brucella* typing tests any isolate that was differ in even one test was excluded from further consideration as member of the genus *Brucella*. So the obtained isolates were Gramnegative, coccobacilli, arranged singly, in short chain pairs, with small groups, negative for haemolysis on blood agar, and it neither grow nor perform lactose fermentation on MacConkey agar, positive for nitrate reduction oxidase, catalase and urease, negative for MR-VP, gelatinase, Citrate utilization and indol production. Out of 91 aborted cows, 7 (13.18%) were positive by culture, whereas out of 59 aborted buffalos, 3 (6.77%) were positive for culture (table 1 and 2). Out of 15 aborted fetuses from cows and buffalos 4 from aborted cow's fetuses and 2 from buffalo's aborted fetus were positive by culture. Out of 12 uterine fluid swabs 2 from aborted cows and 1 from aborted buffalo were positive by culture. Out of 122 milk samples positive 1 from aborted cows, was positive by culture. Uterine swabs and milk samples where firstly cultured on the selective media, so *Brucella* colonies were recognized first by colonial morphology then subcultured to obtain pure culture before submitted to the bacteriological and biochemical test. According to the results of Pearson Chi- Square tests, isolates from cows specimens were significantly higher (P<0.05) than that isolated from buffalos (figure 2).

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been shown that aborted cattle negative for brucellosis registered high intensity for IHC staining of 3β-HSD compared with that positive for brucellosis (fig. 3c). Statistical analysis revealed that the total results of negative scores (1, 2 and 3) in positive cases was significantly higher (P<0.01) than that of negative. Where the comparison between positive scores (4 and 5) showed that negative cases was significantly higher (P<0.01) than that positive. These scores differences were seen

**Figure 6.** IHC staining results. A & B: cow placenta negative for brucellosis, specific staining of chorionic plate with DAB chromogen (brown) and counterstained with Hematoxylin (blue) (A:100x, B:400x). C: cow placenta positive for brucellosis; stained by DAB chromogen (brown) and counterstained with Hematoxylin (blue)

The primer pair used in this study succeeded in the amplification of a 223-bp fragment from *Brucella* isolates cultures that were studied, meanwhile, the DNA extracted from culture harboring *Brucella*'s DNA, so that they yielded predicted 223-bp fragment. All *Brucella* strains that studied

**Figure 4-23.** Agarose gel electrophoresis for PCR products of Brucella isolates, where 223bp PCR products

**Clinical observation:** Clinical findings observed in aborted cattle were in agreement with other researchers (13, 14). Abortion is a frequent complication of brucellosis in animals, where placental localization is believed to be associated with erythritol, a growth stimulant for *B. abortus.*  Whether the rate of abortions from brucellosis exceeds rates associated with bacteremia from other bacterial causes is unclear. In any event, prompt diagnosis and treatment of brucellosis dur-

in both chorionic villi and chorionic plate.

notice, non IHC reaction for 3BHSD enzyme .100x.

with PCR have same 223-bp fragment. (figure 8).

appear as positive results.

**4. Discussion**

**Molecular detection of Brucella spp by PCR technique:**

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#### **Steroid hormones assessments:**

Cows and buffalos positive for *Brucella* infection showed insignificant differences (P>0.05) of serum estradiol concentration compared with that of non infected (463.92±176.57 pg/ml for infected versus 589.66±235.67 pg/ml for non infected). Serum cortisol in infected animals revealed insignificant higher concentration (P>0.05) than non infected animals (78.36±12.14 nmol/l versus 65.48±13.38 nmol/l, respectively). on the other hand, serum progesterone concentration revealed significant decrease (P<0.05) in infected animals (0.495±0.13 ng/ml) compared with (18.468±6.26 ng/ml) in non infected group. This decrement of progesterone concentration reflected on the progesterone/ estradiol balance (P/E Ratio) which reach (0.107%) in infected group compared with (3.132%) in non infected group (figure 2).

**Fig 2.** Mean ±SE of steroid hormones levels in aborted cattle.

#### **Immunohistochemical assay:**

Results of IHC analysis demonstrated positive staining for 3β-HSD in placentas of cattle, in which the localization was found in both chorionic villi and chorionic plate (fig. 3a and b). It has

**Figure 6.** IHC staining results. A & B: cow placenta negative for brucellosis, specific staining of chorionic plate with DAB chromogen (brown) and counterstained with Hematoxylin (blue) (A:100x, B:400x). C: cow placenta positive for brucellosis; stained by DAB chromogen (brown) and counterstained with Hematoxylin (blue) notice, non IHC reaction for 3BHSD enzyme .100x.

#### **Molecular detection of Brucella spp by PCR technique:**

The primer pair used in this study succeeded in the amplification of a 223-bp fragment from *Brucella* isolates cultures that were studied, meanwhile, the DNA extracted from culture harboring *Brucella*'s DNA, so that they yielded predicted 223-bp fragment. All *Brucella* strains that studied with PCR have same 223-bp fragment. (figure 8).

**Figure 4-23.** Agarose gel electrophoresis for PCR products of Brucella isolates, where 223bp PCR products appear as positive results.

### **4. Discussion**

**Clinical observation:** Clinical findings observed in aborted cattle were in agreement with other researchers (13, 14). Abortion is a frequent complication of brucellosis in animals, where placental localization is believed to be associated with erythritol, a growth stimulant for *B. abortus.*  Whether the rate of abortions from brucellosis exceeds rates associated with bacteremia from other bacterial causes is unclear. In any event, prompt diagnosis and treatment of brucellosis during pregnancy can be lifesaving for the fetus (1). It has been postulated that generalized suppression of adaptive immune response mainly occurred during pregnancy. This immune suppressed state prevent maternal rejection of the fetus but has unfortunate consequence of increasing maternal susceptibility to certain infectious agents (15).

International Conference on Applied Life Sciences (ICALS2012)

**Immunohistochemistery assay:** The decrement in the expression of 3β-HSD enzyme in brucella infected animals may attribute to utilization of this enzyme by *Brucella* itself, which may results in the decrement of progesterone biosynthesis. Or due to infection of placenta with *Brucella* that may caused damage in placental tissue. This results were in agreement with that recorded by Samartino *et al*. (22). From the present findings, it can be demonstrated that *B.* abortus may traffics from a phagosome compartment towards the endoplasmic reticulum of the host cell; where

**Brucella detection by PCR**: All *Brucella* strains that were studied with PCR have the same 223 bp fragment. Results of PCR were the same as that obtained by Baily *et al.* (10) whom used PCR amplification contained a single pair of oligonucleotide primers designed to amplify a 223 bp product. Fekete *et al*. (24) used PCR in the diagnosis of brucellosis and described it as specific,

[2] Sauret, J.M., Vilissova, N., (2002). Human brucellosis review. The Journal of the American Board

[3] Boschiroli, M.L., Foulongne, V., O'Callaghan, D., (2001). Brucellosis: a worldwide zoonosis.

[4] Ashford, D. A., J. Lingappa, C. Woods, H. Noll, B. Neville, and B. A. Perrkins. (2004). Adverse events in human associated with accidental exposure to the livestock brucellosis vaccine RB51.

[5] Garin-Bastuji B., Blasco J.M., Grayon M., Verger J.M., Brucella melitensis infection in sheep:

[6] Alonso-Urmeneta B., Marín C., Aragón V., Blasco J.M., Díaz R., Moriyón I., Evaluation of Lipopolysaccharides and Polysaccharides of different Epitope Structures in the Indirect Enzyme-Linked Immunosorbent Assay for Diagnosis of Brucellosis in Small Ruminants and Cattle, Clin.

[7] Cloeckaert, A.; Tibor, A. and Zygmunt. M. S.(1999). Brucella outer membrane lipoproteins share antigenic determinants with bacteria of the family Rhizobiaceae. Clin. Diagn. Lab. Immunol.

[8] Marín C., Moreno E., Moriyón I., Diáz R., Blasco J.M., Performance of competitive and indirect enzyme-linked immunosorbent assays, gel immunoprecipitation with native hapten polysaccharide and standard serological tests in diagnosis of sheep brucellosis, Clin. Diagn. Lab.

[9] Alton, G.G.; Jones, L.M. and Pietz, D.E. (1975). Laboratory techniques in brucellosis 2nd . Ed.

[10] Baily, G.G.; Krahn, J.B.; Drasar, B.S. and Stoker, N.G. (1992). Detection of Brucella melitensis and Brucella abortus by DNA amplification. J. Tropical Med. Hyg. (95) No. 4 p. 271-275.

sensitive and simple and could become a routine diagnostic test for brucellosis.

[1] WHO/OIE/FAO/CDS. (2006). Brucellosis in humans and animals. 7:1-66.

the organism has an optimal environment for replication (23).

**5. References**

6:627-9.

of Family Practice 15, 401–406.

Vaccine. 22: 3435-3439.

Current Opinion in Microbiology 4, 58–64.

present and future, Vet. Res. 29 (1998) 255-274.

Geneva. Monograph. Series No. 55. ch. 2 p. 113.

Diagn. Lab. Immunol. 5 (1998) 749-754.

Immunol. 6 (1999) 269- 272.

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**Bacterial isolation and identification:** *Brucella* strains isolated from cattle were obtained from aborted fetuses and vaginal discharge and were compared to that isolated from milk samples. The number of *Brucella* organisms in milk and colostrums samples was lower than that in abortion material, fetus stomach, fetal fluids and membranes, also milk samples is highly contaminated with other organisms. These results were in agreement with that mentioned by OIE (11). On the other hand, bacteriological and biochemical test of *Brucella* isolates were same as that advised by Alton *et al*. (12) and Qunin *et al*. (13).

**Serological results:** Serological examination performed by RB test in the present study gave positive result in some aborted animals but were negative for ELISA and bacterial isolation, also it gave negative result in buffalo aborted before 2 month or more. This diversity may attributed to the infection with microorganisms other than *brucella* spp. This infection are likely to cause cross-reactions in serological tests with smooth *Brucella* antigens and give false positive for RB. These results indicate that RB test is not confirmative test for diagnosis of brucellosis. Although RB test is known to have many false positive or negative results, but generally it is simple, rapid and can be used as screening method for infection (16). For confirmation of brucellosis, ELISA technique is the suitable and precise detectable test. It has been recorded that antibodies to *Brucella* appear in the serum within 1-2 weeks of infection. The initial response is the appearance of IgM isotype (which can be easily detected by RB) followed by a switch to IgG, after a while titers of both immunoglobulins classes increase. Distinct most of the usual serological tests, ELISA is effective in detecting all immunoglobulins (antibodies) classes and sub-classes important in diagnosis and appears to be the most sensitive serological test (17). IgM is produced soon after infection but declines quickly when production of IgG increases. IgM reacts non specifically in many serological tests and can cause high rates of false positive reactions. IgG1 is consistently produced at high levels in *Brucella*-exposed cattle sera and has a high affinity and specificity for *Brucella* antigens particularly the O-chain.

**Steroid hormones interference:** It has been registered, in the present study, that the results of reproductive hormones (E and P) concentrations in aborted cows and buffalos positive for brucellosis were in orchestration with that registered in immunohistochemical assays. Although estradiol concentration slightly decreased in brucella-infected aborted animals, but progesterone concentration sharply decreased. The important significant point was that related to the decrement of P/E ratio. On the other hand, immunohistochemical results revealed that 3β-HSD enzyme expression on trophoblast registered significant decrease, where 3β-HSD considered as main enzyme in progesterone biosynthesis that play important role in maintenance of pregnancy (18, 19, 20). Gorvel and Moreno (21) showed that in trophoblasts, *B. abortus* induces steroid biosynthesis and modulates the metabolism of prostaglandin precursors, favoring bacterial growth. These changes mimic to some extent what happens during parturition and are likely to contribute to the abortion.

**Immunohistochemistery assay:** The decrement in the expression of 3β-HSD enzyme in brucella infected animals may attribute to utilization of this enzyme by *Brucella* itself, which may results in the decrement of progesterone biosynthesis. Or due to infection of placenta with *Brucella* that may caused damage in placental tissue. This results were in agreement with that recorded by Samartino *et al*. (22). From the present findings, it can be demonstrated that *B.* abortus may traffics from a phagosome compartment towards the endoplasmic reticulum of the host cell; where the organism has an optimal environment for replication (23).

**Brucella detection by PCR**: All *Brucella* strains that were studied with PCR have the same 223 bp fragment. Results of PCR were the same as that obtained by Baily *et al.* (10) whom used PCR amplification contained a single pair of oligonucleotide primers designed to amplify a 223 bp product. Fekete *et al*. (24) used PCR in the diagnosis of brucellosis and described it as specific, sensitive and simple and could become a routine diagnostic test for brucellosis.

## **5. References**


[11] OIE Manual of diagnostic tests and vaccines for terrestrial animals, 5th edition, (2009) part 2, section 2.3, chapter 2.3.1.

International Conference on Applied Life Sciences (ICALS2012)

**Greenhouse Experiments of Symbiotic** 

Nisa Rachmania Mubarik 1,\*, Hanum Habibah<sup>2</sup>

2 Postgraduate School of Bogor Agricultural University, Bogor, Indonesia

Jalan Agatis, IPB Dermaga, Bogor, Indonesia

Corresponding author, Tel/ fax: +62-251-8622833

Email: nrachmania@ipb.ac.id; mubariknisa@yahoo.com

and without nitrate control and the reference strain, USDA 110.

**Plant**

University,

**Abstract**

tiveness.

**1. Introduction** 

gen in soybean plant [1].

\*

**Effectiveness of Acid-Aluminium Tolerance** 

1 Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural

**Bradyrhizobium Japonicum Strains on Soybean** 

The aim of the research was to study the symbiotic effectiveness of seven strains of acid-aluminium tolerance *Bradyrhizobium japonicum* on soybean plant cultivar Slamet. The research conducted in the greenhouse and used complete randomized design with seven inoculation treatments, two controls and one reference strain which used the nutrient solution at pH 4.5. Each treatment had three replications. The all of parameters were measured at 37 days after planting (DAP). Result of the experiments showed that mutant Bj 11 (19) inoculated to soybean plant had the highest symbiotic effectiveness. The treatment of Bj 11 (19) could increase the dry weight of t upper crop (64,88%), N-uptake (190,88%), and symbiotic effectiveness (65,87%) better than treatments with

**Keywords:** soybean, acid-aluminium tolerance, Bradyrhizobium japonicum, symbiotic effec-

Availability of sufficient nitrogen is one of the keys to increase the productivity of soybean plants. Soybean plants generally take nitrogen from the air by root-nodule bacteria and then the bacteria convert nitrogen into ammonia that is needed for plant growth. *Bradyrhizobium japonicum* is one species of slow-growing nodule bacteria which is very important to uptake atmospheric nitro-

Acid soils usually cause problems in soybean production, such as consist of low phosphorus and high aluminium [2] that strongly inhibit the growth of symbiotic nitrogen fixation bacteria on soybean plants [3]. The failure of nodulation under acid soil conditions is common, especially in soils of pH less than 5.0 [4]. B. japonicum is more tolerant at low pH, around pH 4.0-4.5 than

> © 2012 Mubarik et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

provided the original work is properly cited.

Turkey, September 10-12, 2012

and Aris Tri Wahyudi<sup>1</sup>

<sup>337</sup> ISALS


## **Greenhouse Experiments of Symbiotic Effectiveness of Acid-Aluminium Tolerance Bradyrhizobium Japonicum Strains on Soybean Plant**

Nisa Rachmania Mubarik 1,\*, Hanum Habibah<sup>2</sup> and Aris Tri Wahyudi<sup>1</sup>

1 Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University,

Jalan Agatis, IPB Dermaga, Bogor, Indonesia

2 Postgraduate School of Bogor Agricultural University, Bogor, Indonesia

\* Corresponding author, Tel/ fax: +62-251-8622833

Email: nrachmania@ipb.ac.id; mubariknisa@yahoo.com

#### **Abstract**

The aim of the research was to study the symbiotic effectiveness of seven strains of acid-aluminium tolerance *Bradyrhizobium japonicum* on soybean plant cultivar Slamet. The research conducted in the greenhouse and used complete randomized design with seven inoculation treatments, two controls and one reference strain which used the nutrient solution at pH 4.5. Each treatment had three replications. The all of parameters were measured at 37 days after planting (DAP). Result of the experiments showed that mutant Bj 11 (19) inoculated to soybean plant had the highest symbiotic effectiveness. The treatment of Bj 11 (19) could increase the dry weight of t upper crop (64,88%), N-uptake (190,88%), and symbiotic effectiveness (65,87%) better than treatments with and without nitrate control and the reference strain, USDA 110.

**Keywords:** soybean, acid-aluminium tolerance, Bradyrhizobium japonicum, symbiotic effectiveness.

## **1. Introduction**

Availability of sufficient nitrogen is one of the keys to increase the productivity of soybean plants. Soybean plants generally take nitrogen from the air by root-nodule bacteria and then the bacteria convert nitrogen into ammonia that is needed for plant growth. *Bradyrhizobium japonicum* is one species of slow-growing nodule bacteria which is very important to uptake atmospheric nitrogen in soybean plant [1].

Acid soils usually cause problems in soybean production, such as consist of low phosphorus and high aluminium [2] that strongly inhibit the growth of symbiotic nitrogen fixation bacteria on soybean plants [3]. The failure of nodulation under acid soil conditions is common, especially in soils of pH less than 5.0 [4]. B. japonicum is more tolerant at low pH, around pH 4.0-4.5 than

© 2012 Mubarik et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the fast growing nodule bacteria, such as Rhizobium [5]. Previous research by Endarini et al. [6] had managed to get acid aluminium tolerant B. japonicum from several locations in Indonesia [6]. The results showed that BJ 11 isolate has the highest tolerance on acid and had a good ability to grow on pH 4.5 media. Some of the strains showed more competitive than reference strain, USDA 110, in testing of the effectiveness symbiotic at greehouse. Furhermore, Wahyudi et al. [7] had constructed several strains of acid-aluminium tolerance B. japonicum with increased symbiotic effectiveness through transposon TN5 mutagenesis, such as Bj 11 (20) and KDR 15 (37). Some mutants showed the ability to form root nodules more than the wild-type strains viz. Bj 11 (5), Bj 11 (19), Bj 11 (20), and KDR 15 (37).

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cell. ml-1 of *B. japonicum*. N-free nutrient solution and nutrient solution contained KNO3

**Plant Maintenace at Greenhouse.** During soybean plants growing in the greenhouse regularly added with sterile nutrient solution into the bottom of Leonard bottle. Since 10 days after planting (DAP), plants sprayed with fungicide (1g/L) once a week. Harvesting plants were done by cutting plants at the cotyledon to the former boundary separating the top and plant roots. The roots are removed from the bottle and carefully cleaned of sand and charcoal then washed.

**Test of Nitrogenase Activity.** Nitrogenase activity was measured by acetilen reduction using gas chromatography. Each of root and nodule soybean plant put into incubation bottle and then sealed with a rubber cover. The next stage was to capture gas from the bottle as much as 2 ml and replaced it with the injection of 2 ml acetylene gas. The bottles were incubated for 30 minutes and then 0.1 ml was taken for gas injected into the gas chromatography Shimadzu 17A. There are three triplicates for each treatment. Ethylene gas produced was calculated based on peak areas on paper chromatograms. Nitrogenase activity was defined as the total amount of ethylene

**Symbiotic Effectiveness Test.** Symbiotic effectiveness values (SE) was obtained by formula of Gibson [10] SEN (Symbiotic Effectiveness N) = percentage of dry weight of plants inoculated test

age of dry weight of plants inoculated test strain to dry weight of plants treated with reference

**Test of N Total Plant.** N Total number of plant referred to the N total number of the canopy.

**Bacterial Isolate Growth.** Isolates were able to grow on YMA which were added with 0.0025% congo red and 50 mg/ml rifampicin after 7 days incubated on room temperature. Morphology of *B. japonicum* colonies were mucoid, not quite able to absorb congo red, and curve elevated (Fig.1)

**Fig 1.** The growth of Bradyrhizobium japonicum Bj 11 (19) on YMA media + 0.0025% congo red + 50 mg/ml

formed per number of plants per hour in units of µmol [8].

Amount of N content was determined by Kjeldahl method [8].

strain to dry weight of plants treated with KNO3

strain, USDA 110.

**3.1. Results**

**3. Results and Discussion**

rifampicin ten days after inoculation.

control was added every second day. All treatment plants were maintained until 37 DAP.

108

Turkey, September 10-12, 2012

and SER (Symbiotic Effectiveness R) = percent-

<sup>339</sup> ISALS

as

The efforts to obtain potential strains are still wide open in agricultural research. The purpose of the research was aimed to study the symbiotic effectiveness of seven strains of acid-aluminium tolerance Bradyrhizobium japonicum on soybean plant cultivar Slamet.

## **2. Materials and Methods**

#### **2.1. Materials**

Acid tolerant isolates *B. japonicum* were used in the study viz. Bj 11 (wt), KDR 15 (wt), Bj 13 (wt), Bj 11 (5), Bj 11 (19), Bj 11(20), KDR 15 (37), and USDA 110. All bacteria were collected at IPB Culture Collection, Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University. Soybean seeds cultivar Slamet were obtained from Research Institute for Food Crops and Genetic Resources, Bogor, Indonesia.

#### **2.2. Methods**

**Experimental Design.** All the data collected in greenhouse were analyzed using complete randomized block design using Statistical Analysis System (SAS) and the means at p (0.05) level of significance. The experiment was arranged into seven inoculation treatments, two controls and one standard strain which used the nutrient solution at pH 4,5. Each treatments were made in three replications. Growth parameters such as height of plant, dry-weight of upper crop, dryweight of nodules, number of nodules, N uptake of plants, nitrogenase activity of root nodule, and symbiotic effectiveness were determined.

**Medium and Inoculants Preparation**. *Bradyrhizobium japonicum* isolates were grown on Yeast Mannitol Agar (YMA) for 7-8 days at room temperature. The YMA media consist of mannitol (10 g/L), K2 HPO4 (0.5 g/L), MgSO4 . 7H2 O (0.2 g/L), NaCl (0.2 g/L), yeast extract (0.5 g/L), added with 0.0025% congo red and rifampicin (50 mg/ml). The isolates were resistant to rifampicin [7] & [8]. Then they were subcultured into Yeast Mannitol Broth (YMB) and incubated for seven days with 125 rpm at room temperature.

**Soybean Seed Inoculation.** Soybean seeds were selected based on size and healthiness (able to shoot). Seed surface were sterilized using 95% alcohol for 10 seconds and 5% H2 O2 for five minutes then rinsed seven times using sterilized water. The seeds are germinated in a petri dish at room temperature without light. Two days old sprouts grown in Leonard jar, pots filled with sand [7] which filled with N-free nutrient solution of pH 4.5 [9]. Each sprout was inoculated with 108 cell. ml-1 of *B. japonicum*. N-free nutrient solution and nutrient solution contained KNO3 as control was added every second day. All treatment plants were maintained until 37 DAP.

**Plant Maintenace at Greenhouse.** During soybean plants growing in the greenhouse regularly added with sterile nutrient solution into the bottom of Leonard bottle. Since 10 days after planting (DAP), plants sprayed with fungicide (1g/L) once a week. Harvesting plants were done by cutting plants at the cotyledon to the former boundary separating the top and plant roots. The roots are removed from the bottle and carefully cleaned of sand and charcoal then washed.

**Test of Nitrogenase Activity.** Nitrogenase activity was measured by acetilen reduction using gas chromatography. Each of root and nodule soybean plant put into incubation bottle and then sealed with a rubber cover. The next stage was to capture gas from the bottle as much as 2 ml and replaced it with the injection of 2 ml acetylene gas. The bottles were incubated for 30 minutes and then 0.1 ml was taken for gas injected into the gas chromatography Shimadzu 17A. There are three triplicates for each treatment. Ethylene gas produced was calculated based on peak areas on paper chromatograms. Nitrogenase activity was defined as the total amount of ethylene formed per number of plants per hour in units of µmol [8].

**Symbiotic Effectiveness Test.** Symbiotic effectiveness values (SE) was obtained by formula of Gibson [10] SEN (Symbiotic Effectiveness N) = percentage of dry weight of plants inoculated test strain to dry weight of plants treated with KNO3 and SER (Symbiotic Effectiveness R) = percentage of dry weight of plants inoculated test strain to dry weight of plants treated with reference strain, USDA 110.

**Test of N Total Plant.** N Total number of plant referred to the N total number of the canopy. Amount of N content was determined by Kjeldahl method [8].

## **3. Results and Discussion**

#### **3.1. Results**

**Bacterial Isolate Growth.** Isolates were able to grow on YMA which were added with 0.0025% congo red and 50 mg/ml rifampicin after 7 days incubated on room temperature. Morphology of *B. japonicum* colonies were mucoid, not quite able to absorb congo red, and curve elevated (Fig.1)

**Fig 1.** The growth of Bradyrhizobium japonicum Bj 11 (19) on YMA media + 0.0025% congo red + 50 mg/ml rifampicin ten days after inoculation.

**Number and Dry-Weight of Nodules.** Inoculation of variuos strains of *B. japonicum* on soybean cultivar Slamet showed variation number of nodule between 9-21 nodule per plant. The highest number of nodule was found in the plant inoculated with mutant strain, Bj 13 (wt) (Table 1). Most of nodules were located on the secondary roots. The range of nodule dry-weight was 0.0089-0.0440 g per plant. The highest nodule dry-weight presented in soybean plant inoculated by BJ 11 (5) (Table 1).

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**Treatment**

**nodule** 

**nodule**

**Height of** 

**activity** 

**of upper**

**Dry-weight** 

 **N uptake**

 **(mg N** 

**SEN(%)**

**SER (%)**

**plant -1 )**

**Nitrogenase** 

**plant (cm)**

**(µmol 2** 

 **crop** 

**plant -1**

 **(g plant -1 )**

**hour -1 )**

**(nodule** 

 **(g plant -1)**

**plant -1)**

Bj 11 (20) Bj 11 (19)

Bj 11 (5) Bj 11 (wt) Bj 13 (wt)

Inoculation of acid-aluminium tolerant *Bradyrhizobium japonicum* lead to good nodulation, vegetative growth, and symbiotic effectiveness of soybean cultivar Slamet at pH 4.5. Mutant of *Bradyrhizobium japonicum* strain Bj 11 (19) could increase the dry weight of the upper crop (64,88%), the

KDR 15(37)

KDR 15(wt)

USDA 110

Control N Control NO

**Tabel 1.** against N/R

0

d

0

d

37,3

e

0 Effect of inoculation of B. japonicum on soybean cultivar Slamet at 37 DAP using N-free solution at pH 4,5 + A150 µM

Numbers on the same column followed by the same letter were not significantly different based on Duncan Multiple Range Test (

=no detection, N:without inoculation consist of KNO3 0,05%, N0: without inoculation and without KNO3 0,05%, Symbiotic Effectiveness (SE)

c

0,4561

e

6,91

d

83,88

d

77,55 b

α = 0.05). 0

0

d

0

d

46,0

d

0

c

0,5509 de

13,58

c

100,00

cd

96,26 ab

12

bc

0,0241

c

63,0

b

12,21

a

0,6164

d

13,63

c

114,92

cd

100,00 ab

20

ab

0,0373

ab

63,6

ab

10,23

b

0,8031

ab

18,12

ab

146,54

ab

140,35 ab

9

c

0,0089

d

48,8

d

9,38

b 0,5342

e

7,72

d

97,30

d

92,19 b

21

a

0,0262

bc

60,7

c

9,79

b

0,7963

b

16,08

bc

145,86

ab

136,60 ab

17

abc

0,0397

a

71,4

a

12,54

a

0,8447

ab

16,88

ab

155,37

ab

144,55 ab

10

c

0,0440

a

67,8

ab

12,79

a

0,8317

ab

18,65

ab

151,48

ab

144,05 ab

16

abc

0,0377

ab

69,3

ab

10,17

b

0,9083

a

20,10

a

165,87

a

156,78 a

17

abc

0,0338

abc

54,4

d

10,44

b

0,7407

bc

16,03

c

137,01

bc

124,85 ab

**Number** 

**4. Conclusions**

**of** 

**Dry-weight of** 

Turkey, September 10-12, 2012

<sup>341</sup> ISALS

**Height of Plant and Dry-weight of Upper Crop.** All treatments were inoculated with *B. japonicum* strains showed height plant higher than control N without inoculation, except Bj 11 (20) and KDR 15 (37). In general, the dry-weight of upper crop showed significantly different with control without inoculation and without added with 0.05% KNO3 .

**Nitrogenase Activitity.** Inoculation of various strains of *B. japonicum* on soybean cultivar Slamet were significantly influence on the activity of nitrogenase. Mutant strain Bj 11 (5) had a higher nitrogenase activity (12,79 mmol 2 plant-1)and significantly different than the other strains except Bj 11 (wt) and reference strain, USDA 110 (Table 1).

**Symbiotic Effectivenes.** The highest symbiotic effectiveness was found in the plant inoculated with mutant strain Bj 11 (19) of 165,87% compared to control N effectivenes and 156.78% compared to USDA 110. While strain KDR 15 (37) showed the lowest symbiotic effectivenes only 97,30% (Table 1).

**N Uptake.** In general, N uptake in plants inoculated with *B. japonicum* strains were significantly different with control treatments, except strain KDR15 (37). Maximum N uptake of the plant was noticed with the strain Bj 11 (19) up to 20,10 mg N plant-1 and significantly different with control treatments (Table 1).

#### **3.2. Discussion**

The symbiotic interaction between soybean and root nodule bacteria played an important role in increasing the plant growth of soybean plant. Effectivity of a root nodule bacteria in fixing nitrogen were affected by the compatibility between bacteria and the soybean plant [11]. Data on the effect of inoculation acid-aluminium tolerance *B. japonicum* on nodulation and vegetative growth of soybean plant (Table 1) showed that inoculation of root nodule bacteria could increase height of could increase plant, and dry weight of upper-crop up to 37 DAP. Increasing in nodule dry weight could increase N fixination and the plant growth [12]. The nodule dry weight was positively correlated with the ability of plants to fix N and dry weight of the shoot. In the study, three strains viz. Bj 11 (wt), Bj 11 (5), and Bj (19) showed the best on height of plants, dry weight of upper crop, and dry weight of nodule. The highest symbiotic effectiveness, dryweight of upper crop, and N uptake was found in the soybean plant inoculated with Bj 11 (19) compared to plant inoculated with other strains and the reference strain, USDA 110. Bj 11 (19) was proposed to be useful isolate for soybean plant on acid soil pH 4.5. The success or failure of inoculation depends on the competitive nodulation ability against indigenous bradyrhizobia under natural conditions [4].


#### **4. Conclusions**

Inoculation of acid-aluminium tolerant *Bradyrhizobium japonicum* lead to good nodulation, vegetative growth, and symbiotic effectiveness of soybean cultivar Slamet at pH 4.5. Mutant of *Bradyrhizobium japonicum* strain Bj 11 (19) could increase the dry weight of the upper crop (64,88%), the

against N/R

N-uptake (190,88%), symbiotic effectiveness (65,87%) better than treatments with and without nitrate and the standard strain, USDA 110.

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**A Gravity Model Analysis of Egypt's Trade and** 

This paper aims to study the economic effects of trade flows between Egypt and some economic blocs, where study confine to AFTA agreement particularly a Arab interface business and agreements of each bloc COMESA and EU generally because of strength opportunities for these blocs by using descriptive analysis and Gravity Model (GM). The major results confirmed the efficiency of the model in explaining Egyptian trade flow for the three previous blocs. Based on the above results, the study recommends continuing to increase the volume of foreign trade, in

**Keywords**: Economic Effects of Trade Flows, Economic Blocks, Gravity model approach

Trade among Arab countries (ACs) has been consistently weak in spite of several efforts to engage into different forms of regional economic integration. The most important attempts to achieve Arab economic integration were the agreement of 1953 on Transit Trade, the Common Market attempt of 1964, and the agreement of 1981 on the facilitation and development of trade, all signed under the auspices of the Arab League. These attempts, in addition to about 135 bilateral trade-related agreements, were not capable of stretching inter-trade beyond its peak of 10

In 1994, the Intra-trade of ACs as percentage of their total exports was around 8.3 percent. This rate compares unfavorably with the corresponding rates of many regional groupings from both Developed and Developing countries. The latter ratios were 69.9 percent for APEC, 61.7 percent for EU, 47.6 percent for NAFTA, and 11.6 percent for EFTA. For regional groupings from the Developing countries these rates were 18.2 percent for MERCOSUR (Latin America), 12.0 percent

These rates are not strictly comparable across groupings. Difference in the degree of development, size, and weight in international trade of the different countries of the groupings, explain to a great extent the observed variation between these regional groups. This can be said, however, the extent of Intra-Arab trade is arguably weaker than what it should have been given the common historical, religious, social, cultural, and language characteristics shared by these countries. Many factors were presented to explain the weakness of Intra-Arab trade and the obvious failure of previous Arab regional agreements to stimulate trade among Arab countries. These factors

Department of Economic & Rural Development, Suez Canal University, El-Arish, Egypt

**Some Economic Blocks**

addition to reducing the constraints faced by Intra-Arab trade.

El-Sayed M. A.

**1. Introduction**

percent of the total trade of ACs [1].

**Abstract**

© 2012 El-Sayed; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the

original work is properly cited.

1 (1) UNCTAD (1997): Handbook of International Trade and Development Statistics.

for UEMOA (West Africa), and 21.2 percent for ASEAN (South-East Asia).(1)

Turkey, September 10-12, 2012

<sup>343</sup> ISALS

#### **5. Acknowledgements**

This research was funded by Incentive Programs for Applied Research the Ministry of Research and Technology, Republic of Indonesia to NRM. Previous research was supported by competitive grants, DGHE, Ministry of Education, Republic of Indonesia to ATW.

#### **6. References**


## **A Gravity Model Analysis of Egypt's Trade and Some Economic Blocks**

El-Sayed M. A.

Department of Economic & Rural Development, Suez Canal University, El-Arish, Egypt

#### **Abstract**

This paper aims to study the economic effects of trade flows between Egypt and some economic blocs, where study confine to AFTA agreement particularly a Arab interface business and agreements of each bloc COMESA and EU generally because of strength opportunities for these blocs by using descriptive analysis and Gravity Model (GM). The major results confirmed the efficiency of the model in explaining Egyptian trade flow for the three previous blocs. Based on the above results, the study recommends continuing to increase the volume of foreign trade, in addition to reducing the constraints faced by Intra-Arab trade.

**Keywords**: Economic Effects of Trade Flows, Economic Blocks, Gravity model approach

#### **1. Introduction**

Trade among Arab countries (ACs) has been consistently weak in spite of several efforts to engage into different forms of regional economic integration. The most important attempts to achieve Arab economic integration were the agreement of 1953 on Transit Trade, the Common Market attempt of 1964, and the agreement of 1981 on the facilitation and development of trade, all signed under the auspices of the Arab League. These attempts, in addition to about 135 bilateral trade-related agreements, were not capable of stretching inter-trade beyond its peak of 10 percent of the total trade of ACs [1].

In 1994, the Intra-trade of ACs as percentage of their total exports was around 8.3 percent. This rate compares unfavorably with the corresponding rates of many regional groupings from both Developed and Developing countries. The latter ratios were 69.9 percent for APEC, 61.7 percent for EU, 47.6 percent for NAFTA, and 11.6 percent for EFTA. For regional groupings from the Developing countries these rates were 18.2 percent for MERCOSUR (Latin America), 12.0 percent for UEMOA (West Africa), and 21.2 percent for ASEAN (South-East Asia).(1)

These rates are not strictly comparable across groupings. Difference in the degree of development, size, and weight in international trade of the different countries of the groupings, explain to a great extent the observed variation between these regional groups. This can be said, however, the extent of Intra-Arab trade is arguably weaker than what it should have been given the common historical, religious, social, cultural, and language characteristics shared by these countries.

Many factors were presented to explain the weakness of Intra-Arab trade and the obvious failure of previous Arab regional agreements to stimulate trade among Arab countries. These factors

<sup>1 (1)</sup> UNCTAD (1997): Handbook of International Trade and Development Statistics.

© 2012 El-Sayed; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

range from mere economic factors, such as difference in economic systems, similarity of production structure and traded goods, lack of adequate transportation infrastructure compounded by distance, overprotection, heavy reliance on trade taxes, the lack of convertibility of Arab currencies, lack of market information, weakness of marketing strategies, and poor competitiveness of products.

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slavia or the former Soviet Union), they may still have closer trade ties than otherwise (history matters). Thirdly, if countries share a common border, transaction costs may be reduced beyond the mere distance factor, translating into a higher bilateral trade. Finally, the accession to a free trade arrangement may stimulate trade among the constituent countries, as the rise of Spanish

In view of their simplicity and high explanatory power, gravity models have been applied to the particular case of CEE countries in several studies. [8] and [9], two of the most influential early studies in the field, showed that trade of the CEE countries with developed countries has been only a fraction of potential trade. [9] suggested that actual trade with the EU12 was up to 5 times smaller than potential trade for Bulgaria and former Czechoslovakia in 1989. Some CEE transition economies were found to be much closer to equilibrium (this is the case of Hungary, with a ratio of potential to actual trade of 1.8), while countries like Romania and Albania, which did not participate in the Council of the Mutual Economic Assistance, started trade liberalization with regional trade structures closer to the gravity predictions estimated by Baldwin. [10] found that Romania achieved a significantly higher actual share of trade with the EU than predicted by the model in 1996. [11] and) [12] suggest a rapid convergence towards trade potential levels in non-EU countries which have a trade agreement with the EU (so-called associated countries). [13] and [14]found that trade between the EU15 and the CEE countries was close to the predicted level at the end of the1990s. Meanwhile, [15] found that trade potentials were largely exhausted by the end of the decade (in the longer-run however, trade flows could increase in larger proportions

After the theoretical foundation of gravity model had been established, in the 1990s, further studies concentrated on its empirical application. [16] Formulated a more complex and advanced form of gravity equation where he particularly emphasized the role of geographical factors, such as distance, landlockedness and population, as determinants of bilateral trade flows. He also included regional trading blocs, such as APEC, NAFTA and Mercosur, in his gravity equation in order to estimate the impact of regional integration on bilateral trade flows. In a similar way, [17] tried to analyze the impact of economic integration, as embodied by the LAIA, the Andean Pact, and CACM, in Latin American countries' Intra-regional trade flows, based on the gravity-type

Furthermore, they do not present a broader comparison with other regions. Such comparison would bolster the results and put them in perspective. However, with the increasing importance of geographical factors in international trade theory, the gravity model started to attract a re-

Works by [19], [20], [4] and [21] greatly contributed to the establishment of a theoretical foundation for the gravity model by showing that the gravity equation can be derived from a number of

There are two competing models of international trade that provide theoretical justification for the gravity model. They are the Differentiated Products Model and the Heckscher-Ohlin Model. [5] and [19] tried to identify the relationship between the bilateral trade flows and the product of two countries' GDPs by utilizing the Differentiated Products Model. According to Krugman

trade with other euro area countries in the second half of the 1980s indicates.

once all structural adjustment is completed).

awakening interest in the 1980s [18].

different international trade models.

equation.

Turkey, September 10-12, 2012

<sup>345</sup> ISALS

Other factors are of an institutional nature. These include colonial links or the moral commitment to a well established partner (North African Arab countries to Europe, and Middle Eastern countries to the U.S. and Europe), the poor preparation of and lack of commitment to the regional agreements, the lack of adequate trade financing schemes at the regional level, the low quality of bureaucracy, and lengthy trade-related procedures. Last but not least, trade among Arab countries is very sensitive to political events and relationships among these countries.

These impediment factors notwithstanding, Arab leaders have unanimously taken the decision, during their summit of June 1996 in Cairo, to revive the 1981 agreements and create an Arab Free Trade Area (AFTA). The unanimous decision bears witness to the commitment of Arab countries to reinforce trade among each other as a means of facing the fierce competition in international markets entailed by a rampant globalization.

## **2. Research problem and objective**

The agreements of AFTA which came into effect January 1, 1998, entailed the elimination of nontariff barriers and the reduction of tariff rates on goods traded among ACs by an average of 10 percent a year, over a period of ten years. Although, the general feeling is that AFTA has been well prepared for and unambiguously committed to, many suspect that most of the factors that are behind the failure of previous agreements are still present. Arguably, this minimizes the chance of a breakthrough in Intra-Arab trade.

This paper presents an objective evaluation of the potential for success in AFTA agreements based on a model accounting for most of the dimensions involved in explaining Intra-Arab trade flows.

## **3. Methodology**

Gravity models, which were originally proposed by [2] and [3], have become one of the most commonly used workhorse models to analyze patterns in international trade. By analogy with Newton's theory of gravitation, these models express bilateral trade as a function of two key variables: the economic size of the two countries engaged in trade and the distance between them. Accordingly, in their most general form these models suggest that the magnitude of trade between two countries depends on the supply conditions in the source country, the demand conditions in the host country (and other factors which may stimulate or hinder bilateral trade); they are consistent with standard models of international trade (see [4] [5] [6]).

As regards other factors, four variables are commonly added (see, e.g. [7]: Firstly, it is likely that countries sharing the same language trade more with each other than otherwise. This may be partly related to historically established trade ties. A common language dummy could for instance explain the relatively high levels of Spain's trade with its former colonies in Latin America. Secondly, if two countries were part of the same territory (such as the countries of former Yugoslavia or the former Soviet Union), they may still have closer trade ties than otherwise (history matters). Thirdly, if countries share a common border, transaction costs may be reduced beyond the mere distance factor, translating into a higher bilateral trade. Finally, the accession to a free trade arrangement may stimulate trade among the constituent countries, as the rise of Spanish trade with other euro area countries in the second half of the 1980s indicates.

In view of their simplicity and high explanatory power, gravity models have been applied to the particular case of CEE countries in several studies. [8] and [9], two of the most influential early studies in the field, showed that trade of the CEE countries with developed countries has been only a fraction of potential trade. [9] suggested that actual trade with the EU12 was up to 5 times smaller than potential trade for Bulgaria and former Czechoslovakia in 1989. Some CEE transition economies were found to be much closer to equilibrium (this is the case of Hungary, with a ratio of potential to actual trade of 1.8), while countries like Romania and Albania, which did not participate in the Council of the Mutual Economic Assistance, started trade liberalization with regional trade structures closer to the gravity predictions estimated by Baldwin. [10] found that Romania achieved a significantly higher actual share of trade with the EU than predicted by the model in 1996. [11] and) [12] suggest a rapid convergence towards trade potential levels in non-EU countries which have a trade agreement with the EU (so-called associated countries). [13] and [14]found that trade between the EU15 and the CEE countries was close to the predicted level at the end of the1990s. Meanwhile, [15] found that trade potentials were largely exhausted by the end of the decade (in the longer-run however, trade flows could increase in larger proportions once all structural adjustment is completed).

After the theoretical foundation of gravity model had been established, in the 1990s, further studies concentrated on its empirical application. [16] Formulated a more complex and advanced form of gravity equation where he particularly emphasized the role of geographical factors, such as distance, landlockedness and population, as determinants of bilateral trade flows. He also included regional trading blocs, such as APEC, NAFTA and Mercosur, in his gravity equation in order to estimate the impact of regional integration on bilateral trade flows. In a similar way, [17] tried to analyze the impact of economic integration, as embodied by the LAIA, the Andean Pact, and CACM, in Latin American countries' Intra-regional trade flows, based on the gravity-type equation.

Furthermore, they do not present a broader comparison with other regions. Such comparison would bolster the results and put them in perspective. However, with the increasing importance of geographical factors in international trade theory, the gravity model started to attract a reawakening interest in the 1980s [18].

Works by [19], [20], [4] and [21] greatly contributed to the establishment of a theoretical foundation for the gravity model by showing that the gravity equation can be derived from a number of different international trade models.

There are two competing models of international trade that provide theoretical justification for the gravity model. They are the Differentiated Products Model and the Heckscher-Ohlin Model. [5] and [19] tried to identify the relationship between the bilateral trade flows and the product of two countries' GDPs by utilizing the Differentiated Products Model. According to Krugman

& Helpman, under the imperfect substitute model, where each firm produces a product that is an imperfect substitute for another product and has monopoly power in its own product, consumers show preference for variety. When the size of the domestic economy (or population) doubles, consumers increase their utility, not in the form of greater quantity but of greater variety. International trade can provide the same effect by increasing consumers' opportunity for even greater variety. Therefore, when two countries have similar technologies and preferences, they will naturally trade more with each other in order to expand the number of choices available for consumption.

International Conference on Applied Life Sciences (ICALS2012)

XCR: Export Concentration Ratio of country i measured as the share of the three most important

ATFD81: Dummy variable taking the value one if both countries, i and j, have signed the Arab

The country was proved (i) Egypt, with changes of countries (j = 1, 2, ... n) in gravity equation.

(1) Multiplying GDP: multiplying GDP represent the economic size of the two countries, also was as representative of the productive possibility and market size, so the larger countries - with large production possibility - are the most likely to reach economies of scale leading to increased exports of competitive advantage. Also have large domestic markets possibility to absorb more imports, so the increase multiplied GDP is possibility to lead to increase the volume of bilateral trade and it is expected that the coefficient is estimated to be greater than zero. Since the GDP of Egypt remained constant, multiplying GDP depends upon GDP of the partner countries (j) and

(2) Multiplying GDP Per Capita: the variable GDP Per Capita represent the level of income and/ or the purchasing power of imported and exported countries, with the installation of GDP per capita in Egypt, will this variable to explain what If the flow of Egyptian trade depends upon the

(3) Geographical Distance: the distance is a variable resistant to a trade where represent trade barriers such as transportation costs, time, cultural differences and barriers to market access…etc. And the distance used in this study is (Great Circle Distance) between the Egyptian capital and the capitals of countries (trading partners). It is worth noting that many previous studies have been translated coefficient of geographical dimension, it's flexibility of trade in respect of the absolute level of the geographical dimension, where is the volume of trade greater between pairs of countries that are far from the rest of the world [22]. And coefficient distance measures the relative distances of the States, where the lack of distance coefficient refer to trade with nations far more than compared with the more closer. While the increase refer to trade with the more convergence faster than those away. And it is noted that the notion of relative distance (relative dimension) is significant in the case of States (N×N). whereas, in the case of the model used in this study. (N×1) measured all distances in absolute terms for Egypt, so it is expected that the

(4) Border: it's a dummy variable illustrated that the whether the countries share the same or contiguous borders, and this variable takes the picture the correct one if two countries share a

Also was used all variables in the form of the natural logarithm without dummy variable.

this included the lack of impact or influence of the domestic market neutral (internal).

Trade Facilitation and Development agreement of 1981, and zero otherwise. Mji: Flows of imports of country i from country j, in millions of U.S. dollars.

commodities in the total value of its exports.

**4. Results & Discussion** I- Description of GM variables:

level of income of trading partners.

coefficient of the distance be less than zero.

Table (1), illustrated that the GM variable definition.

common border and zero otherwise.

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<sup>347</sup> ISALS

In this paper, we use the log-linear form to estimate bilateral import flows among some economic blocs over average 2008-2010, to give an order of magnitude for the impact of the main factors often advanced in explaining the weakness of Intra-Arab trade.(2) Thus, the gravity equation to be estimated is as follows: (3)

Log(Mij)= ββ1Log(GNPi\*GNPj) + β1Log(GNPi\*GNPj) +β3Log(DISTANCEij) + β4Log(INEQGNPC) + β5PARTNER + β6COSINEij + β7POLFACT +β8Log(XRCi) + β9Log(Mji) + β10ATFD81 + β11BORDER + CONSTANT

Mij: Flow of imports of country j from country i, in millions of U. S. dollars.

GNP: Gross National Product of country i or j in millions of U.S. dollars.

GNPC: GNP per capita in millions of U. S. Dollars.

DISTANCE: Distance in kilometers between the capitals of countries i and j. BORDER: Dummy variable taking the value of unity if i and j share a common border and zero otherwise.(4)

PARTNER: Dummy variable taking the value of unity if i and j are members of GCC or AMU and zero otherwise.

INEQGNPC: Measure of GNP per capita inequality between countries i and j.(5)

POLFACT: Dummy variable taking the value of unity in case of border closing, political disagreement or event affecting normal diplomatic and commercial relations between countries i and j.

COSINE: Measure of trade correspondence between the export structure of country i and the import structure of country j.(6)

$$CONSIDEij = \sum\_{\pi} E\_{\pi} M\_{jk} \sqrt{\sum\_{\pi} E^{\pi} \mu \sum\_{\pi} M^{\pi^2} \mu}$$

<sup>(2)</sup> Arab countries, excluding Palestine, are: Algeria, Bahrain, Comoros, Djibouti, Egypt, Iraq, Jordan, Kuwait, Lebanon Libya, Mauritania, Morocco, Oman, Qatar, Saudi Arabia, Somalia, Sudan, Syria, Tunisia, U.A.E. &Yemen.

<sup>(3)</sup> Frankel et al. (1995) and Cyrus (1996) followed a closer specification.

<sup>(4)</sup> GCC stands for the Gulf Cooperation Council formed in 1981 grouping Bahrain, Kuwait, Qatar, Oman, Saudi Arabia, and the United Arab Emirates. AMU stands for the Arab Maghreb Union formed in 1989 and grouping Algeria, Libya, Mauritania, Morocco, and Tunisia.

<sup>(5)</sup> Balassa (1986), Balassa and Bauwens (1987), and Bergstrand (1990) used this measure of inequality. For a given variable x it is given by: 1+[xlog(x)+(l-x)log(l-x)l/log(2). It has the advantage of depicting relative rather than absolute inequality and falls between zero and one.

<sup>(6)</sup> The cosine measure indicates the cosine of the angle between the export vector of country i and the import vector of country j and is given by:

where Eik stands for exports of commodity k by country i and Mjk for imports of commodity k by country j.

XCR: Export Concentration Ratio of country i measured as the share of the three most important commodities in the total value of its exports.

ATFD81: Dummy variable taking the value one if both countries, i and j, have signed the Arab Trade Facilitation and Development agreement of 1981, and zero otherwise.

Mji: Flows of imports of country i from country j, in millions of U.S. dollars.

## **4. Results & Discussion**

I- Description of GM variables:

The country was proved (i) Egypt, with changes of countries (j = 1, 2, ... n) in gravity equation. Also was used all variables in the form of the natural logarithm without dummy variable.

(1) Multiplying GDP: multiplying GDP represent the economic size of the two countries, also was as representative of the productive possibility and market size, so the larger countries - with large production possibility - are the most likely to reach economies of scale leading to increased exports of competitive advantage. Also have large domestic markets possibility to absorb more imports, so the increase multiplied GDP is possibility to lead to increase the volume of bilateral trade and it is expected that the coefficient is estimated to be greater than zero. Since the GDP of Egypt remained constant, multiplying GDP depends upon GDP of the partner countries (j) and this included the lack of impact or influence of the domestic market neutral (internal).

(2) Multiplying GDP Per Capita: the variable GDP Per Capita represent the level of income and/ or the purchasing power of imported and exported countries, with the installation of GDP per capita in Egypt, will this variable to explain what If the flow of Egyptian trade depends upon the level of income of trading partners.

(3) Geographical Distance: the distance is a variable resistant to a trade where represent trade barriers such as transportation costs, time, cultural differences and barriers to market access…etc. And the distance used in this study is (Great Circle Distance) between the Egyptian capital and the capitals of countries (trading partners). It is worth noting that many previous studies have been translated coefficient of geographical dimension, it's flexibility of trade in respect of the absolute level of the geographical dimension, where is the volume of trade greater between pairs of countries that are far from the rest of the world [22]. And coefficient distance measures the relative distances of the States, where the lack of distance coefficient refer to trade with nations far more than compared with the more closer. While the increase refer to trade with the more convergence faster than those away. And it is noted that the notion of relative distance (relative dimension) is significant in the case of States (N×N). whereas, in the case of the model used in this study. (N×1) measured all distances in absolute terms for Egypt, so it is expected that the coefficient of the distance be less than zero.

(4) Border: it's a dummy variable illustrated that the whether the countries share the same or contiguous borders, and this variable takes the picture the correct one if two countries share a common border and zero otherwise.

Table (1), illustrated that the GM variable definition.


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ln (x2) GDP Per Capita -0.038 -0.185 0.781 2.664 -0.794 -1.880

ln (x3) Distance -0.001 -4.389\*\* -0.001 -5.229 -0.00008 -0.224

Ln (x4) Border -0.277 -0.478 -2.147 -1.207 0 65535

(1) The Egyptian bilateral trade with AFTA was increased about 1% by increased of GDP of the partner countries by about 1% (equivalent). While the trade volume bilateral Egyptian with COMESA and EU were estimated about 1.1, 1% respectively, indicating the importance of EU as

(2) The variable GDP per capita is not significant in the case of the three agreements, which refer to the flow of Egyptian trade does not depend upon the income level of trading partners, but

(3) The distance variable or geographic dimension was statistically significant and corresponding with economic logic in influencing the volume of Egyptian trade bilateral in framework AFTA. But it is not significant in the case of the agreement with COMESA and EU, which requires the development of land, sea and air transportation networks between ACs, including more than the speed of passage of goods and reduce transport costs, where the results indicated that increasing the distance between Egypt and its trading partners within the framework of AFTA was estimated

(4) The border variable was not statistically significant which led to decline from important of

This paper has reviewed most recent factors in Egyptian Trade with some Economic blocks. It was discussed that GDP & Distance only approach that maximizes the trade between Arab countries. It is yet anticipated future works are required to find the statistical significant of AFTA sample size using the other approaches to achieve this on the rest of Economic Blocs. Furthermore, the current approach may be extended for the rest of other factors of Egyptian trade such as GDP per capita, Border, and Language.

about 1% would lead to a reduction of the flow of the bilateral trade by about 1.9%.

common border between in framework AFTA as factor, determined of trade exchange.

No. of Observation 21 22 27

\*\* Significant at level 0.01 \* Significant at level 0.05" ( ) refers to standard Error

Source: Compute from Unified Arab Economic Report 2010.

major trade partner for Egypt.

**5. Summary and conclusion**

**Table 3.** Results of GM for some Economics blocs: Average 2007-2009

depends on the economic size of trading partners even more.

R-squared 0.89 0.82 0.45 R-Adjusted 0.86 0.78 0.34 F- test 31.381 19.469 6.284

(0.204) (0.293) (0.422)

(0.0002) (0.0003) (0.0004)

(0.578) (1.778) (0.000)

**AFTA COMESA EU Coeff. T-stat. Coeff. T-stat. Coeff. T-stat.**

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**Table 1.** GM Variables Definition

II- Descriptive Statistics for the Basic Sample:

While, table (2) showed that the Descriptive Statistics for the Basic Sample: Average 2007-2009.


**Source: Compute** from Unified Arab Economic Report 2010.

**Table 2.** Descriptive Statistics for the Basic Sample: Average 2007-2009

III- Empirical Results of GM:

The empirical results showed that in table (3). The first column in the table represents estimates for coefficient. Results obtained from running T-Statistics are re-ported in columns two.

The aggregate performance of the model in this table looks good value for the coefficient of determination was estimated at about 0.86 in the case of the agreement with AFTA, which means that GM, efficient in explaining the flow of bilateral trade to Egypt.

Results indicated that the GM to study the most important variables affecting on the volume of Egyptian bilateral trade in framework AFTA.



\*\* Significant at level 0.01 \* Significant at level 0.05" ( ) refers to standard Error Source: Compute from Unified Arab Economic Report 2010.

**Table 3.** Results of GM for some Economics blocs: Average 2007-2009

(1) The Egyptian bilateral trade with AFTA was increased about 1% by increased of GDP of the partner countries by about 1% (equivalent). While the trade volume bilateral Egyptian with COMESA and EU were estimated about 1.1, 1% respectively, indicating the importance of EU as major trade partner for Egypt.

(2) The variable GDP per capita is not significant in the case of the three agreements, which refer to the flow of Egyptian trade does not depend upon the income level of trading partners, but depends on the economic size of trading partners even more.

(3) The distance variable or geographic dimension was statistically significant and corresponding with economic logic in influencing the volume of Egyptian trade bilateral in framework AFTA. But it is not significant in the case of the agreement with COMESA and EU, which requires the development of land, sea and air transportation networks between ACs, including more than the speed of passage of goods and reduce transport costs, where the results indicated that increasing the distance between Egypt and its trading partners within the framework of AFTA was estimated about 1% would lead to a reduction of the flow of the bilateral trade by about 1.9%.

(4) The border variable was not statistically significant which led to decline from important of common border between in framework AFTA as factor, determined of trade exchange.

#### **5. Summary and conclusion**

This paper has reviewed most recent factors in Egyptian Trade with some Economic blocks. It was discussed that GDP & Distance only approach that maximizes the trade between Arab countries. It is yet anticipated future works are required to find the statistical significant of AFTA sample size using the other approaches to achieve this on the rest of Economic Blocs. Furthermore, the current approach may be extended for the rest of other factors of Egyptian trade such as GDP per capita, Border, and Language.

#### **6. References**

[1] A. Al-Shibani, "Arab trade liberalization: The Arab free trade area," *the General Secretariat of the General Union of Chambers of Commerce, Industry, and Agriculture in the Arab Countries*, 1997, pp. 67-114.

International Conference on Applied Life Sciences (ICALS2012)

**Competitive Interaction of Common** 

Vahid Sarabi\*, Mehdi Nassiri Mahallati, Ahmad Nezami

**Emergence and Density**

high densities (16 and 20 plants per m2

Iran

**Abstract**

per m2

**1. Introduction**

and Mohammad Hasan Rashed Mohassel

**Lambsquarters (***Chenopodium album* **L.)** 

**and Maize** *(Zea mays* **L.) at Different Time of** 

Department of Agronomy, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad,

Common lambsquarters (*Chenopodium album* L.) is an important weed of maize fields in Iran. In order to study the effect of common lambsquarters relative time of emergence on single-cross 704 maize (*Zea mays* L.), an experiment was designed with different density levels of the weed. The experimental design was a split plot based on a randomized completed block design with three replications. The emergence time of lambsquarters was considered as main plots with three levels: emergence of the weed 14 days earlier, 7 days earlier than maize and simultaneously with maize. Density of weed was the subplot treatment with six levels: 0, 4, 8, 12, 16, and 20 plants

. The results showed that height and leaf area index of maize decrease with earlier emer-

) led to maximum reduction. In contrast, height and LAI

gence time and increasing density of lambsquarters, so that 14 days earlier emergence of weed at

of lambsquarters increased with earlier emergence time especially at high densities. Common lambsquarters was a stronger competitor when emerged 14 days earlier than maize. Maize yield decreased more than 70% in the 7 and 14 days earlier emergence of lambsquarters at high density. Totally, it can be state that relative time of weed emergence compare to its density had maximum effect on maize growth. In addition, controlling lambsquarters before maize emerging at any

Common lambsquarters is the most prevalent weed of soybean [*Glycine max* (L.) Merr.] and maize (*Zea mays* L.) cropping systems in the upper Midwest of the USA [3, 6, 9] and it is one of the most competitive annual broadleaf weeds in maize production in Ontario, Canada [15]. Common lambsquarters causes significant yield damage in many cropping systems because of its rapid growth characteristics, competition for nutrients [12, 14], prolific seed production [6], and seed germination, under a wide range of environmental conditions [10, 11]. Weed density

> © 2012 Sarabi et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the

original work is properly cited.

densities is recommend preventing of maize growth and yield loss.

**Keywords:** Corn, density, emergence time, interference, leaf area, yield loss

Turkey, September 10-12, 2012

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## **Competitive Interaction of Common Lambsquarters (***Chenopodium album* **L.) and Maize** *(Zea mays* **L.) at Different Time of Emergence and Density**

Vahid Sarabi\*, Mehdi Nassiri Mahallati, Ahmad Nezami and Mohammad Hasan Rashed Mohassel

Department of Agronomy, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

#### **Abstract**

Common lambsquarters (*Chenopodium album* L.) is an important weed of maize fields in Iran. In order to study the effect of common lambsquarters relative time of emergence on single-cross 704 maize (*Zea mays* L.), an experiment was designed with different density levels of the weed. The experimental design was a split plot based on a randomized completed block design with three replications. The emergence time of lambsquarters was considered as main plots with three levels: emergence of the weed 14 days earlier, 7 days earlier than maize and simultaneously with maize. Density of weed was the subplot treatment with six levels: 0, 4, 8, 12, 16, and 20 plants per m2 . The results showed that height and leaf area index of maize decrease with earlier emergence time and increasing density of lambsquarters, so that 14 days earlier emergence of weed at high densities (16 and 20 plants per m2 ) led to maximum reduction. In contrast, height and LAI of lambsquarters increased with earlier emergence time especially at high densities. Common lambsquarters was a stronger competitor when emerged 14 days earlier than maize. Maize yield decreased more than 70% in the 7 and 14 days earlier emergence of lambsquarters at high density. Totally, it can be state that relative time of weed emergence compare to its density had maximum effect on maize growth. In addition, controlling lambsquarters before maize emerging at any densities is recommend preventing of maize growth and yield loss.

**Keywords:** Corn, density, emergence time, interference, leaf area, yield loss

#### **1. Introduction**

Common lambsquarters is the most prevalent weed of soybean [*Glycine max* (L.) Merr.] and maize (*Zea mays* L.) cropping systems in the upper Midwest of the USA [3, 6, 9] and it is one of the most competitive annual broadleaf weeds in maize production in Ontario, Canada [15]. Common lambsquarters causes significant yield damage in many cropping systems because of its rapid growth characteristics, competition for nutrients [12, 14], prolific seed production [6], and seed germination, under a wide range of environmental conditions [10, 11]. Weed density

© 2012 Sarabi et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

is a crucial factor in crop-weed competition. Crook and Renner [5] observed a 20% reduction in soybean yield where lambsquarters was present throughout the entire growing season at a density of four plants m-2. Sibuga and Bandeen [13] found that maize yield reduced by as much as 58% when lambsquarters density reached 277 plants m-2.

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Where *LAI* is maize leaf area, *LAIWF* is estimated leaf area index in weed-free plots, *D* is weed density (plants m-2), *I* is the percent leaf area index loss per unit weed density as *D* approaches

The relative leaf area of lambsquarters at the beginning of senescence was determined from

*<sup>+</sup> <sup>=</sup>* (2)

Where *LW* is lambsquarters relative leaf area, *LAIW* is the leaf area index of weed and *LAIC* is the leaf area index of maize. The relationship between maize yield loss and weed relative leaf area

1 − <sup>+</sup> <sup>1</sup> <sup>=</sup> (3)

with weed and *LW* is the relative leaf area of weed. Another version of the model was derived from the empirical model introduced by Cousens [4]. This model includes an extra parameter for

*W*

The result of this research showed that earlier emergence time and density of lambsquarters has remarkable effect on maize leaf area index. Maximum maize leaf area index (3) was obtained in the same emerging at low density of weed except of weed free check. In contrast, the lowest LAI (0.4 and 0.3) was observed when maize emergence was delayed 14 days at 16 and 20 plants m-2, respectively (Figure 1). Based on the coefficients of Equation 1, reduction in maize leaf area index per unit weed density as *D* approaches zero at 14 and 7 days earlier emergence of lambsquarters (*I*) was 17.63 and 9.09 percent respectively, while, in simultaneous emergence of weed was 2.71 percent. These coefficients show that first plant of lambsquarters impose more competition pressure to maize at the earlier emergence time especially in 14 days earlier emergence of weed. Also, maximum reduction in maize leaf area index was obtained at high densities in 14 and 7 days earlier emergence of weed than maize, so that maize leaf area index reduced by almost 100 percent (Table 1). Hence, the time of weed emergence is very important in competition between cropweed, as earlier emergence of weed would be led to reduction of leaf area index of crop close to

(4)

is the predicted proportional maize yield loss, *q* is the damage coefficient associated

*W C W*

( ) *<sup>W</sup> W <sup>L</sup> q L qL <sup>Y</sup>*

*W*

1 1

*<sup>L</sup> <sup>m</sup> q qL <sup>Y</sup>*

 <sup>+</sup> <sup>−</sup>

*<sup>W</sup> LAI LAI LAI*

zero, and *A* is the percent leaf area index loss as *D* approaches infinity.

*L*

the maximum yield loss caused by weed (*m*) (Equation 4):

100 percent and consequently the maximum yield loss.

*L*

=

Equation 2:

Where *Y<sup>L</sup>*

was determined using Equation 3 [8]:

**3. Results and discussion**

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<sup>353</sup> ISALS

The timing of weed emergence relative to crop is important to crop growth and yield. Yield losses are usually high when weeds emerge earlier or at the same time as the crop [1, 2, 7]. Common lambsquarters is known as one of the most troublesome weeds in maize field of Iran. The majority of studies record effects of weeds on yield in the simultaneous emergence time or times after crop emergence, whereas weeds usually cause to yield loss when emerge before crops and interference with crops during the growing season. Therefore, the objectives of this study were to (1) evaluate the importance and influence of earlier emergence time of weed at different densities on maize yield and leaf area index and (2) determine the competitiveness of lambsquarters and its effects on maize yield when emerged at different times and densities.

#### **2. Materials and methods**

An experiment was conducted in 2006 growing season at the Agricultural Faculty of Ferdowsi University of Mashhad, Iran. The experiment was a split plot based on randomized complete block design with three replications. Emergence time of lambsquarters as E1 , E2 and E3 (emergence of the weeds 14 or 7 days earlier and simultaneously with maize emergence, respectively) were assigned to main plots and density of weed at 6 levels of D0 , D1 , D2 , D3 , D4 and D5 (0, 4, 8, 12, 16 and 20 plants m-2) as subplot. Maize seed were planted 1.5 to 2 cm deep in 70-cm between and 20-cm in-row spacing at density of 7 plants m-2. The field was moldboard-plowed, harrowed and cultipacked in the spring to be prepared for planting. Starter fertilizer was broadcasted at a rate of 300-200-200 kg ha-1 N-P-K before planting based on local recommendations and was incorporated by cultivation and shallow disking for seedbed preparation. Nitrogen fertilizer was applied three times, 50% before planting, 25% at the six-to eight- leaf stage, and 25% at the ten-to twelve-leaf stage of maize. Based on a pre-test it was determined that emergence took 15 days for lambsquarters and 7 days for maize. We adjusted sowing dates of lambsquarters to achieve emergence 14 and 7 days prior to maize emergence and simultaneously with maize. Common lambsquarters emergence times were based on visual estimates of 50% emerged plants. At the initiation of lambsquarters senescence, five-tagged maize and five plants of lambsquarters near to them were clipped at the soil surface, sectioned, and placed in cloth bags. After measuring plants leaf area with leaf area meter (Delta-T Devices, Cambridge, England), plants were dried to constant weight in an oven for 70 h at 75°C, and dry weights were recorded.

The relationship between lambsquarters density and maize leaf area index was analyzed separately for each time of emergence by using a nonlinear hyperbolic model described by Cousens [4]. Equation 1 was used to describe relationship between lambsquarters density and maize leaf area index:

$$LAI = LAI\_{WF} \left[ 1 - \frac{D}{100 \left( 1 + \frac{D}{A} \right)} \right] \tag{1}$$

Where *LAI* is maize leaf area, *LAIWF* is estimated leaf area index in weed-free plots, *D* is weed density (plants m-2), *I* is the percent leaf area index loss per unit weed density as *D* approaches zero, and *A* is the percent leaf area index loss as *D* approaches infinity.

The relative leaf area of lambsquarters at the beginning of senescence was determined from Equation 2:

$$L\_{W} = \frac{LAI\_{W}}{LAI\_{W} + LAI\_{C}} \tag{2}$$

Where *LW* is lambsquarters relative leaf area, *LAIW* is the leaf area index of weed and *LAIC* is the leaf area index of maize. The relationship between maize yield loss and weed relative leaf area was determined using Equation 3 [8]:

$$Y\_L = \frac{qL\_w}{1 + (q-1)L\_w} \tag{3}$$

Where *Y<sup>L</sup>* is the predicted proportional maize yield loss, *q* is the damage coefficient associated with weed and *LW* is the relative leaf area of weed. Another version of the model was derived from the empirical model introduced by Cousens [4]. This model includes an extra parameter for the maximum yield loss caused by weed (*m*) (Equation 4):

$$Y\_L = \frac{qL\_w}{1 + \left(\frac{q}{m} - 1\right)L\_w} \tag{4}$$

#### **3. Results and discussion**

The result of this research showed that earlier emergence time and density of lambsquarters has remarkable effect on maize leaf area index. Maximum maize leaf area index (3) was obtained in the same emerging at low density of weed except of weed free check. In contrast, the lowest LAI (0.4 and 0.3) was observed when maize emergence was delayed 14 days at 16 and 20 plants m-2, respectively (Figure 1). Based on the coefficients of Equation 1, reduction in maize leaf area index per unit weed density as *D* approaches zero at 14 and 7 days earlier emergence of lambsquarters (*I*) was 17.63 and 9.09 percent respectively, while, in simultaneous emergence of weed was 2.71 percent.

These coefficients show that first plant of lambsquarters impose more competition pressure to maize at the earlier emergence time especially in 14 days earlier emergence of weed. Also, maximum reduction in maize leaf area index was obtained at high densities in 14 and 7 days earlier emergence of weed than maize, so that maize leaf area index reduced by almost 100 percent (Table 1). Hence, the time of weed emergence is very important in competition between cropweed, as earlier emergence of weed would be led to reduction of leaf area index of crop close to 100 percent and consequently the maximum yield loss.

**Figure 1.** Observed and estimated maize leaf area index at the beginning of lambsquarters senescence as a function of weed density at the 14 days (■), 7 days (▲) earlier emergence of weed and simultaneous (●) emergence time. Regression lines were fitted using Equation 1.

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**Parameter estimates a**

<sup>b</sup> 1.64 ± 0.27 ― 0.99

E-7 1.1 ± 0.62 ― 0.92 E0 0.28 ± 0.15 ― 0.93

E-14 1.3 ± 0.14 0.79 ± 0.89 0.99

E-7 0.83 ± 0.3 0.75 ± 0.27 0.92 E0 0.04 ± 0.23 0.15 ± 0.83 0.93

**<sup>a</sup>q,** represents the damage coefficient associated with lambsquarters; m, represents the maximum yield loss

**Table 2.** Estimated parameters obtained from maize yield loss as a function of the relative leaf area of the lambs-

[2] R. E. Blackshaw, E. H. Stoble, and A. R. W. Sturko. Effects of seeding dates and densities of green foxtail (*Setaria viridis*) on the growth and productivity of spring wheat (*Triticum aestivum*). *Weed* 

[3] C. Boerboom. Ready to tackle lambsquarters? University of Wisconsin Extension. 2009.http:// ipcm.wisc.edu/WCMNews/tabid/53/EntryId/686/Ready-to-Tackle-Lambsquarters.aspx.

[4] R. Cousens. An empirical model relating crop yield to weed and crop density and a statistical

[5] T. M. Crook, and K. A. Renner. Common lambsquarters (*Chenopodium album*) competition and

comparison with other models. *J. Agr. Sci.* 1985, **105**: 513-521.

time of removal in soybeans (*Glycine max*). *Weed Sci.* 1990, **38**: 358–364.

[1] R. J. Aldrich. Predicting crop yield reduction from weeds. *Weed Technol.* 1987, **1**: 199–206.

**q m R2**

) describes the fit of each model to the observed data.

is the same emergence of this weed with maize, respectively.

densities showing that emerging lambsquarters in this time and 14 days earlier than maize was

These results show that crop yield would be reduce more than 70%, when weed emerged earlier than crop even though competitiveness of crop is the same as weed. Hence, this weed should be controlled in the earlier emergence time to prevent greater yield loss and in the same emerging time to prevent more plants of weed reaching reproductive growth and developing seed bank.

led to yield reduction by 75 and 79 percent, respectively (Table 2).

**Lambsquarters relative time of emergence**

E-14

caused by weed. The coefficient of determination (R<sup>2</sup>

**b E-14,** E-7 are 14 and 7 days earlier emergence and E0

quarters for the first, second and third emergence times.

**Model**

One-parameter model

Two-parameter model

**4. References**

*Sci.* 1981, **29**: 212–217.

Accessed: February 3, 2010.

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<sup>355</sup> ISALS

**Figure 2.** Relation between estimated yield loss in maize and relative leaf area of lambsquarters at the initiation of weed senescence for the 14 days (■), 7 days (▲) earlier emergence of weed and simultaneous (●) emergence time. Common lambsquarters densities were zero, 4, 8, 12, 16 and 20 plants m-2.


**a LAIWF,** represents predicted weed-free leaf area index ± SE of maize; I, represents leaf area index loss ± SE as weed density approaches zero; A, represents leaf area index loss ± SE at high weed densities. The coefficient of determination (R2) describes the fit of each model to the observed data.

**b E-14,** E-7 are 14 and 7 days earlier emergence and E0 is the same emergence of this weed with maize, respectively.

**Table 1.** Observed weed-free maize leaf area index and rectangular hyperbola parameter estimates for the first, second and third lambsquarters emergence times as a function of weed density.

Relative damage coefficient indicated that lambsquarters in 14 days earlier emergence was a stronger competitor than maize, so that at this time of emergence, *q* was larger than one (1.64 and 1.3 for one and two parameter model, respectively) and a convex curve is found above the diagonal line. In contrast, this coefficient was smaller than one (0.28 and 0.04) for both parameter model and a concave curve is found under the diagonal line (Table 2 and figure 2). Hence, lambsquarters was not good competitor when emerged simultaneously with maize and crop was the stronger competitor. Coefficients also showed that maize and weed had equals competition ability at the 7 days earlier emergence, but maximum yield loss (*m*) was obtained at high

densities showing that emerging lambsquarters in this time and 14 days earlier than maize was led to yield reduction by 75 and 79 percent, respectively (Table 2).

These results show that crop yield would be reduce more than 70%, when weed emerged earlier than crop even though competitiveness of crop is the same as weed. Hence, this weed should be controlled in the earlier emergence time to prevent greater yield loss and in the same emerging time to prevent more plants of weed reaching reproductive growth and developing seed bank.


**<sup>a</sup>q,** represents the damage coefficient associated with lambsquarters; m, represents the maximum yield loss caused by weed. The coefficient of determination (R<sup>2</sup> ) describes the fit of each model to the observed data.

**b E-14,** E-7 are 14 and 7 days earlier emergence and E0 is the same emergence of this weed with maize, respectively.

**Table 2.** Estimated parameters obtained from maize yield loss as a function of the relative leaf area of the lambsquarters for the first, second and third emergence times.

#### **4. References**


[6] F. Forcella, R. G. Wilson, K. A. Renner, J. Dekker, R. G. Harvey, D. A. Alm, D. D. Buhler, and J. Cardina. Weed seed banks of the U.S. Maize Belt: magnitude, variation, emergence, and application. *Weed Sci.* 1992, **40**: 636–644

International Conference on Applied Life Sciences (ICALS2012)

, Dr. Abdelbaky Mousa

, Abdelmagid Hassan Abdelmagid<sup>2</sup>

Research aims to evaluate the biological control of weeds for the culture of fingerlings fish grass carp, by highlighting the economic, environmental and social, based on this research to the economic analysis descriptive and quantitative to estimate and explain the phenomena related to the subject matter on the basis of measurements studies feasibility and evaluation of economic projects, and noted the results of research to the return of the production of fingerlings of about 1.02, 1.36, 2.635, million pounds in cases production current expectancy and full capacity respectively. The yield indirect has been estimated at about 46.28, 61.7, 1119.55, one million pounds for the cases of three respectively were estimated payback period for the project in case of return of direct 45, 18.5 4, the year of the three cases respectively while the estimated internal rate of return of 2% , 5%, 25% of the cases of three The yield indirect has increase than 100% and recommended that the results of research that denominated biological led to raising the efficiency of use of water resources, increase production fish, increase the agricultural area, to increase agricultural production, reduce water loss 0.71 at about 1.033 billion cubic meter annually as equivalent to 1.9% of Egypt's share of the Nile River where led to provide the amount of irrigation water in the light of these results, the study recommends the need to expand the dynamic resistance of aquatic weeds, especially fish farming

**Keywords**: the internal rate of return for the project (IRR),the percentage of benefits tocosts (B / C Ratio), Net present value( NPV), payback period(PBP). Water losses by evaporation transpiration (E.T):

Aquatic weeds are considered one of the most dangerous problems that weaken the efficiency of irrigation and drainage networks because of the resulting obstruction of the flow of water and it does not reach to the ends of waterways in addition to the loss of large amounts of water by evaporation transpiration. The more spread aquatic weeds, the more snails spread disease-causing deadly epidemic of health are common in many developing countries.The issue of environment protection and preservation of various types of pollution is considered one of the most important contemporary issues facing both the developed and developing countries alike. Therefore, it was necessary to look for better ways to weed and maintain the environment at the same time. It had

**Environmental and Economic Evaluation for** 

**the Breeding of Grass Carp in Egypt's Water** 

<sup>1</sup> Department of Agricultural Economics, Tanta University, Tanta, Egypt

**Channels**

**Abstract**

Abdelbaky Elshaib<sup>1</sup>

Congratulations and grasses.

**1. Introduction**

Ahmed Mohammed Ahmed<sup>1</sup>

<sup>2</sup>National Water Research Center, Cairo, Egypt

© 2012 Ahmed et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

provided the original work is properly cited.

Turkey, September 10-12, 2012

<sup>357</sup> ISALS


## <sup>357</sup> ISALS
