**Contribution to the Study of the Impact of Phosphate Fertilizer on Biochemical Parameters of** *Triticum durum*

Sabrina Bouchelaghem\* , Djebar M. R

Laboratory of Toxicology and cellular bioenergetics, University of Annaba, Algeria \*Corresponding author, Email: Sabrina\_bouchelaghem@yahoo.fr

#### **Abstract**

In this study we are interested in assessing the impact of different regimes of NPK and its effect on wheat Triticum durum. The first results show that the presence of fertilizer causes a decrease in the percentage and speed of germination and an inhibition of growth of wheat. On the metabolic level, the NPK caused a significant increase in mean levels of proline and soluble sugars, and inhibition of protein synthesis.

**Keywords**: NPK, Proline, Protein, Soluble sugar, *Triticum durum*.

#### **1. Introduction**

Adequate fertilization is a prerequisite for modern agriculture in order to meet high yields and optimum quality of crops. The vast majority of mineral elements essential to plant development, is only necessary in minute amounts, which may be provided by most soils without supplements. However, some key elements that are phosphorus (P), potassium (K), sulfur (S) and nitrogen (N), are frequently in short supply in the soil to allow optimal growth of crops. Despite its abundance, atmospheric nitrogen is unusable for most plants. The only plants able to use nitrogen from the air are those that develop a symbiotic relationship with microorganisms in nodules on plant roots. Unlike phosphorus and potassium, plants need large amounts of nitrogen, nitrogen representing 3-4% of their dry matter. Moreover the efficiency of nitrogen fertilization on crop yields contributed to the development of modern agriculture more intensive nitrogen. In addition to heavy loads caused by this item of expenditure, the massive inflow of nitrogen fertilizers started in the middle of last century affects the natural nitrogen cycle, which is not without consequences for the environment and health.

The objective of this study is to evaluate the effects of NPK fertilizer nitrogen on physiological parameters, biochemical and metabolic ones on wheat *Triticum durum*.

## **2. Materials and methods**

#### **2.1. Biological material**

The experimental material used in our work is wheat (Triticum durum) Geta come from hard JTGC (demonstration farm and seed production of Guelma).

© 2012 Bouchelaghem 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.

#### **2.2. Determination of soluble sugars:**

The total soluble sugars were determined by the method of Burnett and Schield [2],

#### **2.3. Determination of proline.**

Proline is determined by the method of Monnoveux and Nemmar, [3].

#### **3. Results**

#### **3.1. Effects of NPK on the physiological parameters of germination and growth**

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The results of the eff ects of treatment with NPK on the mean levels of proline are shown in Figure 3.

In this section we determined the eff ects of NPK on germination percentage, there is a slight decrease in germination percentage especially in the plan (0.5:0.4:0.1) (20%). Our result is in perfect agreement with the work of **[4]** (Savoie and Smith, 1997) which showed that germination was

This disturbance observed in wheat seeds is due to the eff ects of substances contained in the manure on the outbreak of various enzyme activities originally starting the germination process.

In pea *Pisum sativum*, the nature reserve is mainly protein, up to 40% **[7]**. Firstly, it has been shown in *Medicago truncatula* (another legume) that a massive fl ow of nitrogen is released into the seed during germination. The high concentrations of nitrogen (as ammonium free) are toxic to cells that

Indeed, in a second step, it is also proposed that metabolism of amino acids is very active during germination. They would not only protein synthesis, but also provide nutrients for the growth of

The determination of proline stems and roots exposed to NPK shows a strong increase of proline, this could explain as stress response. This ability of plants to the synthesis and accumulation of proline is not specifi c only to wheat, it is also for many glycophytes, in pea **[8]**, the barley Hor-

The results we obtained for the mean levels of sugars showed increasing rates for wheat. This could be due to osmotic stress in response to treatment with NPK. **[12]**. Many studies confi rm the accumulation of high levels of soluble sugars in diff erent types of plants under stress condi-

need to be cleared. The nitrogen is assimilated to form amino acids: this is detoxifi cation.

*3.2.2. Effect of NPK on the average content of proline:*

**Figure 3.** Eff ect of NPK on the average rate of proline roots from wheat seeds.

The work of other authors **[5, 6]** as support the fi ndings in our work.

new tissue and the synthesis of sugars in the seed.

deum vulgare L. **[9]**, beans **[10]** and Nicotiana tabacum **[11]**.

tions: water **[13, 14]**. ; saline **[15, 16]**, osmotic **[17]** and metal **[18]**.

**4. Discussion and Conclusion**

reduced with increasing nitrogen.

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*3.1.1. Effects on the germination percentages.*

**Figure 1.** Eff ects of diff erent regimes of NPK on the average rate of seed germination of wheat germination time = 96h. p = 0.031.

#### **3.2. NPK effects on biochemical parameters of germination.**

#### *3.2.1. Effect of NPK on the average content of sugar*

The results of treatment eff ects by the various regimes of NPK on average contents of total sugars in roots and leaves of wheat seeds are shown in Figures 2.

#### **Figure 2**

#### *3.2.2. Effect of NPK on the average content of proline:*

The results of the eff ects of treatment with NPK on the mean levels of proline are shown in Figure 3.

**Figure 3.** Eff ect of NPK on the average rate of proline roots from wheat seeds.

#### **4. Discussion and Conclusion**

In this section we determined the eff ects of NPK on germination percentage, there is a slight decrease in germination percentage especially in the plan (0.5:0.4:0.1) (20%). Our result is in perfect agreement with the work of **[4]** (Savoie and Smith, 1997) which showed that germination was reduced with increasing nitrogen.

This disturbance observed in wheat seeds is due to the eff ects of substances contained in the manure on the outbreak of various enzyme activities originally starting the germination process. The work of other authors **[5, 6]** as support the fi ndings in our work.

In pea *Pisum sativum*, the nature reserve is mainly protein, up to 40% **[7]**. Firstly, it has been shown in *Medicago truncatula* (another legume) that a massive fl ow of nitrogen is released into the seed during germination. The high concentrations of nitrogen (as ammonium free) are toxic to cells that need to be cleared. The nitrogen is assimilated to form amino acids: this is detoxifi cation.

Indeed, in a second step, it is also proposed that metabolism of amino acids is very active during germination. They would not only protein synthesis, but also provide nutrients for the growth of new tissue and the synthesis of sugars in the seed.

The determination of proline stems and roots exposed to NPK shows a strong increase of proline, this could explain as stress response. This ability of plants to the synthesis and accumulation of proline is not specifi c only to wheat, it is also for many glycophytes, in pea **[8]**, the barley Hordeum vulgare L. **[9]**, beans **[10]** and Nicotiana tabacum **[11]**.

The results we obtained for the mean levels of sugars showed increasing rates for wheat. This could be due to osmotic stress in response to treatment with NPK. **[12]**. Many studies confi rm the accumulation of high levels of soluble sugars in diff erent types of plants under stress conditions: water **[13, 14]**. ; saline **[15, 16]**, osmotic **[17]** and metal **[18]**.

#### **5. References**

[1] Kaur J. C. Duffu . 1989. The effect of naf on cereal seed germination and seeding growth. Plant Cell and environnement. 12:154-161.

International Conference on Applied Life Sciences (ICALS2012)

**Frost Occurrence Risk Management for** 

, Reza Hosseini<sup>2</sup>

, Mohammad-Saber Fallah Nezhad<sup>1</sup>

This work develops a statistical model to assess the frost risk in Rafsanjan, one of the largest Pistachioproduction regions in the world. These models can be used to estimate the probability that: a frosthappens in a given time-period in the year; a frost happens after 10 warm days in the growing season etc.These probability estimates then can be used for: (1) assessing the agroclimate risk of investing in thisindustry; (2) pricing of weather derivatives. Autoregressive models with different seasonal componentsand lags are compared using AIC, BIC, AICc and cross validation criterions. The optimal model is AR(1)with 12 terms from Fourier series. The long-term trends are also accounted for and estimated from data.The optimal models are then used to simulate future weather from which the probabilities of appropriatehazard events for pistachio yield are

**Keywords:** Pistachio; Frost; Weather derivative; Minimum temperature; Time-varying autore-

The greater Rafsanjan area in north of Kerman Province in Iran is a region with the largest pistachioproduction in the world and most of the region's economy relies on pistachio production. In therecent years the most important risk factor for pistachio producers and industry (e.g. farmers,distributers) has been frosts that have destroyed a large proportion of the yield. Therefore methodsthat can estimate the probability of such events is useful. In particular such methods can: (1) assess the agroclimate risk of investing in this industry; (2) pricing of weather derivatives. In factweather derivatives, which may be created as part of a risk management program, can be writtenin terms of the attainment or non-attainment of specific target-values stipulated in the contract.Temperature-related trades account for 80% of the transactions among all weather derivatives [1]. Most of the work in this area has focused on HDD/CDD (heating degreedays/cooling degree days) (e.g. [2,3]). In this paperwe focus on the occurrence of frosts an issue recently

The models developed in this paper can be applied to estimate: the probability that a given period is frost-free; the probability that a given day is the start of a long frost-free period;the distribution of the length of the frost-free period and so on. The same model can be used tocompute the

**Pistachio Industry in Rafsanjan**

1 Department of industrial engineering, Yazd University, Iran 2 Division of Biostatistics, University of Southern California, USA

Alireza Hosseini<sup>1</sup>

**Abstract**

estimated.

gressivecoefficients.

**1. Introduction**

Yahya Zare Mehrjardi<sup>1</sup>

© 2012 Hosseini 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.

considered in [4], foragricultural crops in Canada.

Turkey, September 10-12, 2012

,

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## **Frost Occurrence Risk Management for Pistachio Industry in Rafsanjan**

Alireza Hosseini<sup>1</sup> , Mohammad-Saber Fallah Nezhad<sup>1</sup> , Yahya Zare Mehrjardi<sup>1</sup> , Reza Hosseini<sup>2</sup>

1 Department of industrial engineering, Yazd University, Iran

2 Division of Biostatistics, University of Southern California, USA

#### **Abstract**

This work develops a statistical model to assess the frost risk in Rafsanjan, one of the largest Pistachioproduction regions in the world. These models can be used to estimate the probability that: a frosthappens in a given time-period in the year; a frost happens after 10 warm days in the growing season etc.These probability estimates then can be used for: (1) assessing the agroclimate risk of investing in thisindustry; (2) pricing of weather derivatives. Autoregressive models with different seasonal componentsand lags are compared using AIC, BIC, AICc and cross validation criterions. The optimal model is AR(1)with 12 terms from Fourier series. The long-term trends are also accounted for and estimated from data.The optimal models are then used to simulate future weather from which the probabilities of appropriatehazard events for pistachio yield are estimated.

**Keywords:** Pistachio; Frost; Weather derivative; Minimum temperature; Time-varying autoregressivecoefficients.

#### **1. Introduction**

The greater Rafsanjan area in north of Kerman Province in Iran is a region with the largest pistachioproduction in the world and most of the region's economy relies on pistachio production. In therecent years the most important risk factor for pistachio producers and industry (e.g. farmers,distributers) has been frosts that have destroyed a large proportion of the yield. Therefore methodsthat can estimate the probability of such events is useful. In particular such methods can: (1) assess the agroclimate risk of investing in this industry; (2) pricing of weather derivatives. In factweather derivatives, which may be created as part of a risk management program, can be writtenin terms of the attainment or non-attainment of specific target-values stipulated in the contract.Temperature-related trades account for 80% of the transactions among all weather derivatives [1]. Most of the work in this area has focused on HDD/CDD (heating degreedays/cooling degree days) (e.g. [2,3]). In this paperwe focus on the occurrence of frosts an issue recently considered in [4], foragricultural crops in Canada.

The models developed in this paper can be applied to estimate: the probability that a given period is frost-free; the probability that a given day is the start of a long frost-free period;the distribution of the length of the frost-free period and so on. The same model can be used tocompute the

© 2012 Hosseini 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.

probability that a given day of the year is the beginning of the growing season (the first day that the mean temperature is higher than 5 degrees for 5 consequent days) as well as thelength of the growing season which are important for agricultural applications. For example in thisstudy we estimate the probability of a useful event: "*the minimum temperature goes below zero atleast one day in the period March 27th-April 20th*". This is an important event because it coincideswith the general flowering time of pistachio trees. Throughout this paper, temperature is measuredin degrees Celsius. Let us denote the minimum temperature series by {Y (t)}, t = 0,1,2,…, wheret denotes time. We let F to be the investor's defined frost which we take it to be zero in this work.Then we can define the binary frost process:

International Conference on Applied Life Sciences (ICALS2012)

The estimation of the parameters is done by maximizing the (partial) likelihood of the data asdiscussed in [4]. The nice property of the gaussian error assumption is themaximization can be done in closed form exactly in the same way as minimizing the mean-squareerror of classical regression problem originally solved by Gauss and therefore the estimation is fastin statistical packages

In the above we introduced several autoregressive models of: (1) various lags; (2) various seasonal complexity (number of Fourier terms); (3) various long-term trends. Therefore we need to usesome criteria to select an optimal model. The problem of model selection is an important onein statistical theory and application. Various criteria are suggested in the literature for example:AIC in [5]; BIC in [6] and AICc in [7]. Denote thelikelihood of the data by L (in this paper the "partial

Since n in our data is large compared to k, AIC and AICc are very close. When we compared themodels using these criteria, AIC and AICc give rise to the same optimal model while BIC pickeda simpler model. In Table 1 we have compared these optimal models using cross-validation errorand cross-validated correlation. The cross-validation proceeds by: (1) taking an existing data pointout; (2) fitting the model; (3) predicting the value of the point we took out (validation). Then thecross-validation error (CVE) is the mean square error of the predictions and the crossvalidationcorrelation (CVR) is the correlation between the predictions and the observed. Table 1 shows thatwhile the CVE and CVR are very close for the two models, the model picked by AIC/ AICc slightlyoutperforms the one picked by BIC and therefore we use that model for estimation.

**Tab 1.** We compare the optimal model picked by AIC and AICc (first row) with the optimal model picked by

Previous section found an optimal fit to the data from which estimating the probability of anydesired (possibly complex) event is possible by performing multiple simulations. In order to find out the probability of frost in any given day during 2011-2012 we have done 10000 simulationsfrom the model for 2011-2011 and then for each day we have calculated the proportion of frostdays (number of frost days divided by 10000). The results are plotted in Figure 1.As we pointed

BIC, (second row) using cross validation error and cross-validated correlation.

**4. Applications in frost risk assessment**

such as "R"(a free widely used software by statisticians and practitioners).

likelihood"), the number of covariates by pand the sample size by n. Then we have

**3. Statistical model selection**

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<sup>295</sup> ISALS

$$Y\_F(t) = \begin{cases} 1 & Y(t) \le F \text{ (deg C)}\\ F & Y(t) > F \text{ (deg C)}. \end{cases}$$

In order to study frosts we can use these approaches among others: (a) Fit the continuous-valued Markov model to the Y (t) chain; (b) Fit a binary Markov model to the YF (t) chain. Hosseinisuggests using binary Markov models to avoid assumptions regarding the distribution of temperature and gain robustness for modeling frosts in Alberta, Canada[4]. They show time-varyinghigh-order Markov models with complex seasonal structure are needed and therefore their computations become challenging. Here we investigate Method (a) in fitting such chains and calculatingthe probabilities of frost events. The advantages of Method (a) are: (1) Thefitting can be donewith standard packages such as R with less computational problems; (2) only this method canestimate the probability of complicated events. One such complicated event is: *"the temperaturein March-April is above 5 (deg C) for at least 3 consecutive days and is below zero after".* A comparison of the two methods in terms of estimation when they are both applicable is left to futureresearch.

#### **2. Data and statistical models**

The data in this study are daily minimum temperature values collected at Rafsanjan weather station from 1992 to 2010. At the moment we do not have access to more data from other stationsin the area but we hope to acquire those data for future studies to offer more local predictions. Inorder to model frost occurrences, we introduce statistical models for minimum daily temperaturein Rafsanjan. Several features of the temperature process should be considered in modeling: (1) seasonal trends over time; (2) long-term trends (possibly a result of global warming or volcanicevents etc) (3) dependence over time. Let {Y (t)}, t = 0,1,2,…, T denote the daily minimumtemperature process in centigrade, where t denotes the day starting from March 1st 1992 toDecember 28th 2010. Here we consider autoregressive models with a seasonal component andvarious lags:

$$Y(t) = \mu(t) + \epsilon(t), \quad \mu(t) = a\_0(t) + \sum\_{i=1}^{r} a\_i Y(t-i)$$

Where µ(t)=E{Y(t)|Y(t-1),Y(t-2),…} is the conditional mean of minimum temperatureat time t; ε(t) are independent identically distributed normal errors ε(t) ~ N(0,δ2 ); a0 (t) is thefixed trend coefficient; a1 , a2 ,…,a<sup>r</sup> are autoregressive coefficients. We allow a0 (t) to include bothseasonal and long-term effects by using a Fourier series with period, , and a quadratictrend:

$$a\_0(t) = \{\alpha\_0 + \sum\_{j=1}^k \alpha\_j \cos(j\omega t) + \beta\_j \sin(j\omega t)\} \quad + \quad \{\gamma\_1 t + \gamma\_2 t^2\},$$

The estimation of the parameters is done by maximizing the (partial) likelihood of the data asdiscussed in [4]. The nice property of the gaussian error assumption is themaximization can be done in closed form exactly in the same way as minimizing the mean-squareerror of classical regression problem originally solved by Gauss and therefore the estimation is fastin statistical packages such as "R"(a free widely used software by statisticians and practitioners).

### **3. Statistical model selection**

In the above we introduced several autoregressive models of: (1) various lags; (2) various seasonal complexity (number of Fourier terms); (3) various long-term trends. Therefore we need to usesome criteria to select an optimal model. The problem of model selection is an important onein statistical theory and application. Various criteria are suggested in the literature for example:AIC in [5]; BIC in [6] and AICc in [7]. Denote thelikelihood of the data by L (in this paper the "partial likelihood"), the number of covariates by pand the sample size by n. Then we have

$$AIC = 2p - 2\ln(L),\ \ AICc = AIC + \frac{2k(k+1)}{n-k-1},\ \ BIC = p\log(n) - 2\ln(L).$$

Since n in our data is large compared to k, AIC and AICc are very close. When we compared themodels using these criteria, AIC and AICc give rise to the same optimal model while BIC pickeda simpler model. In Table 1 we have compared these optimal models using cross-validation errorand cross-validated correlation. The cross-validation proceeds by: (1) taking an existing data pointout; (2) fitting the model; (3) predicting the value of the point we took out (validation). Then thecross-validation error (CVE) is the mean square error of the predictions and the crossvalidationcorrelation (CVR) is the correlation between the predictions and the observed. Table 1 shows thatwhile the CVE and CVR are very close for the two models, the model picked by AIC/ AICc slightlyoutperforms the one picked by BIC and therefore we use that model for estimation.


**Tab 1.** We compare the optimal model picked by AIC and AICc (first row) with the optimal model picked by BIC, (second row) using cross validation error and cross-validated correlation.

#### **4. Applications in frost risk assessment**

Previous section found an optimal fit to the data from which estimating the probability of anydesired (possibly complex) event is possible by performing multiple simulations. In order to find out the probability of frost in any given day during 2011-2012 we have done 10000 simulationsfrom the model for 2011-2011 and then for each day we have calculated the proportion of frostdays (number of frost days divided by 10000). The results are plotted in Figure 1.As we pointed out in the introduction because the flowering time of different varieties ofpistachios in Rafsanjan is generally between March 27th to April 20th, it is important to investigate the frost-occurrence during this period which we call the hazard period. Figure 2 shows thedistribution of the "number of frost days" during the hazard period of 2012, where the frequencyout of 10000 of any "number of frost days" is plotted. We observe that while it is most likely thatno frost occurs in that period, there is a considerable probability that there are at least one frosts.This probability turns out to be about 9 percent which is a plausible number with our experience of pistachio damages caused by frosts in the past 20 years.

International Conference on Applied Life Sciences (ICALS2012)

This paper developed and compared several statistical models to estimate the probability of hazardfrost events for pistachio industry in Rafsanjan. Despite the importance of such risk factors, nosystematic studies and estimations of these risks are available in this region as far as we know;this paper is one of the first attempts in developing methods that can assess such risks. Assessingthe probabilities of the hazard events are useful in estimating the risk of investing in this industryfrom production to distribution and exporting. However here we have not investigated other riskfactors such as: extremely high temperature during summer; heavy short-time rain during floweringperiod; slow but long rain during the flowering time. For future studies we plan to acquire thedata for precipitation, maximum temperature and developing models that assess

Another important aspect of assessing the risk is relating the risk factors to the losses inyield or monetary values involved. For this study we relied on expert knowledge (by interviewingfarmers and agriculture engineers) to define our hazard period. However if for example data foryield per km2 becomes available for enough number of years and/or locations, one can develop astatis-

[1] Cao, M. and Wei, J. Weather derivatives, valuation and market price of weather risk The Journal

[2] Richards, T. J., Manfredo, M. R. and Sanders, D. R. Pricing Weather Derivatives American Journal

[3] Benth, F. E., and Benth, J. S. Volatility of temperature and pricing of weather derivatives

[4] Hosseini.R, Le.N, Zidek.J,2012. Time-Varying Markov Models for Binary Temperature Series in Agrorisk Management. Journal of Agricultural Biological and Ecological Statistics, Vol. 17,Num.

[5] Kedem, B. and Fokianos, K. Regression Models for Time Series Analysis, Wiley Series in

[6] [Akaike, H. A new look at the statistical model identi\_cation. IEEE Transactions on

[7] Schwartz, G. Estimating the dimension of a model. Annals of Statistics, 6:461-464, 1978.

[8] Brockwell, P.J., and Davis, R.A. Time Series: Theory and Methods, 2nd edition Springer,2009.

tical model to relate the weather events to the losses in the yield in the same model.

We are indebted to Mr. islami for providing the data for this study.

of Future Markets, 24(11):1065-1089, 2004.

Quantitative Finance, 7(5):553-561, 2007.

AutomaticControl, AC- 19:716-723, 1974.

Probability and Statistics, 2002.

of Agricultural Economics, 86(4):1005-1017, 2004.

**5. Summary and conclusion**

these other riskfactors.

**6. Acknowledgements**

**7. References**

2,283-305.

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**Fig 1.** Estimated daily frost probability for 2011-2012 from the model, obtained using 1000 simulations of future weather.

**Fig 2.** Distribution of frost days during the Hazard period (March 27th to April 20th 2012). This is based on 10000 simulations of the future chains. The probability of at least one frost based on this simulations is 0.0872 which is about 9 percent.

## **5. Summary and conclusion**

This paper developed and compared several statistical models to estimate the probability of hazardfrost events for pistachio industry in Rafsanjan. Despite the importance of such risk factors, nosystematic studies and estimations of these risks are available in this region as far as we know;this paper is one of the first attempts in developing methods that can assess such risks. Assessingthe probabilities of the hazard events are useful in estimating the risk of investing in this industryfrom production to distribution and exporting. However here we have not investigated other riskfactors such as: extremely high temperature during summer; heavy short-time rain during floweringperiod; slow but long rain during the flowering time. For future studies we plan to acquire thedata for precipitation, maximum temperature and developing models that assess these other riskfactors.

Another important aspect of assessing the risk is relating the risk factors to the losses inyield or monetary values involved. For this study we relied on expert knowledge (by interviewingfarmers and agriculture engineers) to define our hazard period. However if for example data foryield per km2 becomes available for enough number of years and/or locations, one can develop astatistical model to relate the weather events to the losses in the yield in the same model.

## **6. Acknowledgements**

We are indebted to Mr. islami for providing the data for this study.

## **7. References**


International Conference on Applied Life Sciences (ICALS2012)

**Study on Pollen Germination and Pollen Tube** 

**Growth of Five Iranian Apricot Cultivars on in** 

This research was conducted as factorial experiment based on CRD in three replication with two factors including: cultivars in 5 levels and counting time after culture in three levels (10, 24 and 48 hours). The aims was evaluation of pollen germination and pollen tube growth. Therefore, pollen grain was collected from unopened flowers of Iran apricot cultivars including : jahangiri, azghandi, royal, shahrood 49, shahrood 18. Pollens was stored in desiccators and cultured in a medium including %15 sucrose, 100 ppm boric acid and %0.5 agar. The result was showed wide range of percentage of germination and pollen tube growth between cultivars. highest and least pollen tube length were observed in shahrood 49 (1287µ m) and royal ( 812 µ m). there was not any significant relationship between germination percentage and pollen tube growth. The range of germination was % 20-54. and 24 hours was enough for obtain maximum germination.

Apricot is one of important fruit at world. and is very important in Iran too. and this tree are capable for irregular yielding and is limit for ecological adaptation and can use for researchers. Irregular yielding has some reasons and can omit proper varieties. Self incompatibility is an important factor And reduce yielding which proceed from lack of compatible pollen. (11, 24). Incompatibility report at apricot different varieties at all world (1,4,11,19). It is one of important big difficults. limiting at adaptation is other difficult at Apricot produce. every cultivar need to special condition (2, 9, 24 ). Apricot need to cold winter and warm summer (2, 15) ovule longevity, anther Reception, pollen tube growth in pistil, and temperature are important at apricot produce (3, 5, 8). pollen tube growth and germination speed is very important. at low speed, yielding reduce (8). Health in pollen is important too (13). For successful Zygosis, 20- 30 pollen is necessary (10). in vitro condition is best ways for this studies. for this experiment, pollen collection from flower anther Flower must be at the balloon stage. in turkey cultivars, sucrose reports: 10-15 % (18). At Euguslavia varieties reports :agar 1%, sucrose 15%, germination rate 14.9-88.7%. (6). and in armenia varieties report 54.9 – 72.65 % (11). At some Iranian varieties germination percentage reported 33.2 – 51.42 % (12). At different varieties need to different temperature (17, 23 ). low and high temperature is Harmful for germination (8). proper temperature for pollen germination in apricot Suggest 10 – 20 (8, 20). Hajilo suggested temperature 15 -20 for pollen Germination in

**Keywords:** apricot, iran cultivars, pollen germination, pollen tube growth, in vitro

**Vitro Condition**

Islamic Azad University, Bam Branch, Iran

Reza Kamrani

**1. Introduction**

**Abstract**

© 2012 Kamrani; 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.

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## **Study on Pollen Germination and Pollen Tube Growth of Five Iranian Apricot Cultivars on in Vitro Condition**

Reza Kamrani

Islamic Azad University, Bam Branch, Iran

#### **Abstract**

This research was conducted as factorial experiment based on CRD in three replication with two factors including: cultivars in 5 levels and counting time after culture in three levels (10, 24 and 48 hours). The aims was evaluation of pollen germination and pollen tube growth. Therefore, pollen grain was collected from unopened flowers of Iran apricot cultivars including : jahangiri, azghandi, royal, shahrood 49, shahrood 18. Pollens was stored in desiccators and cultured in a medium including %15 sucrose, 100 ppm boric acid and %0.5 agar. The result was showed wide range of percentage of germination and pollen tube growth between cultivars. highest and least pollen tube length were observed in shahrood 49 (1287µ m) and royal ( 812 µ m). there was not any significant relationship between germination percentage and pollen tube growth. The range of germination was % 20-54. and 24 hours was enough for obtain maximum germination.

**Keywords:** apricot, iran cultivars, pollen germination, pollen tube growth, in vitro

#### **1. Introduction**

Apricot is one of important fruit at world. and is very important in Iran too. and this tree are capable for irregular yielding and is limit for ecological adaptation and can use for researchers. Irregular yielding has some reasons and can omit proper varieties. Self incompatibility is an important factor And reduce yielding which proceed from lack of compatible pollen. (11, 24). Incompatibility report at apricot different varieties at all world (1,4,11,19). It is one of important big difficults. limiting at adaptation is other difficult at Apricot produce. every cultivar need to special condition (2, 9, 24 ). Apricot need to cold winter and warm summer (2, 15) ovule longevity, anther Reception, pollen tube growth in pistil, and temperature are important at apricot produce (3, 5, 8). pollen tube growth and germination speed is very important. at low speed, yielding reduce (8). Health in pollen is important too (13). For successful Zygosis, 20- 30 pollen is necessary (10). in vitro condition is best ways for this studies. for this experiment, pollen collection from flower anther Flower must be at the balloon stage. in turkey cultivars, sucrose reports: 10-15 % (18). At Euguslavia varieties reports :agar 1%, sucrose 15%, germination rate 14.9-88.7%. (6). and in armenia varieties report 54.9 – 72.65 % (11). At some Iranian varieties germination percentage reported 33.2 – 51.42 % (12). At different varieties need to different temperature (17, 23 ). low and high temperature is Harmful for germination (8). proper temperature for pollen germination in apricot Suggest 10 – 20 (8, 20). Hajilo suggested temperature 15 -20 for pollen Germination in

© 2012 Kamrani; 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.

apricot (12). Negation suggested sucrose 15 % agar 0.6 %and boric Acid 100 ppm (21). In other experiment use agar 1 – 6 %. Sucrose 10 – 20 %, Boric acid 0.1 % and temperature 15 – 20 %centigrade degree was considered. After (10 -24 and 48 )hours measure germination percent and measure pollen tube Length after 48 hour. (8, 22, 25).

International Conference on Applied Life Sciences (ICALS2012)

**S.V DF MS**

Variety 4 59583.43

Error 8 11911.081

According to table :shortest pollen tube length belong to "royal" And longest pollen tube length belong to "shahrood 49" and at this experiment Pollen tube length was 812 – 1287 micro millimeter(18). Iran apricots are different with each other and can use at plant breeding and have incompatibility. yielding at incompatibility varieties need to pollination by a proper variety (21). suggestion this experiment carry out by other varieties such as turkey cultivars and other place. that's on aportunity to thanks dr. bouzary and dr. abdolbaghi and mr. tavoosi that they were my

[2] Bahly. G. H. and L. F. Hough. apricots.i n:j.janic and j.n.moore.advencesin fruit breeding. purdue

[3] Burgos. L. and J. Egea. apricot embryo-sac development in relation to fruit set. J.Hort.sci. 1993 [4] Burgos. L. T. Berenguer and j.Egea.self and cross compatibility among apricot cultivars.

[5] Burgos. L. T. Berenguer and j.Egea.embryo sac development in pollination and non pollination

[8] Egea,J.,burgos,L.Zoroa,N.andEgea,L..influences of temperature on the invitro germination of

[11] Guleryuz,M.and I.Bolat.investigation on characteristics of apricot cultivars in Erzincan urkey.

[12] Hajillo, j. and gerigourian.the effect of different temperature on apricot pollen germination.

[13] Javadi, t and arzani, k,the effect of protection duration on iranian olive pollen in vitro germination.

[9] Faust,M.physiology of temperate zon fruit trees.john wiley and sons,new York.USA. 1989 [10] Gerriero, R and Bartolini,s.flower biology in apricot :main aspects and problems. 1993

[6] Duric, B.pollen germination in some apricot varieties in vojvodina.CAB abstract 1991 [7] Egea, j. andL.Burgos.apricot breeding for quality and self compatibility. acta.hort.. 1999

Total 14

[1] Anonymous. fao state database results.available on the www. fao. org. 2001

university press.west lefa yet Uindiana. V.S.A. 1979

flowers of two apricot cultivars. J. Hortscience. 1995

pollen of apricot. horticultural science. 1992

Iranian scientistics and technology journal. (2000)

permanent guide in doing this reaserch.

**Table 2.** 

**4. References** 

hortscience. 1993

acta horticulture.1999

seed and seedling journal. (1999)

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### **2. Material and methods**

This research was done at seed and plant improvement research institute of karaj in iran. All of cultivars are Iran native. Trees were six years old. The Root stocks of this trees are wild apricot**.** Temperature average in this city is 13.8 Centigrade degree. The Maximum of annual temperature is 26 centigrade degree on july and the minimum of annual temperature is -12 centigrade degree on anuary. The highest Temperature was 42 centigrade degree from thirty years old and The lowest temperature was -12 centigrade degree from thirty years old. Annual rain average is 260 illimeters. The lowest annual rain is 100 millimeters On april. This research was conducted as factorial experiment based on CRD in three replication with two factors including : cultivars in 5 levels including : jahangiri, azghandi, royal, shahrood 49, shahrood 18. and counting time after culture in three levels (10, 24 and 48 hours). Data analysis with "mstatc". For this research in 5 Iran cultivars, collect pollen from flower anther and Put at Desicator and protect in refrigerator at 3-5 centigrade degree. For Pollen culture use Sterile Petridishes. Medium was included sucrose 15 %. agar 0.5 % acid boric 100 pm. and then pollens scatter on cultivation field And put in incubator at 20 centigrade degree and normal light. for estimation of pollen germination percentage at three time (10, 24, and 48) hours counted. Then get out petridishes from incubator and add some drops chloroform that Determined germination (8). at end by a microscope that connect to computer Take 15 photo from different view. and save in computer. at any view counted Total pollens and total germinated pollens that pollen tube length was equation or longer of pollen thickness. (21, 16). 45 counting use for estimate germination speed and percentage. pollens distributed low numbers in the same ways. High amount of pollen causes germination (14) and petridishes with pollen above 50 discount.

#### **3. Summary and conclusion**


Table of data analysis shows difference between apricot varieties. Germination percentage average at 5 apricot Varieties was 20 – 54 %. Pollen tube growth is different with each other.


#### **Table 2.**

According to table :shortest pollen tube length belong to "royal" And longest pollen tube length belong to "shahrood 49" and at this experiment Pollen tube length was 812 – 1287 micro millimeter(18). Iran apricots are different with each other and can use at plant breeding and have incompatibility. yielding at incompatibility varieties need to pollination by a proper variety (21). suggestion this experiment carry out by other varieties such as turkey cultivars and other place. that's on aportunity to thanks dr. bouzary and dr. abdolbaghi and mr. tavoosi that they were my permanent guide in doing this reaserch.

#### **4. References**


[14] Kwack,b.h..the effect of calcium on pollen germination. Proceeding of the American society of horticulture science. 1965

International Conference on Applied Life Sciences (ICALS2012)

**Influence of Congruent-Incongruent Teaching** 

**and Learning Style on Agricultural and Natural** 

Department of Agricultural Economics, Faculty of Agriculture, Kurdistan University, Sanandaj,

Based on Kolb's theory of learning and teaching a Post-test Group Design was applied to investigate the effect of congruent/ incongruent teaching and learning styles on students' performance at the faculties of Agriculture and Natural Resources at Kurdistan University, Iran in 2007. Using SPSS software descriptive statistics, descriptive statistics and T- test was employed to analyze the data. Results showed that: 1- the dominant learning styles among male and female students are Accommodator and Assimilator, respectively; 2- the main teaching style among teachers is Assimilator which corresponds to female students learning style; 3- Congruency between teachers' learning style and students' learning style has a positive significant effect on students' performance. The result could be used to improve the quality of teaching and learning at the faculties of Agriculture and Natural Resources at

**Keywords:** Higher education in agriculture, Learning-style, Teaching-style, Experiential learn-

An extensive amount of literature is available pertaining to learning styles and teaching styles and whether the match or mismatch between these two affects student performance. Of those studies which measured the effects of teaching approach on student achievement, mixed results have been reported. While a paucity of research exists in education which identifies a significant relationship between a teacher's teaching style and a student learning style and student performance [See for example 19, 16, 4, 10, 9, 3, 2 &8], there is some

Although the influence of match and mismatch between teaching and learning style on student performance has received deep consideration in different countries and different parts of higher education system, the situation in Iran especially in Higher Agricultural Education System is not satisfying. Literature shows that most of the teachers in Iranian Higher Agricultural Education System (IHAES) pay less attention, if any, to their student learning style when defining their strategies to teaching. This is while most research shows a positive

**Resources Student Performance**

Farzad Eskandari

the University of Kurdistan, Iran.

ing, Student performance.

**1. Introduction**

Iran

**Abstract**

© 2012 Eskandari; 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.

other that rejects this hypothesis [See for example, 7 & 18].

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## **Influence of Congruent-Incongruent Teaching and Learning Style on Agricultural and Natural Resources Student Performance**

## Farzad Eskandari

Department of Agricultural Economics, Faculty of Agriculture, Kurdistan University, Sanandaj, Iran

#### **Abstract**

Based on Kolb's theory of learning and teaching a Post-test Group Design was applied to investigate the effect of congruent/ incongruent teaching and learning styles on students' performance at the faculties of Agriculture and Natural Resources at Kurdistan University, Iran in 2007. Using SPSS software descriptive statistics, descriptive statistics and T- test was employed to analyze the data. Results showed that: 1- the dominant learning styles among male and female students are Accommodator and Assimilator, respectively; 2- the main teaching style among teachers is Assimilator which corresponds to female students learning style; 3- Congruency between teachers' learning style and students' learning style has a positive significant effect on students' performance. The result could be used to improve the quality of teaching and learning at the faculties of Agriculture and Natural Resources at the University of Kurdistan, Iran.

**Keywords:** Higher education in agriculture, Learning-style, Teaching-style, Experiential learning, Student performance.

#### **1. Introduction**

An extensive amount of literature is available pertaining to learning styles and teaching styles and whether the match or mismatch between these two affects student performance. Of those studies which measured the effects of teaching approach on student achievement, mixed results have been reported. While a paucity of research exists in education which identifies a significant relationship between a teacher's teaching style and a student learning style and student performance [See for example 19, 16, 4, 10, 9, 3, 2 &8], there is some other that rejects this hypothesis [See for example, 7 & 18].

Although the influence of match and mismatch between teaching and learning style on student performance has received deep consideration in different countries and different parts of higher education system, the situation in Iran especially in Higher Agricultural Education System is not satisfying. Literature shows that most of the teachers in Iranian Higher Agricultural Education System (IHAES) pay less attention, if any, to their student learning style when defining their strategies to teaching. This is while most research shows a positive

relationship between congruent teaching- learning styles and student achievement. Many IHAES teachers assume if their students do not show a satisfying performance on exams, it is because of variables related to students not because of quality and style of teaching.

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**variable frequency percentage**

Accommodator 105 35

Assimilator 80 26.7 Convergent 59 19.7 Divergent 56 18.6

Accommodator 70 51.1 Assimilator 18 13.1 Convergent 33 24.1 Divergent 16 11.7

Accommodator 35 21.5 Assimilator 62 38 Convergent 26 16 Divergent 40 24.5

According to the data in table 1, the dominant learning styles among male and female students are Accommodator and Assimilator, respectively. In other words, male students at the faculty of agriculture prefer to learn through real experience and by doing while females have a preference

> **Teaching style Frequency Percentage** Assimilator 9 39.1 Convergent 6 26.1 Divergent 6 26.1 Accommodator 2 8.7

As data in table 2 shows, the dominant teaching style among teachers is Assimilator (39.1 percent

of the participants) which is more congruent with learning styles of female students.

for learning through working on abstract ideas and theoretical issues. Profile of teaching style preferences of teachers is shown in table 2.

Learning style of the total sample

Learning style of male students

Learning style of female students

**Table 1.** Learning style of the sample

**Table 2.** Teaching style of teachers

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As with agricultural education the researcher could not find any practical research testing the relationship between learning- teaching style and student performance. Currently, research on relation between learning and teaching style and student performance in Iranian colleges of agriculture is very limited. If teachers in Iranian colleges of agriculture are to recognize and appreciate differences in students' learning style and meet the challenge set forth by Anderson and Adams [1], an expansion of this research area is essential.As a result, the purposes of this study were to identify the teaching styles of agricultural education instructors and the learning styles of their students in specific field areas, to determine if a match existed between the two, and to determine if relationships existed between student success and style match or mismatch.

Research questions studied were as follows:


## **2. Methods**

To achieve the purpose of the study a post-test group quasi-experimental design was employed. The subjects of the study were 23 agricultural teachers, 2000 students and 23 courses from faculty of agriculture at the University of Kurdistan, Iran.

All teachers at the faculty were studied. But with the student population, stratified and systematic sampling techniques were applied resulting in selecting 300 students from different areas of study at the faculty. For each teacher one course was selected.

The Kolb's Learning Styles Inventory (1999) and the Kolb's Teaching StylesInventory (1999) were used to obtain a profile of the teaching styles of the instructorsand the learning styles of their students.

Descriptive statistics and T-test were employed to analyze the data.

## **3. Result**

Data related to learning style of the student sample is presented in table 1.


**Table 1.** Learning style of the sample

According to the data in table 1, the dominant learning styles among male and female students are Accommodator and Assimilator, respectively. In other words, male students at the faculty of agriculture prefer to learn through real experience and by doing while females have a preference for learning through working on abstract ideas and theoretical issues.

Profile of teaching style preferences of teachers is shown in table 2.


#### **Table 2.** Teaching style of teachers

As data in table 2 shows, the dominant teaching style among teachers is Assimilator (39.1 percent of the participants) which is more congruent with learning styles of female students.


Descriptive statistics for congruent / incongruent teaching-learning style at the studied faculty is demonstrated in table 3.

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day's instructors with a new direction to take toward developing a more personalized form of

In this article we have discussed the significance of matching teaching and learning styles in IHAES and provided some empirical evidence to indicate that IHAES students exhibit distinctive learning style characteristics. To understand and respect individual's diverse learning styles, We suggest that teachers employ instruments to identify students' learning styles and provide diverse instructional strategies to address their differences, and that teachers plan lessons to match students' learning styles while at the same time encouraging students to diversify their learning style preferences. By doing this we can assist our students in becoming more effective learners Like other research this study had some limitations. This study was limited to only one faculty at one university in Kurdistan province, Iran. This study should bereplicated in other higher agricultural education institutions across the country with a larger population to gain a better picture of the relationship between congruent-incongruent learning and teaching style and student performance as well as to compare withthe findings of this study and previous research.

Also, this study did not focus on extraneous variables. A similar study should be conducted which incorporates student characteristics, social variables, socioeconomic levels, and gender to

[1] Anderson, J.A., & Adams, M. 1992. Acknowledging the learning styles of diverse student populations: Implications for instructional design. In L. L. Border & N. Van Note Chism (Eds.), New Directions for Teaching and Learning, (pp. 19-33). San Francisco: Jossey-Bass Publishers, Inc.

[2] Cafferty, J. 1980. Learning styles.Chicago, IL: National Association of Nursing Education. ERIC

[3] Campbell, B. W. 1989. A study of the relationship between teachers' and students' learning styles and students' achievement in business communications. Unpublished doctoral dissertation,

[4] Canfield, A. A., & Canfield, J. S. 1988. Canfield Instructional Styles Inventory (ISI) Manual. Los

[5] Cano, J. And Garton, B. L. 1994. The Relationship Between Agriculture PreserviceTeachers'Learning Styles and Performance in a Methods of Teaching Agriculture Course. Journal of Agricultural

[6] Carthey, J. H. 1993. Relationships between learning styles and academic achievement and brain hemispheric dominance and academic performance in business and accounting courses.

[7] Cupkie, L. 1980. The effects of similarity of instructor preferred teaching style and student preferred learning style on student achievement in selected courses in a metropolitan community

[8] Danile, A., Rasmussenm C., Jackson, J. and Brenner, D. 1984. Cognitive style as a predictor of

college. Unpublished doctoral dissertation, university of Missouri-Kansas city.

Unpublished master's thesis, Winona State University, Minnesota.

achievement: multivariate analysis. ERIC document, NO. ED248217.

determine if these variables significantly affect style match and student success.

instruction.

**5. References** 

document, Service number: ED 2495533.

University of Illinois at Urbana-Champaign.

Angeles, CA: Western Psychological Services.

Education. 35(2), PP. 6-10.

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**Table 3.** Descriptive statistics for congruent/ incongruent teaching-learning style

Table 3 shows that for most students (60.3%) the teaching style of the teacher does not match their learning style. In other words for almost two third of students teachers at the faculty do not consider the learning style of them when planning for teaching strategies. Table also shows that the congruent group has a higher average final exam score (15.70) compared to incongruent group. To test whether this difference in final exam score is accidental or because of the match and mismatch between teachers teaching style and student learning style, T-test was applied.

Result considering whether matching or mismatching between teaching style of teacher and learning style of student influences student performance is exhibited in table 4.


**Table 4.** T-test result for congruent / incongruent teaching-learning style and student performance

Based on the data exhibited in table 4, congruency between teachers' learning style and students' learning style has a positive significant effect on students' performance. So, one can conclude that the higher average final exam score gained by congruent student group is due to the match between teachers teaching styles and students learning styles.

## **4. Conclusions and Implications**

It can be concluded from this study that a positive relationship exists between congruent teaching- learning styles and student performance hence confirming the theory established by researchers that style match will produce higher performance by students as measured by final exam scores and course grades [14, 6, 11& 15]. The findings are congruent with research by Cano et al.[5]; Welborn, [19]; Cafferty, [2]; Daniel etal, [8]; Witkin et al.[20]; and Koppleman [12] that found a match between teacher's teaching style and student's learning style will cause in a more higher significant performance by learners.

Iranian agricultural educators can use the outcomes of this study to assess the importance that their teaching styles may have to the learning of their students. This study shows that student learning will be improved when the instructor's teaching style and the students' learning style match [13 &10]. Henson and Borthwick contended that "assessing learning styles provides today's instructors with a new direction to take toward developing a more personalized form of instruction.

In this article we have discussed the significance of matching teaching and learning styles in IHAES and provided some empirical evidence to indicate that IHAES students exhibit distinctive learning style characteristics. To understand and respect individual's diverse learning styles, We suggest that teachers employ instruments to identify students' learning styles and provide diverse instructional strategies to address their differences, and that teachers plan lessons to match students' learning styles while at the same time encouraging students to diversify their learning style preferences. By doing this we can assist our students in becoming more effective learners

Like other research this study had some limitations. This study was limited to only one faculty at one university in Kurdistan province, Iran. This study should bereplicated in other higher agricultural education institutions across the country with a larger population to gain a better picture of the relationship between congruent-incongruent learning and teaching style and student performance as well as to compare withthe findings of this study and previous research.

Also, this study did not focus on extraneous variables. A similar study should be conducted which incorporates student characteristics, social variables, socioeconomic levels, and gender to determine if these variables significantly affect style match and student success.

#### **5. References**


[9] Dyer, J. E. and Osborne, E.W. 1996. Effects of teaching approach on achievement of agricultural education students with varying learning style. Journal of Agricultural Education, 37(4), PP. 38-45.

International Conference on Applied Life Sciences (ICALS2012)

*Tagetes erectus* **– A Tool for the Management of** 

Tomato (*Lycopersicum esculentum* Mill.) is among the most economically valuable vegetable crops in the world. It is estimated that diseases reduce tomato production to a greater extent worldwide. Natural plants derivative compounds contribute a lot in fight against pathogens. The current study indicates the pathogenic potential of *Alternaria alternata* FCBP-573 against tomato. RAPD analysis confirmed that *A. alternata* FCBP-573 had variability in its genetic constitution with other two isolates; this disparity in genetic constitution might be a cause to stir up more pathogenicity in this isolate. Therefore, *A. alternata* FCBP-573 was selected and subjected to biological control by *Tagetes erectus* L. In antifungal bioassays plant parts of *T. erectus* with 1, 2, 3, & 4% conc. of aqueous, methanol and n-hexane extracts of each part were evaluated against *A. alternata* FCBP-573. Results revealed that growth of *A. alternata* FCBP-573 was greatly inhibited at 4% conc. of methanol extract followed by aqueous and n-hexane extract. Among different plant parts tested, root extract exhibited more promising results by causing 81-92% reduction in biomass. The study concludes that aqueous and organic extracts of ornamentals have potential to obstruct dreadful effect of pathogenic fungi by suppressing their growth. *T. erectus* conferred vital and surprisingly

**Keywords:** Tomato, *Alternaria alternata, Tagetes erectus,* aqueous, organic extracts, antifungal bioassays.

Tomato is among the economically essential and nutritious vegetable crops in the world. The average yield of tomato in Pakistan is 10.1 tons/ha (Anonymous, 2005). There are about 200 known diseases of tomato, of which 30 are economically important (Jones et al., 1997). Early blight of tomato, stem canker, black mold rot, leaf spot and black shoulder disease is caused by the fungus *Alternaria alternata* f sp. *Lycopersici, so a single pathogen proves economically destructive*. To protect the plants from diseases and pathogens, chemical control methods are exercised. Usage of biodegradable materials as effective micro-organisms and plant extracts from different parts are being used during last few years for plant disease control (Shafique et al., 2011). Numerous studies conducted in Pakistan revealed a wide spectrum prospects of using extracts of plants for biological control of pathogenic fungi (Shafique et al., 2011). On the basis of above mentioned investigations, *Tagetes erectus* (marigold) was selected to investigate antifungal activity of its various parts against *Alternaria alternata*.

The pure cultures of *A. alternata* FCBP- 573, FCBP- 479 and FCBP-349 isolated from tomato plants were acquired from FCBP, Institute of Agricultural Sciences, University of the Punjab, Lahore.

*Alternaria alternata* **Strains of Tomato**

Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan

stable compounds having inhibitory potential against *A. alternata* FCBP-573.

Sobiya Shafique, Shazia Shafique

**Abstract** 

**1. Introduction**

**2. Materials and Methods**

© 2012 Shafique.; 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

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## *Tagetes erectus* **– A Tool for the Management of**  *Alternaria alternata* **Strains of Tomato**

## Sobiya Shafique, Shazia Shafique

Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan

#### **Abstract**

Tomato (*Lycopersicum esculentum* Mill.) is among the most economically valuable vegetable crops in the world. It is estimated that diseases reduce tomato production to a greater extent worldwide. Natural plants derivative compounds contribute a lot in fight against pathogens. The current study indicates the pathogenic potential of *Alternaria alternata* FCBP-573 against tomato. RAPD analysis confirmed that *A. alternata* FCBP-573 had variability in its genetic constitution with other two isolates; this disparity in genetic constitution might be a cause to stir up more pathogenicity in this isolate. Therefore, *A. alternata* FCBP-573 was selected and subjected to biological control by *Tagetes erectus* L. In antifungal bioassays plant parts of *T. erectus* with 1, 2, 3, & 4% conc. of aqueous, methanol and n-hexane extracts of each part were evaluated against *A. alternata* FCBP-573. Results revealed that growth of *A. alternata* FCBP-573 was greatly inhibited at 4% conc. of methanol extract followed by aqueous and n-hexane extract. Among different plant parts tested, root extract exhibited more promising results by causing 81-92% reduction in biomass. The study concludes that aqueous and organic extracts of ornamentals have potential to obstruct dreadful effect of pathogenic fungi by suppressing their growth. *T. erectus* conferred vital and surprisingly stable compounds having inhibitory potential against *A. alternata* FCBP-573.

**Keywords:** Tomato, *Alternaria alternata, Tagetes erectus,* aqueous, organic extracts, antifungal bioassays.

## **1. Introduction**

Tomato is among the economically essential and nutritious vegetable crops in the world. The average yield of tomato in Pakistan is 10.1 tons/ha (Anonymous, 2005). There are about 200 known diseases of tomato, of which 30 are economically important (Jones et al., 1997). Early blight of tomato, stem canker, black mold rot, leaf spot and black shoulder disease is caused by the fungus *Alternaria alternata* f sp. *Lycopersici, so a single pathogen proves economically destructive*. To protect the plants from diseases and pathogens, chemical control methods are exercised. Usage of biodegradable materials as effective micro-organisms and plant extracts from different parts are being used during last few years for plant disease control (Shafique et al., 2011). Numerous studies conducted in Pakistan revealed a wide spectrum prospects of using extracts of plants for biological control of pathogenic fungi (Shafique et al., 2011). On the basis of above mentioned investigations, *Tagetes erectus* (marigold) was selected to investigate antifungal activity of its various parts against *Alternaria alternata*.

## **2. Materials and Methods**

The pure cultures of *A. alternata* FCBP- 573, FCBP- 479 and FCBP-349 isolated from tomato plants were acquired from FCBP, Institute of Agricultural Sciences, University of the Punjab, Lahore.

Pathogenicity test was performed according to Grogan et al. (1975). Conidial suspension of 2.0 × 105 conidia/mL of all the isolates was primed as described by Noomrio and Dahot (1992). Disease rating scale was made on the basis of disease incidence and disease severity. Disease severity was calculated by following formula and screening of the most pathogenic isolate was carried out:

International Conference on Applied Life Sciences (ICALS2012)

served. Thus, A. alternata FCBP-573 was distinguished by the presence of two DNA fragments with an approximate size of 700 and 900 bp produced by the same primer which was not evident in other two. It was inferred from the analysis of amplicons that this difference in genetic framework could be a reason for more pathogenicity in A. alternata FCBP-573 (Fig. 1). The dendrogram was constructed on the basis of pattern of amplifications in RAPD analysis by using

highest similarity was found to be ~ 94.11% among *A. alternata* FCBP-479B and *A. alternata* FCBP-349. While isolate *A. alternata* FCBP-573A showed 21% similarity with both the other isolates. The studies reveal that *A. alternata* FCBP-573 induced maximum infection in host plant and exhibited about 79% genetic disparity from other two strains so this most pathogenic isolate was selected

UPGMA

Percent Similarity 4 20 36 52 68 84 100

Aqueous fractions of root extracts of *T. erectus* exhibited the most promising results in suppressing the fungal growth. A marked decrease of 90% in biomass production was recorded at 4% conc. Aqueous fractions of shoot extracts of *T. erectus* exhibited significant but less inhibitory potential in suppressing the fungal growth than aqueous root extract. However, 4% conc. caused significant reduction of about 44% in mycelial growth. The antifungal activities of aqueous flower extracts of

**3.3. Biological Control of A. alternata FCBP-573 through Aqueous Extracts**

Percent Similarity

software MVSP32 3.12 version (Fig. 2). In dendrogram analysis the

for subsequent biocontrol through *T. erectus.*

**Fig 1.** RAPD DNA fragment amplification with primer RAPD-7 showing genetic constitution of *A. alternata i*solates. Lane M indicates DNA marker, A: *A. alternata* FCBP-573, B: *A. alternata* FCBP-479 and

C: *A. alternata* FCBP-349.

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FCBP-573

**Fig 2.** Dendrogram showing similarities among

different isolates of *A. alternata.*

FCBP-349

FCBP-479

 x 100 Total area of plant Infected area of plant Disease severity (%) <sup>=</sup>

 The genomic DNA of isolates of *A. alternata* was mined using CTAB method (Saghai-Maroof et al., 1984). The RAPD amplification conditions were optimized by following method described by Williams et al. (1990). Five primers RAPD-6 (GATGACCGCC), RAPD-7 (TGTCTGGGTG), RAPD-8 (GTTGCCAGCC), RAPD-9 (GAACGGACTC), and RAPD-10 (TCGCCAGCCA) were used in RAPD analysis.

Fresh samples of *T. erectus* were collected from PU Lahore Aqueous extract (20% w/v) was prepared according to Bajwa *et al.* (2007). The protocol of Alkhail (2005) was used to prepare plant extracts in methanol and n-hexane. Aqueous and organic solvent extract bioassays were carried out in liquid 2% malt extract (ME) medium and incubated at 28±3 ºC for 7 days. Their dry weight yield was determined after 24 h oven drying at 60 ºC according to Bajwa et al. (2006). The rate of fungal biomass increase or decrease was determined from the dried biomass*.* Percentage reduction in fungal biomass was calculated as:

> 100 Growth in control Growth in control - Growth in treatment Growth inhibition (%) <sup>=</sup> <sup>×</sup>

All the data was analyzed by analysis of variance (ANOVA), following this, Duncan's Multiple Range (DMR) test (Steel and Torrie, 1980) was applied to separate the treatment means.

### **3. Results**

#### **3.1. Pathogenicity Test**

*A. alternata* FCBP-573 of was proved the most pathogenic (Plate 1), as it induced symptoms of dark brown to black canker with concentric zonation on stems near soil line or aboveground. The cankers became enlarge slowly and plants died. Foliar symptoms were visualized in the form of curling and pointed necrotic lesions on lowest leaflets or in later stages, complete necrosis of leaflets on sides of the midrib was noticed (Plate 1). On the basis of symptoms disease rating scale was developed that is as follows. On the basis of symptoms, *A. alternata* FCBP-573 exhibited maximum pathogenicity towards tomato plants, so was screened out as the most pathogenic strain.

#### **3.2. Molecular Analysis**

In RAPD analysis primer RAPD-7 amplified the genome of all isolates while remaining four primers (RAPD-6, RAPD-8, RAPD-9 and RAPD-10) provided no any amplification (Fig. 1). At 225 bp, a unique band was primed by A. alternata FCBP-573 and A. alternata FCBP-479. At 400- 500 bp level except isolate A. alternata FCBP-573, in other both isolates amplifications were observed. Thus, A. alternata FCBP-573 was distinguished by the presence of two DNA fragments with an approximate size of 700 and 900 bp produced by the same primer which was not evident in other two. It was inferred from the analysis of amplicons that this difference in genetic framework could be a reason for more pathogenicity in A. alternata FCBP-573 (Fig. 1). The dendrogram was constructed on the basis of pattern of amplifications in RAPD analysis by using software MVSP32 3.12 version (Fig. 2). In dendrogram analysis the

highest similarity was found to be ~ 94.11% among *A. alternata* FCBP-479B and *A. alternata* FCBP-349. While isolate *A. alternata* FCBP-573A showed 21% similarity with both the other isolates. The studies reveal that *A. alternata* FCBP-573 induced maximum infection in host plant and exhibited about 79% genetic disparity from other two strains so this most pathogenic isolate was selected for subsequent biocontrol through *T. erectus.* UPGMA

**Fig 1.** RAPD DNA fragment amplification with primer RAPD-7 showing genetic constitution of *A. alternata i*solates. Lane M indicates DNA marker, A: *A. alternata* FCBP-573, B: *A. alternata* FCBP-479 and C: *A. alternata* FCBP-349.

**Fig 2.** Dendrogram showing similarities among different isolates of *A. alternata.*

## **3.3. Biological Control of A. alternata FCBP-573 through Aqueous Extracts**

Aqueous fractions of root extracts of *T. erectus* exhibited the most promising results in suppressing the fungal growth. A marked decrease of 90% in biomass production was recorded at 4% conc. Aqueous fractions of shoot extracts of *T. erectus* exhibited significant but less inhibitory potential in suppressing the fungal growth than aqueous root extract. However, 4% conc. caused significant reduction of about 44% in mycelial growth. The antifungal activities of aqueous flower extracts of

*T. erectus* in terms of growth inhibition potential were recorded highest at 3% concentration. This suppression in biomass production was in the range of 40-57%.The results of aqueous fractions conclude that root extracts of *T. erectus* exhibited more promising results as compared to aqueous shoot and flower extract as it suppressed the growth of *A. alternata* FCBP-573 up to 90% while shoot and flower extract induced only 44% and 57% biomass production, respectively.

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Control 1% 2% 3% 4%

 **root Extarct shoot Extract flower Extract**

c d b c

cd de

a b

b b

**0 0.5 1 1.5 2 2.5 3**

a

b

e

**Fig 4.** Effect of various conc.s of different parts of methanolic extracts of T. erectus on the biomass of A.

In Pathogenecity test *A. alternata* FCBP-573 induced maximum characteristic symptoms. These results were found in agreement with the work conducted by Gilchrist and Grogan (1974) who reported same trend of disease development in tomato by *A. alternata*. Presently, RAPD analysis was carried out to evaluate the isolates of *A. alternata* for their genetic diversity. In dendrogram analysis, isolate *A. alternata* FCBP-479 and *A. alternata* FCBP-349 showed 79% disparity with *A. alternata* FCBP- 573. In previous study, Roberts et al. (2000) also reported a high genetic variation

Pathogenicity test and RAPD analysis confirmed the strong pathogenic potential of *A. alternata*  FCBP-573 so subsequently in present study, root, shoot and flower extracts of *T. erectus* in different solvents were examined. Presently, the results indicated that aqueous fractions of root extracts of *T. erectus* effectively inhibited the growth of *A. alternata* FCBP-573*.* A marked decrease in biomass production (90%) was recorded at 4% conc. of aqueous root extracts because at higher concentration, thiophenes concentration also increased. It is similar to the work done by Riaz et al. (2008) who observed 54-79% suppression in biomass by employing various concentrations of aqueous fractions of *T. erectus.* In present study, Methanol extracts showed maximum antifungal stress at 4% conc. of root, shoot and flower extracts causing a decline of about 92, 86 and 82%, respectively, in biomass. Working on parallel line Bajwa et al. (2008) reported maximum antifungal activity of methanol extract of rice varieties on *M. phaseolina* and *A. rabiei*. The n-hexane root extract showed the highest antifungal activity against *A. alternata* FCBP-573. A gradual decrease in biomass production was observed with increase in extract conc. At 4% conc. root, shoot and flower extracts exhibited the strongest antifungal effect by expressing 81, 64 and 70% growth inhibition. Earlier Daoud et al. (1990) have reported good antifungal activity of *M. azedarach* against *Alternaria, As-*

This study concludes that aqueous and methanolic extracts of *T. erectus* possess potential antifungal compounds against *A. alternata*, which hold strong antimycotic activity and can be used as a perfect approach for future plant disease management programs eliminating fungal spread.

**Dry Biomass (g)**

in *A. alternata* populations at molecular level.

*pergillus* and *Penicillium spp*.

alternata FCBP-573.

**4. Discussion**

Turkey, September 10-12, 2012

c

a

<sup>313</sup> ISALS

#### **3.4. Biological Control of A. alternata FCBP-573 through Methanolic Extracts**

The maximum antifungal stress was induced by 4% root and shoot extract conc. causing 92% and 86% decline in dry biomass., respectively. Antifungal activity of methanolic flower extracts was assayed and its effect on the growth of *A. alternata* FCBP-573 is presented in Fig.4. The data analysis revealed significant reduction in growth of *A. alternata* FCBP-573 with flower extracts of *T. erectus* but the extract fractions showed significant differences in their efficacy. The reduction in biomass was ranged from 28 to 82%.It is indicated from the results that methanolic extracts of root of *T. erectus* exhibited the best potential in suppressing the fungal growth than methanolic shoot and flower extract.

#### **3.5. Biological Control of A. alternata FCBP-573 through n-Hexane Extracts**

The n-hexane extract of root showed the highest antifungal activity against A. alternata FCBP-573. At 4% concentration extract exhibited the strongest antifungal upshot by expressing 81.5% growth inhibition. In case of n-hexane shoot extract a variable pattern of antimycotic activity was observed but 4% concentration of shoot extract was the most effective in suppressing the growth of A. alternata FCBP-573 up to 64%. The data of n-hexane flower extracts revealed a steep and significant reduction in growth by 1 to 4% concentrations in comparison to control. It is indicated from the results that there was insignificant reduction in growth at 3 and 4% concentrations. Maximum arrest in biomass production was evident at 4% concentration as it induced about 57% capture at this dose. The antifungal activities of n-hexane extracts of T. erectus root exhibited more promising results in suppressing the fungal growth than n-hexane shoot and flower extract.

**Fig 3.** Effect of various conc.s of different parts of aqueous extracts of T. erectus on the biomass production of A. alternata FCBP-573.

**Fig 4.** Effect of various conc.s of different parts of methanolic extracts of T. erectus on the biomass of A. alternata FCBP-573.

### **4. Discussion**

In Pathogenecity test *A. alternata* FCBP-573 induced maximum characteristic symptoms. These results were found in agreement with the work conducted by Gilchrist and Grogan (1974) who reported same trend of disease development in tomato by *A. alternata*. Presently, RAPD analysis was carried out to evaluate the isolates of *A. alternata* for their genetic diversity. In dendrogram analysis, isolate *A. alternata* FCBP-479 and *A. alternata* FCBP-349 showed 79% disparity with *A. alternata* FCBP- 573. In previous study, Roberts et al. (2000) also reported a high genetic variation in *A. alternata* populations at molecular level.

Pathogenicity test and RAPD analysis confirmed the strong pathogenic potential of *A. alternata*  FCBP-573 so subsequently in present study, root, shoot and flower extracts of *T. erectus* in different solvents were examined. Presently, the results indicated that aqueous fractions of root extracts of *T. erectus* effectively inhibited the growth of *A. alternata* FCBP-573*.* A marked decrease in biomass production (90%) was recorded at 4% conc. of aqueous root extracts because at higher concentration, thiophenes concentration also increased. It is similar to the work done by Riaz et al. (2008) who observed 54-79% suppression in biomass by employing various concentrations of aqueous fractions of *T. erectus.* In present study, Methanol extracts showed maximum antifungal stress at 4% conc. of root, shoot and flower extracts causing a decline of about 92, 86 and 82%, respectively, in biomass. Working on parallel line Bajwa et al. (2008) reported maximum antifungal activity of methanol extract of rice varieties on *M. phaseolina* and *A. rabiei*. The n-hexane root extract showed the highest antifungal activity against *A. alternata* FCBP-573. A gradual decrease in biomass production was observed with increase in extract conc. At 4% conc. root, shoot and flower extracts exhibited the strongest antifungal effect by expressing 81, 64 and 70% growth inhibition. Earlier Daoud et al. (1990) have reported good antifungal activity of *M. azedarach* against *Alternaria, Aspergillus* and *Penicillium spp*.

This study concludes that aqueous and methanolic extracts of *T. erectus* possess potential antifungal compounds against *A. alternata*, which hold strong antimycotic activity and can be used as a perfect approach for future plant disease management programs eliminating fungal spread.

#### **5. References**

[1] S.M. Ahmed, S.A.M. Abdelgaleil. Antifungal activity of extracts and sesquiterpene lactones from *Magnolia grandiflora* L. (Magnoliaceae). *Int. J. Agric. Biol*. 2005, **4**: 638–642.

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**Biological Control Potential of Parthenium** 

**Hysterophorus Against Fusarium Solani – A** 

Fusarium wilt, caused by *Fusarium solani,* is potential disease of potato in Pakistan. Recent studies have shown that fungal plant pathogens can be controlled by plant products as the plant materials are biodegradable, display structural diversity and complexity. Presently the pathogenic potential and biological control of *Fusarium solani* was studied by inoculating potato plants with three strains of *F. solani,* to screen the most virulent isolate among *F. solani* FCBP-016, *F. solani* FCBP-434 and *F. solani* FCBP-470. It was found through pathogenicity test that *F. solani* FCBP-434 was the most pathogenic among three *F. solani* strains with variation in genetic level. This disparity in genetic constitution might be a cause of its high pathogenicity. Further, antifungal bioassays were conducted to confirm mycotoxic potential of different plant parts i.e., root, shoot and leaf of *P. hysterophorus* against *F. solani* FCBP-434 using 1, 2, 3, and 4% concentrations of the aqueous, methanol and n-hexane extracts. Bioassays revealed that among the three solvents of *P. hysterophorus*, the growth of *F. solani* FCBP-434 was greatly inhibited at 1 & 2% conc. of aqueous and methanol extracts of leaf and stem while in case of n-hexane extract 3 & 4% conc. were proved more effective. Among root extracts, the higher concentrations of aqueous and n-hexane exhibited more promising results by causing reduction of 85 & 74%, respectively, whereas in

**Keywords:** Fusarium wilt, Potato, Biocontrol, *P. hysterophorus,* Aqueous and organic solvents.

Potato (*Solanum tuberosum*) is graded at third level among food after wheat and rice and ranks fifth in Pakistan for its total production [1]. Diseases are the most important factor for its low per acre production. Fusarium wilt is a fungal disease which can be caused by *Fusarium* species, particularly *F*. *solani*. Plant extracts and essential oils show antifungal activity against a wide range of fungi [2]. Therefore, the development of biopesticides has been focused as a viable pest control strategy in recent years. Among these, *Parthenium hysterophorus* is important. The allelopathic and antifungal potential of *P. hysterophorus* is due to release of phytotoxic substances such as caffeic, ferulic, vanillic, chlorogenic, *p-* coumaric and parthenin, *p*-hydroxybenzoic acids, ambrosin and coronopilin [3]. The antifungal activities of root and shoot extracts of two Asteraceous plant species viz. *P. hysterophorus* L and *Ageratum conyzoides* were determined against *Macrophomina phaseolina* (Tassi) Goid., the cause of charcoal rot disease of sunflower (*Helianthus annus* L.). A

**Cause of Fusarium Wilt in Potato**

methanol extract again lower conc. were more inhibitory.

Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan

Shazia Shafique, Sobiya Shafique

**Abstract** 

**1. Introduction**

© 2012 Shafique 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>315</sup> ISALS


## **Biological Control Potential of Parthenium Hysterophorus Against Fusarium Solani – A Cause of Fusarium Wilt in Potato**

Shazia Shafique, Sobiya Shafique

Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan

#### **Abstract**

Fusarium wilt, caused by *Fusarium solani,* is potential disease of potato in Pakistan. Recent studies have shown that fungal plant pathogens can be controlled by plant products as the plant materials are biodegradable, display structural diversity and complexity. Presently the pathogenic potential and biological control of *Fusarium solani* was studied by inoculating potato plants with three strains of *F. solani,* to screen the most virulent isolate among *F. solani* FCBP-016, *F. solani* FCBP-434 and *F. solani* FCBP-470. It was found through pathogenicity test that *F. solani* FCBP-434 was the most pathogenic among three *F. solani* strains with variation in genetic level. This disparity in genetic constitution might be a cause of its high pathogenicity. Further, antifungal bioassays were conducted to confirm mycotoxic potential of different plant parts i.e., root, shoot and leaf of *P. hysterophorus* against *F. solani* FCBP-434 using 1, 2, 3, and 4% concentrations of the aqueous, methanol and n-hexane extracts. Bioassays revealed that among the three solvents of *P. hysterophorus*, the growth of *F. solani* FCBP-434 was greatly inhibited at 1 & 2% conc. of aqueous and methanol extracts of leaf and stem while in case of n-hexane extract 3 & 4% conc. were proved more effective. Among root extracts, the higher concentrations of aqueous and n-hexane exhibited more promising results by causing reduction of 85 & 74%, respectively, whereas in methanol extract again lower conc. were more inhibitory.

**Keywords:** Fusarium wilt, Potato, Biocontrol, *P. hysterophorus,* Aqueous and organic solvents.

#### **1. Introduction**

Potato (*Solanum tuberosum*) is graded at third level among food after wheat and rice and ranks fifth in Pakistan for its total production [1]. Diseases are the most important factor for its low per acre production. Fusarium wilt is a fungal disease which can be caused by *Fusarium* species, particularly *F*. *solani*. Plant extracts and essential oils show antifungal activity against a wide range of fungi [2]. Therefore, the development of biopesticides has been focused as a viable pest control strategy in recent years. Among these, *Parthenium hysterophorus* is important. The allelopathic and antifungal potential of *P. hysterophorus* is due to release of phytotoxic substances such as caffeic, ferulic, vanillic, chlorogenic, *p-* coumaric and parthenin, *p*-hydroxybenzoic acids, ambrosin and coronopilin [3]. The antifungal activities of root and shoot extracts of two Asteraceous plant species viz. *P. hysterophorus* L and *Ageratum conyzoides* were determined against *Macrophomina phaseolina* (Tassi) Goid., the cause of charcoal rot disease of sunflower (*Helianthus annus* L.). A

© 2012 Shafique 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.

measured reduction in *M. phaseolina* biomass was evaluated due to aqueous extracts of different concentrations [4]. On the basis of efficacy of plant extracts, present study has been designed to find out the effective plant product for the management of *F. solani* by using *P. hysterophorus* and molecular characterization of *F. solani.*

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scale after 15 days of incubation at 25 °C. Five levels of pathogenicity were detected on potato plants (Fig. 1). *Fusarium solani* FCBP-434 was proved to be the most pathogenic. Rapid results were obtained with only two primers i.e. A-02 and B-05 and the dendrogram generated from each primer and cluster of primers by MINITAB are presented Fig. 2. The RAPD data obtained with 13 primers was evaluated to analyze the genetic parity or disparity among different genotypes. A dendrogram was constructed on the basis of genetic distances by UPGMA method and two main groups of cluster were identified in the homology tree: F. solani FCBP-016 and F. solani FCBP-470 on one side and F. solani FCBP-434 on the other side (Fig. 3). F. solani FCBP-016 and F. solani FCBP-470 are 100% similar to each other but they show only 44.34% similarity with F. solani FCBP-434. The findings of pathogenicity test and molecular analysis showed that F. solani FCBP-434 have high pathogenic potential so

**Fig 1.** Periodic progression of Fusarium wilt of potato. **Fig 2.** Homology tree constructed by UPGMA

The data on dry biomass production in early growth phase of 7-10 days after incubation (DAI) revealed that all the conc. significantly decreased the fungal biomass as compared to control (Fig. 4). Amongst all concentrations 1% concentration exhibited the most promising results as it caused about 85% reduction in fungal biomass while 62, 52 and 41% decline in fungal biomass was depicted by 2, 3 and 4% aqueous extract, respectively. The response of *F. solani*  in terms of dry biomass production was variable when grown in different concentrations of methanol extract of *P. hysterophorus*. Amongst these 2% concentration was the most effective in suppressing the biomass production up to 76% and 3% concentration was also found to decline fungal biomass production up to 67% In dry fungal biomass production with an increase in concentration of n-hexane extract revealed the maximum antifungal stress by 4% concentration causing a decline of about 66% in the biomass production of *F. solani*. It was followed by 3, 2 & 1% concentration which revealed a significant reduction, in the range of 56,

it was subjected to further biocontrol assays.

**3.2. Biological control through leaf extract**

50 and 44%, respectively.

Turkey, September 10-12, 2012

method for genetic similarity amongst genotypes of

F. solani FCBP-016, 434 and 470.

<sup>317</sup> ISALS

## **2. Methodology**

Pure cultures of *Fusarium solani* FCBP-016, *F. solani* FCBP-434 and *F. solani* FCBP-470 were taken from FCBP, Institute of Agricultural sciences, University of the Punjab, Lahore. They were maintained and subcultured on Malt extract agar medium at 4 °C monthly.

For pathogenecity test the protocol of Grogan et al. [5] was adopted. Pathogenicity test was performed by inoculating the conidial suspension @ 10-15 mL/plant containing 2 x 105 conidia mL-1 on 1-month-old potted potato plants and soil with three selected isolates of the fungus. Disease severity was calculated with the help of following formula.

> x100 Total area of the plant Affected area of <sup>a</sup> plant Disease severity <sup>=</sup>

Screening of the most pathogenic isolate was carried out on the basis of pathogenicity test. The most pathogenic species was isolated and subjected to further biocontrol assays.

The genomic DNA of three different isolates of *F. solani* was extracted by CTAB method [6] with some modifications.

Aqueous extract was primed according to Bajwa et al*.* [7]. While the method of Alkhail [8] was followed for preparation of extract in methanol and n-hexane. The lower conc. of 1, 2, 3 & 4% of aqueous, n-hexane and methanol extracts of leaf, stem and root were prepared by adding appropriate quantity of sterilized distilled water. To make methanol and n-hexane control, 2 mL of methanol and n-hexane were dissolved in sterilized distilled water to make final volume 100 mL, in respective flasks.

Extract bioassays were carried out in liquid medium according to Bajwa et al*.* [9]. Their dry weight yield was determined after 24 h oven drying at 60 °C and percentage inhibition in biomass production was calculated as:

$$\text{Biomass reduction (\%)} = \frac{\text{Biomass in Control - Biomass in extracted treatment}}{\text{Biomass in Control}} \ge 100$$

All the data was subjected to analysis of variance (ANOVA) followed by Duncan's Multiple Range Test to delineate mean differences [10].

#### **3. Results**

#### **3.1. Pathogenecity Test**

The pathogenicity of three isolates of *F. solani* (FCBP-016, FCBP-434, FCBP-470) was assessed which enabled the reproduction of typical symptoms of the disease over the timescale after 15 days of incubation at 25 °C. Five levels of pathogenicity were detected on potato plants (Fig. 1). *Fusarium solani* FCBP-434 was proved to be the most pathogenic. Rapid results were obtained with only two primers i.e. A-02 and B-05 and the dendrogram generated from each primer and cluster of primers by MINITAB are presented Fig. 2. The RAPD data obtained with 13 primers was evaluated to analyze the genetic parity or disparity among different genotypes. A dendrogram was constructed on the basis of genetic distances by UPGMA method and two main groups of cluster were identified in the homology tree: F. solani FCBP-016 and F. solani FCBP-470 on one side and F. solani FCBP-434 on the other side (Fig. 3). F. solani FCBP-016 and F. solani FCBP-470 are 100% similar to each other but they show only 44.34% similarity with F. solani FCBP-434. The findings of pathogenicity test and molecular analysis showed that F. solani FCBP-434 have high pathogenic potential so it was subjected to further biocontrol assays.

**Fig 1.** Periodic progression of Fusarium wilt of potato. **Fig 2.** Homology tree constructed by UPGMA

method for genetic similarity amongst genotypes of F. solani FCBP-016, 434 and 470.

#### **3.2. Biological control through leaf extract**

The data on dry biomass production in early growth phase of 7-10 days after incubation (DAI) revealed that all the conc. significantly decreased the fungal biomass as compared to control (Fig. 4). Amongst all concentrations 1% concentration exhibited the most promising results as it caused about 85% reduction in fungal biomass while 62, 52 and 41% decline in fungal biomass was depicted by 2, 3 and 4% aqueous extract, respectively. The response of *F. solani*  in terms of dry biomass production was variable when grown in different concentrations of methanol extract of *P. hysterophorus*. Amongst these 2% concentration was the most effective in suppressing the biomass production up to 76% and 3% concentration was also found to decline fungal biomass production up to 67% In dry fungal biomass production with an increase in concentration of n-hexane extract revealed the maximum antifungal stress by 4% concentration causing a decline of about 66% in the biomass production of *F. solani*. It was followed by 3, 2 & 1% concentration which revealed a significant reduction, in the range of 56, 50 and 44%, respectively.

**Fig 3.** Effect of various conc.s of aqueous, methanol and n-hexane leaf extracts of P. hysterophorus on dry biomass production of F. solani FCBP-434.

**Fig 4.** Effect of various conc.s of aqueous, methanol and n-hexane stem extracts of P. hysterophorus on dry biomass production of F. solani FCBP-434.

International Conference on Applied Life Sciences (ICALS2012)

**Fig 6.** Effect of various conc.s of aqueous, methanol and n-hexane root extracts of P. hysterophorus on dry

In the present study, pathogenicity test was carried out to assess the pathogenicity of three isolates of *Fusarium solani*. Among the isolates FCBP-434 proved to be more pathogenic and destructed the leaves on infected plants in 15 days after inoculation. Similar results were reported from various host plant - *F. oxysporum* combinations [11]. Molecular analysis of three isolates was also conducted. It was obvious from the results that *F. solani* FCBP-434 had genetic variability than other two isolates. In several studies RAPD fingerprinting technique has been employed to detect mutation, genetic relatedness and genetic variation within and between natural bacterial and human DNA and fungal populations [12, 13]. With the help of these two evidences about high pathogenic ability of *F. solani* FCBP-434, further its biological control assays were conducted. It is obvious from the study that mycelial growth rate was significantly inhibited by antifungal compounds specifically at lower concentrations of aqueous leaf and stem extract. These results are supported from previous investigations in which leaf extracts of *Datura stramonium* have been shown to cause a decline in the development of rust pustules on wheat leaves [14]. Further increase in extract concentration exhibited significant difference in antimycotic activity as compared to 1% extract. In case of aqueous and organic root extract of *P. hysterophorus,* phytotoxins or antifungal compounds inhibited the biomass at higher concentration. The variation in antifungal activity of shoot and root extracts may be attributed to the different chemical nature of the compounds present in these parts [15]. Greater inhibition (42-76%) of growth of *F. solani* at 2-3% conc. of leaf methanolic extract was observed. While in case of stem and root methanolic extracts, lower conc (1-2%) were more effective as compared to higher conc. (3-4%). The pattern of gradually higher production of biomass in response to increasing conc. of aqueous extract was also similar to investigation of Bajwa et al*.* [16]. Comparative effectiveness of n-hexane extracts of selected test species revealed that higher concentrations were relatively more allelopathic thanlower concentrations. These results are supported by the work of Vir and Sharma [17] that employed 10%

conc. of neem oil that exhibited 100% inhibition in *A. niger, D. rostrata* and *M. phaseolina.*

biomass production of F. solani FCBP-434.

**4. Discussion**

Turkey, September 10-12, 2012

<sup>319</sup> ISALS

#### **3.3. Biological control through stem extract**

All extract doses significantly inhibited the growth of the test pathogen in the same manner as depicted by aqueous leaf extract (Fig. 5). The greatest reduction in biomass production (70%) was observed in 1% extract. There was 48-70% reduction in fungal biomass production as noticed due to 1-4% concentrations of the extracts. All regimes of methanol extract of *P. hysterophorus* caused considerable inhibition in biomass production. The maximum inhibition in fungal growth was evidenced by 1% concentration which decreased biomass production upto 80%. While all the other concentrations (2-4%) caused about 15-38% reduction in biomass production, respectively. A variable response of dry biomass production in *F. solani* FCBP-434 was recorded to n-hexane extract of *P. hysterophorus* in different concentrations. *F. solani* FCBP-434 exhibited a significant reduction when exposed to different concentrations of extracts compared to control. The reduction in biomass ranged from 33 to 77%.

#### **3.4. Biological control through root extract**

All the doses of aqueous root extracts significantly retarded growth of the test fungus pathogen (Fig. 6). The fungal biomass suppressed at lower concentration (1-2%) in a range of 14- 45%. While higher concentrations (3-4%) proved the most effective as these caused a reduction up to 67-85%, respectively. In methanol treatment, the lowest concentration (1%) was the most effective in reducing and suppressing the target fungal pathogen up to 77% as compared to higher concentrations. The fungal biomass increased at higher concentrations (2-4%) but still a significant inhibition in biomass production was detected with reference to control.A similar kind of suppressive effect of various concentrations was recorded against target fungal pathogen as exhibited by n-hexane leaf and stem extract. All the concentrations significantly reduced the fungal biomass production. Amongst these 4% concentration was the most effective in suppressing the biomass production up to 74%.

**Fig 6.** Effect of various conc.s of aqueous, methanol and n-hexane root extracts of P. hysterophorus on dry biomass production of F. solani FCBP-434.

#### **4. Discussion**

In the present study, pathogenicity test was carried out to assess the pathogenicity of three isolates of *Fusarium solani*. Among the isolates FCBP-434 proved to be more pathogenic and destructed the leaves on infected plants in 15 days after inoculation. Similar results were reported from various host plant - *F. oxysporum* combinations [11]. Molecular analysis of three isolates was also conducted. It was obvious from the results that *F. solani* FCBP-434 had genetic variability than other two isolates. In several studies RAPD fingerprinting technique has been employed to detect mutation, genetic relatedness and genetic variation within and between natural bacterial and human DNA and fungal populations [12, 13]. With the help of these two evidences about high pathogenic ability of *F. solani* FCBP-434, further its biological control assays were conducted.

It is obvious from the study that mycelial growth rate was significantly inhibited by antifungal compounds specifically at lower concentrations of aqueous leaf and stem extract. These results are supported from previous investigations in which leaf extracts of *Datura stramonium* have been shown to cause a decline in the development of rust pustules on wheat leaves [14]. Further increase in extract concentration exhibited significant difference in antimycotic activity as compared to 1% extract. In case of aqueous and organic root extract of *P. hysterophorus,* phytotoxins or antifungal compounds inhibited the biomass at higher concentration. The variation in antifungal activity of shoot and root extracts may be attributed to the different chemical nature of the compounds present in these parts [15]. Greater inhibition (42-76%) of growth of *F. solani* at 2-3% conc. of leaf methanolic extract was observed. While in case of stem and root methanolic extracts, lower conc (1-2%) were more effective as compared to higher conc. (3-4%). The pattern of gradually higher production of biomass in response to increasing conc. of aqueous extract was also similar to investigation of Bajwa et al*.* [16]. Comparative effectiveness of n-hexane extracts of selected test species revealed that higher concentrations were relatively more allelopathic thanlower concentrations. These results are supported by the work of Vir and Sharma [17] that employed 10% conc. of neem oil that exhibited 100% inhibition in *A. niger, D. rostrata* and *M. phaseolina.*

Thus it can be recommended that use of *P. hysterophorus* against *Fusarium solani* give better results as they are environmental safe alternatives and can be further exploited for formulating integrated disease management.

International Conference on Applied Life Sciences (ICALS2012)

**Antifungal Activity of Essential Oils Extracted** 

**Mold Disease on Citrus Fruit**

Corresponding author, Tel.: +92-42-99231846; fax: +92-42-99231187

Tehmina Anjum\* and Nosheen Akhtar

Email: tehminaanjum@yahoo.com

\*

**Abstract**

**1. Introduction**

during citrus storage.

**From Clove, Cumin and Cinnamon Against Blue** 

Institute of Agricultural Sciences, Quaid-e-Azam Campus, University of the Punjab, Lahore, Pakistan

Essential oils obtained from Cumin seeds, Clove buds and Cinnamon bark was checked for their antifungal potentials against *Penicillium italicum*, causal agent of blue mold disease in citrus fruit. Selected essential oils were checked in different concentrations of 3, 6, 12, 24 and 48µl/ml for their ability to inhibit the mycelial growth of the test fungi. The *in vitro* study revealed that the essential oils of cumin and clove have the potential to inhibit mycelial growth of test fungi completely at concentrations of 12 and 48µl/ml, respectively. Essential oil of cinnamon, however failed to completely inhibit the mycelial growth even at maximum used concentration of 48µl/ml. *In vivo* assays also supported these results. Clove and cumin oils showed complete fungal inhibition at concentration of 24 and 48µl/ml, respectively when applied on citrus fruits. Whereas, cinnamon essential oil could not stop fungal infection even at its highest tested concentration. The study

Fungi cause significant losses in almost all perishables due to post harvest rots. One of these fungi is *Penicillium italicum* Whemer (blue mold) that results in a universal post-harvest disease of almost all kinds of citrus fruit[1]. Chemicals imazalil, sodium ortho-phenyl phenate, and thiabendazole, have been widely used to control this problem [2]. However increase in public concern regarding contamination of perishables with fungicidal residues and proliferation of resistance in the pathogenic population [3] has forced the community to search for environment safe strategies. In the past few years, there has been a huge increase in the search of natural substances such as essential oils and plant extracts as potential antifungal agents [4]. The use of essential oils to control post-harvest fruit diseases have been deeply investigated and is well documented since the volatile compounds may have better applicability as fumigants for control [5]. In the present work, it has been therefore thought desirable to discover the antifungal potencies of essential oils extracted from three different plants against *P. italicum*, a dominant mycotoxin producing fungi

was extended to chemical identification of tested essential oils through GC-MS.

**Keywords:** essential oils, citrus, Penicillium itallicum, antifungal activity

© 2012 Anjum and Akhtar.; 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>321</sup> ISALS

#### **5. References**


## **Antifungal Activity of Essential Oils Extracted From Clove, Cumin and Cinnamon Against Blue Mold Disease on Citrus Fruit**

Tehmina Anjum\* and Nosheen Akhtar

Institute of Agricultural Sciences, Quaid-e-Azam Campus, University of the Punjab, Lahore, Pakistan \* Corresponding author, Tel.: +92-42-99231846; fax: +92-42-99231187 Email: tehminaanjum@yahoo.com

**Abstract**

Essential oils obtained from Cumin seeds, Clove buds and Cinnamon bark was checked for their antifungal potentials against *Penicillium italicum*, causal agent of blue mold disease in citrus fruit. Selected essential oils were checked in different concentrations of 3, 6, 12, 24 and 48µl/ml for their ability to inhibit the mycelial growth of the test fungi. The *in vitro* study revealed that the essential oils of cumin and clove have the potential to inhibit mycelial growth of test fungi completely at concentrations of 12 and 48µl/ml, respectively. Essential oil of cinnamon, however failed to completely inhibit the mycelial growth even at maximum used concentration of 48µl/ml. *In vivo* assays also supported these results. Clove and cumin oils showed complete fungal inhibition at concentration of 24 and 48µl/ml, respectively when applied on citrus fruits. Whereas, cinnamon essential oil could not stop fungal infection even at its highest tested concentration. The study was extended to chemical identification of tested essential oils through GC-MS.

**Keywords:** essential oils, citrus, Penicillium itallicum, antifungal activity

### **1. Introduction**

Fungi cause significant losses in almost all perishables due to post harvest rots. One of these fungi is *Penicillium italicum* Whemer (blue mold) that results in a universal post-harvest disease of almost all kinds of citrus fruit[1]. Chemicals imazalil, sodium ortho-phenyl phenate, and thiabendazole, have been widely used to control this problem [2]. However increase in public concern regarding contamination of perishables with fungicidal residues and proliferation of resistance in the pathogenic population [3] has forced the community to search for environment safe strategies. In the past few years, there has been a huge increase in the search of natural substances such as essential oils and plant extracts as potential antifungal agents [4]. The use of essential oils to control post-harvest fruit diseases have been deeply investigated and is well documented since the volatile compounds may have better applicability as fumigants for control [5]. In the present work, it has been therefore thought desirable to discover the antifungal potencies of essential oils extracted from three different plants against *P. italicum*, a dominant mycotoxin producing fungi during citrus storage.

© 2012 Anjum and Akhtar.; 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.

## **2. Methodology**

#### **2.1. Test Fungi**

The strain of *Penicillium italicum* used in this study was isolated from the decaying citrus fruit (variety: Mandarin). The fungal culture was maintained on Malt Extract Agar (MEA) medium at 4±1<sup>o</sup> C. A 7-14 days old culture of the isolate was used as the source of inoculum and for the preparation of spore suspension for various studies. The spores were removed from the surface of the culture, suspended in 5 ml of sterile distilled water containing 0.05% (v/v) Tween 80 and its concentration was adjusted to 106sp/ml using a haemocytometer for further *in vivo* studies.

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trol fruit was subjected to the same treatments except that sterile distilled water was used instead of essential oil. The treated fruits were labeled, placed in sterilized petri plates and incubated at

Qualitative analysis of the tested essential oils was undertaken by gas chromatography-mass spectroscopy (GC-MS) using a Hewlett-Packard mass detector (model 7890) coupled with mass spectrometer selective detector 5975. Analysis was carried out using a column HP5 mass-selective detector (MSD) (30 m x 0.25 mm; 0.25 µm film thickness), the operating conditions were as follows: Helium was the carrier gas at a flow rate of 1 ml/min. diluted samples (1:100 v/v, in methanol) of 1 µl were injected manually at temperature 250°C. NIST (National Institute of Stan-

In vitro antifungal activity of selected oils were checked through two methods i.e., dilution method and the volatile method (Table 1). A little variation was observed in results of both the assays. In dilution method cumin oil gave best control on the mycelial growth of P. italicum as its lowest concentration of 3µl/ml gave 96% inhibition. This increased with increase in oil concentration and 12µl/ml completely inhibited the fungal growth. The results of cumin oil was followed by clove oil, whose lowest tested concentration i.e., 3µl/ml showed 84% mycelial growth inhibition. However, the complete inhibition of fungal growth was recorded at maximum tested concentration i.e., 48 µl/ml. Cinnamon gave the lowest effect among the

In volatile method clove oil gave best control of P. italicum instead of cumin that showed highest inhibition of mycelial growth in dilution method perhaps because of high volatility of phenols that are abundantly present in clove essential oil. Clove oil in 3, 6 and 12µl/ml concentrations gave 97% control on tested fungi. Increase in oil concentration of clove oil gave similar results as were recorded when the same oil was checked through dilution method. Complete inhibition of mycelial growth was observed at maximum tested concentration. In case of cumin the lowest tested concentration of 3µl/ml could only inhibit *P. italicum* up to 32% in contrast to its significant control of 96% recorded in dilution method. Concentration increased the inhibition percentage up to 98-99%. Romagnoli et al.,[6] reported a strong antifungal activity against dermatophytes and phytopathogens including fungi and yeast. They also found cumin aldehyde, pinenes, and *p*-cymene, and a fraction of oxygenate compounds such as alcohol and epoxides as the most active ingredients of cumin essential oil. Cinnamon oil also depicted better control on tested fungi in volatile method when compared to the dilution method perhaps because it is also rich in volatile phenols live clove essential oil. However, still the inhibition effect of cinnamon essential oil failed to match with the controlling

23±2 °C for two weeks to assess decay and fungal growth symptoms on daily basis.

dards and Technologies) Mass Spectra Library was also used as a reference.

**2.6. Chemical identification of essential oils by GC-MS**

**3. Results and Discussion**

three tested EOs.

capacity of other two tested oils.

**3.1. In vitro antifungal efficacy of essential oil**

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#### **2.2. Extraction of Essential Oils**

Seeds of cumin (Cuminum cyminum L.), clove buds [Syzygium aromaticum (L.) Merrill & Perry], and cinnamon bark (Cinnamomum verum J. Presl) were imperiled to hydrodistillation in a modified Clevenger apparatus for 3-4 hours. Isolated EOs were stored in glass after dehydrating with anhydrous sodium sulphate and were kept in the refrigerator at 4±1oC before use. Different concentrations of plant essential oils (Eos) were prepared by adding 3, 6, 12, 24 and 48 µl of pure Eos in 1 ml of 0.05% (v/v) Tween 80 in case of in vivo and 0.5% (v/v) Tween 80 in case of in vitro experiments.

#### **2.3. In Vitro Antifungal Assay by Agar Dilution Method**

Prepared concentrations of EOs were mixed with sterile molten MEA medium. Thirty milliliters of media containing different concentrations of EOs and 0.5% Tween 80 was poured into each petri plate which was then inoculated with test fungi and incubated for 7 days at 25±1°C.

#### **2.4. In Vitro Volatile Assay to Check Antifungal Activity**

In this method the MEA plates were first inoculated with test fungi and then pure extracted EOs in quantities of 3, 6, 12, 24, and 48 µl were applied on the surface of the sterilized filter paper, which was placed in the lid of the petri plate. After inoculation the plates were incubated in inverted position for seven days at 25±1°C. The zone of inhibition was measured in two directions at right angles to each other. The percentage of mycelial growth inhibition by each essential oil concentration was calculated from the mean colony diameter (cm) on medium without essential oil amendment (control) and from the mean colony diameter (cm) on each essential oil amended plate (zone of growth).

#### **2.5. In vivo antifungal assay**

Fruits were sterilized with 6% sodium hypochlorite solution followed by immersion in sterile distilled water for two minutes and surface sterilization in 70% ethanol for another two minutes. Fruit was wounded (2-wounds per fruit) at the equatorial side with a sterile stainless steel scalpel where each wound was about 4 mm long and 2 mm deep. 15 µl of spore suspension was inoculated into each wound using a micropipette under aseptic conditions. Two hours later, each wound was inoculated with a pre-determined concentration from each plant essential oil. Control fruit was subjected to the same treatments except that sterile distilled water was used instead of essential oil. The treated fruits were labeled, placed in sterilized petri plates and incubated at 23±2 °C for two weeks to assess decay and fungal growth symptoms on daily basis.

#### **2.6. Chemical identification of essential oils by GC-MS**

Qualitative analysis of the tested essential oils was undertaken by gas chromatography-mass spectroscopy (GC-MS) using a Hewlett-Packard mass detector (model 7890) coupled with mass spectrometer selective detector 5975. Analysis was carried out using a column HP5 mass-selective detector (MSD) (30 m x 0.25 mm; 0.25 µm film thickness), the operating conditions were as follows: Helium was the carrier gas at a flow rate of 1 ml/min. diluted samples (1:100 v/v, in methanol) of 1 µl were injected manually at temperature 250°C. NIST (National Institute of Standards and Technologies) Mass Spectra Library was also used as a reference.

## **3. Results and Discussion**

#### **3.1. In vitro antifungal efficacy of essential oil**

In vitro antifungal activity of selected oils were checked through two methods i.e., dilution method and the volatile method (Table 1). A little variation was observed in results of both the assays. In dilution method cumin oil gave best control on the mycelial growth of P. italicum as its lowest concentration of 3µl/ml gave 96% inhibition. This increased with increase in oil concentration and 12µl/ml completely inhibited the fungal growth. The results of cumin oil was followed by clove oil, whose lowest tested concentration i.e., 3µl/ml showed 84% mycelial growth inhibition. However, the complete inhibition of fungal growth was recorded at maximum tested concentration i.e., 48 µl/ml. Cinnamon gave the lowest effect among the three tested EOs.

In volatile method clove oil gave best control of P. italicum instead of cumin that showed highest inhibition of mycelial growth in dilution method perhaps because of high volatility of phenols that are abundantly present in clove essential oil. Clove oil in 3, 6 and 12µl/ml concentrations gave 97% control on tested fungi. Increase in oil concentration of clove oil gave similar results as were recorded when the same oil was checked through dilution method. Complete inhibition of mycelial growth was observed at maximum tested concentration. In case of cumin the lowest tested concentration of 3µl/ml could only inhibit *P. italicum* up to 32% in contrast to its significant control of 96% recorded in dilution method. Concentration increased the inhibition percentage up to 98-99%. Romagnoli et al.,[6] reported a strong antifungal activity against dermatophytes and phytopathogens including fungi and yeast. They also found cumin aldehyde, pinenes, and *p*-cymene, and a fraction of oxygenate compounds such as alcohol and epoxides as the most active ingredients of cumin essential oil. Cinnamon oil also depicted better control on tested fungi in volatile method when compared to the dilution method perhaps because it is also rich in volatile phenols live clove essential oil. However, still the inhibition effect of cinnamon essential oil failed to match with the controlling capacity of other two tested oils.


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**Fig 1.** *In vivo* antifungal activity of **A:** Clove; **B:** Cumin**; C:** Cinnamon essential oils, used at various concentra-

Cumin oil showed complete inhibitory effect at concentration of 48µl/ml while cinnamon oil did not completely inhibit fungal progression even at its highest concentration when applied on citrus fruit. The minimal inhibitory effect of cumin and cinnamon essential oils at lowest tested concentration (3µl/ml) was 88.27 and 83.05 % respectively. Whereas, no significant difference was recorded in inhibitory effects caused by both the cumin and cinnamon essential oils used in

The data presented in Fig. 1B indicated that cumin oil in concentrations of 3 and 6µl/ml resulted in the inhibition of 88.71 and 89.27% respectively and growth started from the 5th day of the inoculation. However the concentrations of 12 and 24µl/ml delayed the mycelial growth in citrus fruit till 6th and 7th day. While the data presented in Fig. 1C shows that the mycelial development of tested fungus was started from 3rd day of inoculation at all tested cinnamon oil concentrations (3-48µl/ml) except the concentration of 24µl/ml that delayed the growth up to 5 days. These results depicted that the efficacy of clove essential oil during in vivo assays followed by

In general, the results obtained from GC-MS analysis of the essential oils used were in accordance to the previous literature. Clove oil shows the presence of eugenol, alpha-terpineol, Isoeugenol and beta-terpinene as its major components. Eugenol has been reported by different

that of cumin, whereas cinnamon oil showed least inhibitory effect.

tions, on mycelial growth of *Penicillium italicum*

higher concentrations of 6 – 48µl/ml.

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\* Values are means (n=3). Mean values followed by different letters within the column are significantly different according to Duncan Multiple Range Test (P<0.05).

**Table 1.** In vitro antifungal activity of plant essential oils, used at various concentrations, on mycelial growth of Penicillium italicum\*

#### **3.2. In Vivo Antifungal Efficacy of selected Essential Oils**

The results of in vivo antifungal efficacy indicated that all the three EOs had a good inhibitory effect on mycelial growth of P. italicum when tested on the surface of the citrus fruit. Yahyazadeh et al.,[7] revealed that essential oil can result in loss of pigmentation in fungal conidia as they became hyaline that may affect virulence of the pathogen; hence a decrease in the incidence of the infection.

Figure 1A, shows that clove essential oil exhibited most pronounced antifungal potentials against P. italicum as it completely inhibited the mycelial growth at concentration of 24µl/ml after 15 days of incubation. At concentration of 3µl/ml the inhibition of P. italicum was 62% and growth started at 4th day of inoculation. Increase in concentration to 6 and 12µl/ml increased fungal inhibition up to 79 and 93% respectively, and also the fungal growth on citrus fruit was delayed till 7th and 11th day.

**Fig 1.** *In vivo* antifungal activity of **A:** Clove; **B:** Cumin**; C:** Cinnamon essential oils, used at various concentrations, on mycelial growth of *Penicillium italicum*

Cumin oil showed complete inhibitory effect at concentration of 48µl/ml while cinnamon oil did not completely inhibit fungal progression even at its highest concentration when applied on citrus fruit. The minimal inhibitory effect of cumin and cinnamon essential oils at lowest tested concentration (3µl/ml) was 88.27 and 83.05 % respectively. Whereas, no significant difference was recorded in inhibitory effects caused by both the cumin and cinnamon essential oils used in higher concentrations of 6 – 48µl/ml.

The data presented in Fig. 1B indicated that cumin oil in concentrations of 3 and 6µl/ml resulted in the inhibition of 88.71 and 89.27% respectively and growth started from the 5th day of the inoculation. However the concentrations of 12 and 24µl/ml delayed the mycelial growth in citrus fruit till 6th and 7th day. While the data presented in Fig. 1C shows that the mycelial development of tested fungus was started from 3rd day of inoculation at all tested cinnamon oil concentrations (3-48µl/ml) except the concentration of 24µl/ml that delayed the growth up to 5 days. These results depicted that the efficacy of clove essential oil during in vivo assays followed by that of cumin, whereas cinnamon oil showed least inhibitory effect.

In general, the results obtained from GC-MS analysis of the essential oils used were in accordance to the previous literature. Clove oil shows the presence of eugenol, alpha-terpineol, Isoeugenol and beta-terpinene as its major components. Eugenol has been reported by different workers to be the most effective component of the clove and cinnamon EOs against various pathogens[8]. Vazquez et al.,[9] reported complete inhibition of P. citrinum by 2000 ppm of eugenol in a liquid medium.

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**Clinical, Serological, Hormonal, Bacteriological** 

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

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

**and Molecular Detection of Brucellosis in** 

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

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

**Aborted Cows and Buffalos**

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

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

**Abstract**

confirmation of brucellosis.

**1. Introduction**

© 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.

Turkey, September 10-12, 2012

<sup>327</sup> ISALS

The major components found in cinnamon oil were eugenol and cinnamaldehyde, whereas cumin oil revealed the presence of gamma-terpinen, cuminaldehyde and 4-carvomenthenol. Singh and Upadhayay[10] showed antifungal activity of Cuminaldehyde against Aspergillus flavus and Aspergillus niger. In a recent study Romagnoli et al.,[6] found cumin aldehyde, pinenes, and p-cymene, and a fraction of oxygenate compounds such as alcohol and epoxides as the most active ingredients of cumin essential oil.

#### **4. References**

