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Article History

Received: 01 October 2018

Accepted: 07 February 2019

Published: 12 February 2019

Characterization of Environmental Factors Conducive for

Ascochyta Blight of Gram and its Chemical Management Safoora Asjad1, Safdar Ali1, Amer Habib1, Muhammad Muntazir Mehdi Khan1, Ahsan Abdullah1,

Anosh Razzaq1, Muhammad Jabran1, Abdul Wahid1, Junaid Asghar1

1. Department of Plant Pathology, University of Agriculture, Faisalabad, Pakistan.

Corresponding author:

Muhammad Muntazir Mehdi Khan

Department of Plant Pathology, University

of Agriculture, Faisalabad, Pakistan

Email: [email protected]

How to Cite?

(Asjad et al., 2019)

Safoora Asjad, Safdar Ali, Amer Habib, Muhammad Muntazir Mehdi Khan, Ahsan Abdullah, Anosh Razzaq, Muhammad

Jabran, Abdul Wahid, Junaid Asghar. Characterization of Environmental Factors Conducive for Ascochyta Blight of Gram

and its Chemical Management. The Int. J. Biol. Res., 2019, 2(1):219-232 (ISSN 2618-1436 (print); ISSN 2618-1444 (online).

(http://www.rndjournals.com/)

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This work is licensed under a Creative Commons Attribution 4.0 International License.

ABSTRACT Gram (Cicer arietinum L.) is an important pulse crop belongs to family Fabaceae. Gram is cultivated worldwide including Europe, Africa, Asia, Australia, South and North America. It is third important pulse crop in world and had great economic importance. It is main source of protein in subcontinent and is grown well even in poor and sandy soil. Gram is vulnerable to many biotic and abiotic stresses. Among the biotic stresses, gram blight that was caused by Aschochyta rabiei which causes severe losses. Favorable environmental conditions further enhance the disease. The present experiment was focused on the management of ascochyta blight by application of chemicals and studied the environmental conditions that cause the disease to spread more. For this purpose, three fungicides were tested in-vitro and best were sprayed in the field. In screening trial four varieties were used. Environmental conditions influence the spread of disease. The disease predictive model was developed for study the influence of environmental variables. A field trial was conducted in research area of Department of Plant Pathology, University of Agriculture, Faisalabad. The experiment following randomize complete block design (RCBD) and the treatments were analyzed statistically by using LSD test. In vitro evaluation of chemicals Gemstar super 325 SC Azoxystrobin+Difenoconazole, Nativo 75 WG, Allete 80%WP was used. Among these Gemstar super 325 SC Azoxystrobin+Difenoconazole and Nativo 75 WG completely inhibits the fungal growth while Allete 80%WP reduces growth when compared to the control. Among the four varieties no one is resistant but 07005 show moderate resistances while others are susceptible to disease. The disease predictive model were developed Y=81.42-122 X1, Y = 73.38 - 1.589 X2, Y = 14.98 + 0.710 X3, Y = 47.42 + 4.87 X4. Where Y= Ascochyta blight, X1= Maximum temperature, X2= Minimum temperature, X3= Relative humidity, X4= Rainfall. All are significant at P<0.05 but negative correlation was observed between maximum temperature (R2=49.56) and minimum temperature (R2=51.49) as the temperature increase the disease decrease but relative humidity R2= (53.16) and rainfall

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R2= (35.72) was positively correlated with disease. Among the treatments Gemstar super 325 SC Azoxystrobin+Difenoconazole was the best one and show significant response in reducing disease. Key words: Gram (Cicer arietinum L.), gram blight INTRODUCTION Gram (Cicer arietinum L.) is usually well-known as chickpea belongs to family leguminosae. It is thought to be the third most essential grain vegetable on the world after dry beans and pea, being broadly cultivated in numerous subtropical and warm-mild places. The family Leguminosae is the third largest family after Orchidaceae and Asteraceae in Spermatophyta and consists of approximately 800 genera and 20,000 species (Lewis et al. 2005; Smy kal et al. 2015). It is thought to be originated in Mediterranean or Himalaya region (Jhorar et al., 1997). Gram was not just an important source of proteins for human and source of animal feed, but also plays important role in nitrogen fixation. (Sharma & Jodha, 1984; Islam et al., 2011). Gram was grown in a wide range of ecological environments in the world. In subcontinent it positions second in food legumes while third in pulses, and is esteemed for its high protein content: 25.3– 28.9% (Berger & Turner, 2007 Gram (Cicer arietimum L.) grasps an important position in vegetable harvests and is the greatest yield producer after wheat and rice for nutrition in Pakistan. (Cowan et al., 1967) revealed that gram is a good source of iron when compared to other. Chickpea being one of the genuine pulse alters in Pakistan, it is commonly grown on the sandy soils of the country where the same harvests of the rabi season can create (Khattak et al., 2007). Gram is a cheap and rich source of protein (20–23% in the grain), oil (3–6%) (carbohydrates, 40%), (Gil et al., 1996) and minerals (P, Mg, Zn, K, Fe and Mn) (Ibrikci et al., 2003) and β-carotene (Milan et al., 2006). Desi and Kabuli are two different types of gram. The Desi types have pinkish flowers, stem have anthocyanin pigmentation, and a thick and coloured seed coat. The Kabuli have white flowers, stem have no anthocyanin pigmentation, and have beige or white coloured seeds, a smooth surface of seed and thin seed coat (Jukanti et al., 2012). The weight of seed was ranges from 0.1 to 0.3g in desi and 0.2 to 0.6g in Kabuli types respectively (Frimpong et al., 2009). Desi types account for about 85% of the entire gram area and are frequently grown in Africa and Asia (Pande et al., 2005). The Kabuli was largely grown in North America, North Africa, West Asia and Europe. There is a increasing demand for gram because of its nutritional value Chickpea is without cholesterol and is a decent source of dietary fibre (DF), vitamins and minerals (Chibbar et al., 2010). Globally, the area of cultivation was 11.67 million hectare which produces 9.31 million tons. India represents roughly 65% of world gram production, second was Pakistan (9.5%) and then Turkey (6.7%), while Africa, Ethiopia are the leading gram producer (Kimurto et al., 2013). Gram is an important winter grain legume sown under rain-fed conditions in Pakistan. It provides a rich and cheap source of vegetable protein for human nutrition (Hulse, 1991). It is an important pulse crop of Pakistan because of successful cultivation during winter in the vast barani area of the country. Gram is grown all over the Pakistan. Ecologically it can be divided into three gram producing regions Southern, Northern and Central regions. Gram is growing on a variety of soils, but saline and alkaline soils are avoided. It is grown on an area of 1052.3 thousand hectares that produces 837 thousand tons with an average yield of 697 kg per ha (Hassan et al., 2012). In Pakistan gram quantitatively accounts as a small portion of country’s total food supply but its qualitative importance is significant as food supplement for the vegetarian diet necessities. Area under cultivation in Pakistan is 1073 thousand hectares and annual production is 842 tons per ha. The average yield of gram is 784 kg per ha. The yield is very low as compared to its potential which could be managed through crop production and integrated pest management (Jabbar et al., 2014). Although many factors contribute towards low gram production but fungal disease blight caused by Ascohcyta rabiei (Pass.) Lab. is the major limiting factor. This disease has been reported in Pakistan as well as in different ram growing parts of the world (Nene et al., 1996). Ascochyta blight, a necrotrophic fungus caused by the pathogen Ascochyta rabiei, is a widespread major foliar disease that causes extensive grain yield losses (up to 100%) (Kimurto et al., 2013). Gram production was seriously dropped by aschochyta blight (Chongo et al., 2001). It is broadly spread and has several physiological races of Ascochyta rabiei. Ascochyta rabiei belongs to order Sphaeropsidales and family Sphaeropsidaceae. Ascochyta blight firstly recognized in 1911 in Subcontinent (Harveson et al., 2010). Crop losses exceeding 50% in South Australia and Victoria. Ascochyta blight has been reported from 29 countries including the Middle East and the Mediterranean and is usually associated with severe reduction in Gram yield and quality, especially


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under cool and wet weather conditions (Kaiser, 1997). The disease is also noticed in Pakistan along with the other gram growing parts of the world (Iqbal et al., 2010). Many epiphytotics of blight disease was described in Pakistan. It arises in nearly all states where gram is grown (Ali et al., 2009). Favorable environmental conditions for the pathogen cause 100% crop loss (Armstrong-Cho et al., 2008). Ascochyta blight is a highly destructive disease in most gram growing areas of the world (Shahid et al. 2008). The pathogen grows asexually on the host plant, while its perfect stage can be recovered from wintering debris (Armstrong et al. 2001; Ahmad et al. 2014). Blight disease affects all parts of the shoot of the plants, producing lesions, and shoot breakage (Pande et al. 2005). The disease significantly reduces seed yields and quality, depending on climatic conditions, yield losses for susceptible cultivars can reach 100 % (Shahid et al. 2008). Economic losses due to blight damage have been substantial in many regions including Australia, Canada, Latin America, southern Europe, the United States of America, and West Asia (Gan et al. 2006). Ascochyta blight infection and disease development occur in the temperature range of 5–30◦C, with an optimum temperature of 200C and relative humidity of >95% (Jhorar et al. 1998). Ascospores are discharged from pseudothecia during periods of rain and may spread over 100m (Trapero-Casas et al. 1996). Similarly, secondary spread of Ascochyta rabiei conidia occurs during rainfall over short distances of∼1m by splash dispersal, whereas the combination of rain and strong wind may spread the conidia up to 10m in the direction of the wind (Shtienberg et al., 2006). Once infection has been established within the field, asexual spores cause secondary spread of the disease. All aerial parts of plant can come under symptomatic stress of ascochyta blight (Shtienberget al., 2000). Emerging seedlings show brown lesions at their base which actually happens due to infected seeds. Progressively, these lesions start increasing in size thus covering and girdling around the stem ultimately resulting in death of the plants. Leaflets bear irregular round to elongated shaped brown lesions showing dark red or dark brown margins. Usually circular lesions with darker margin become visible upon the green pods having concentrically and circularly arranged pycnidia. Involving the serious infections, the lesions prevail to the seeds inside the pods which become shriveled due to fungal fruiting bodies (Islam et al., 2017). Adequate management of ascochyta blight is the key for gram production. Cultural practices such as planting disease free seed, crop rotation with non-host crops and the destruction of infected stubble are all important in reducing the likelihood of an epidemic. Several fungicides proved to be effective for blight control. It can be controlled by fungicide treatment (Nene and Reddy, 1987; Chang et al., 2007). Specific formulations of metalaxyl, captan, thiabendazole, and benomyl are used on gram seed (Wise et al. 2011). On the other hand, systemic fungicides include the quinone outside inhibitors (pyraclostrobin or azoxystrobin), succinate dehydrogenase inhibitor ssuchasboscalidand, since 2007, steroldemethylation inhibitors, such as triazoles (prothioconazole, difenoconazole, etc) (Bahr et al., 2016). Use of chemicals for management of disease was necessary for good production and yield. Cultural practices like healthy seed plantation, crop rotation, destruction of inoculum reduces the chances of disease. But these are not enough for controlling the disease. Therefore, application of fungicides is necessary for suppression of disease (Pande et al., 2005). By considering the above described facts, the field experimental will be lead on gram to achieve these objectives: To screened out different gram cultivar in contradiction of Ascochyta blight disease To check the effectiveness of fungicides in managing Ascochyta blight disease of gram MATERIAL AND METHOD 2.1 In vitro experiment 2.1.1 Collection of diseased samples For the isolation of Ascochyta rabiei, the diseased samples were collected. The selected samples were showing the symptoms of disease. The infected samples show brown lesions in the form of concentric rings. On the inner side lesion was brown and the margin is darker with slightly darker red or brown margins. 2.1.2 Isolation of pathogen Isolation of Ascochyta rabiei is very important to precede the experiment. The isolation of pathogen was carried out from the samples under the controlled condition to avoid contamination. For this purpose, some infected plant parts were taken from diseased plant. Diseased leaves were cut into small pieces 3-5 mm at lesion site. Rinse with water to remove dirt from it and sodium hypochlorite or potassium hypochlorite was used for surface sterilization of leaves surface. These leaves pieces were dried in blotted paper and transfer into the petri plates containing PDA (potato dextrose agar media) with sterilized needle under laminar air flow cabinet to evade contamination. Petri plates were


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sealed and labeled properly with the help of tape and then incubate at 250C. Fungus growth was measured after 36-48 hours after incubation. These plates were observed on daily bases for colony characters on pattern of growth of pathogen. 2.1.3 Mass culturing of Ascochyta rabiei Mass culturing was done by repeated multiplication of isolated culture on the PDA plates. Petri plates were filled one third with PDA medium. A minute piece of fungus was taken from the edge of fungal growth with sterilized needle and placing into the petri plates with PDA in laminar air flow chamber. These petri plates were incubated at room temperature. Fungus growth was measured after 24-36 hours after incubation. These plates were observed on daily bases for colony characters on pattern of growth of pathogen. 2.1.4 Identification of pathogen For the microscopic identification of pathogen slides were prepared. For the preparation of slide first of all place a drop of water on the sterilized glass slide. Then add a small quantity of purified culture in this water drop and cover the slide with the help of cover slip. Then place the slide under microscope. First the pathogen was observed under low magnification of microscope then at high magnification so that the fungus will be clearly visible. 2.2 Evaluation of chemicals 2.2.1 Preparation of fungicidal concentration Three fungicides were tested against Ascochyta rabiei. Three fungicides Gemstar super 325 SC Azoxystrobin+Difenoconazole, Nativo 75 WG, Allete 80%WP will be used for treatment. Two concentrations (250 ppm and 130ppm) of each fungicide R and R/2 were prepared for testing them against Ascochyta rabiei. For preparation of concentration R, the recommended dose of fungicide was used. The recommended dose of Gemstar super 325 SC Azoxystrobin+Difenoconazole, Nativo 75 WG, Allete 80%WP was 1g/L. The recommended dose of each was added into 100ml water for the preparation of standard solution represented by R. R/2 was half of standard solution was prepared by taking 500 ml of the recommended dose into a separate flask. All these concentrations of fungicides were used for conducting inhibitory studies on Ascochyta rabiei. 2.2.2 Poison food technique In vitro evaluation of chemicals was carried by using a poison food technique. Potato dextrose was made by using standard method. After the preparation of PDA, prepare the solution of 1000 ppm as a stock solution of chemicals. From this solution prepare two concentrations of each chemical 130 ppm and 250 ppm, and then pour it to the petri plates. The fungicides containing media was used for testing antifungal activity of fungicide against the mycelial growth of the fungus. Three replicates of each treatment were made under CRD design. These plates were then wrapped under laminar flow chamber and incubate at room temperature and radial growth of the fungus was recorded daily. Effect of these concentrations of selected fungicides was calculated by colony diameter (cm) and by using formula of inhibition percentage of pathogen growth. This formula was described by Venkateswarlu and Sreeramulu (2013). Inhibition %= Diameter of colony-Diameter of colony in treatment ×100/Diameter of colony of control I= (C-T)/C×100 I = inhibition percentage of mycelial growth C = Mycelial growth pathogen in control plate T = Mycelial growth of pathogen in inoculated plate 2.3 Screening of gram varieties against Ascochyta blight disease 2.3.1 Collection of gram varieties The evaluation of gram varieties for resistance against Ascochyta blight was done by using four varieties. These varieties were V.K 15011, 07005, V.N. 15014 and Noor-2013. These varieties of gram were collected from Pulses Research Institute, Ayub Agriculture Research Institute, Faisalabad. 2.3.2 Establishment of nursery The disease screening nursery was established. After measuring and leveling the land, sub-plots were made for each replicate. Three replications were used for the management in the trial. Plants were seeded R × R 25-40cm, P × P is 10cm, replicate row 91cm and replicate length was 243 cm. For the trial Randomize Complete Block Design (RCBD) was used in the field. After establishing the nursery it was screened against Ascochyta blight of gram. The gram varieties were evaluated concerning disease incidence and severity. Fungicides which were found effective from in-vitro evaluation were used in the field.


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2.3.3 Data recording Data were recorded when plants show the symptoms of disease. The varieties was screened out against disease by using the disease incidence and severity formula and the following disease rating scale will be used for the assessment o Table2.1 Disease rating scale Disease incidence for this purpose was calculated by using following formula:

Disease incidence = number of infected plants/total number of plants×100 Disease severity was calculated by using formula: Disease severity = number of infected leaves/total number of leaves×100 2.4 In-vivo evaluation of fungicides against gran blight 2.4.1 Preparation of fungicidal formulation Three fungicides Gemstar super 325 SC Azoxystrobin+Difenoconazole, Nativo 75 WG, Allete 80%WP were used for the control of disease. These three fungicides concentrations were prepared at recommended dose 1g/L. These fungicides were weighed by using electric weight machine for proper measuring of fungicides. Then each fungicide was mixed in 1 litre of water separately. 2.4.2 Incorporation of fungicides Among the treatments three were fungicides and fourth was controlled. Three replicates were made for all the treatments and each variety with four rows. Three fungicides were separately applied in separate row with fourth was controlled. Fungicides were sprayed at an interval of 7 days for the evaluation of their effect on the management of disease. The results of these treatments were recorded on the basis of disease incidence, disease severity and on the basis of disease decrease over control by using formula: Disease incidence (%) = number of infected plants/total number of plants×100 Disease severity (%) = number of infected leaves/total number of leaves×100 Percentage disease decrease over control = Disease in control – Disease in treatment/Disease in control×100 2.5 Environmental data collection from Metrological Department of UAF The data of temperature, rainfall and relative humidity was taken from the Metrological Department of University of Agriculture, Faisalabad. 2.6 Statistical analysis Data were recorded before and after applying fungicides. All the recorded data were processed through statistical test f disease (Rehman et al., 2013). RESULTS 3.1 Characterization of pathogen The fully developed colony of Ascochyta rabiei was observed for 4 days after incubation at room temperature. The colony of Asocochyta rabiei shows whitish color. The fully mature colony of Ascochyta rabiei shows greyish white mycelial growth. 3.2 In-vitro evaluation of fungicides against Ascochyta rabiei by poisoned food technique Three fungicides Gemstar super 325 SC Azoxystrobin+Difenoconazole, Nativo 75 WG and Allete 80%WP were evaluated for their inhibitory effect against Ascochyta rabiei by poisoned food technique. Two concentrations were used for all of these fungicides. The results were recorded for 4 days based on the colony diameter (cm) and percentage of inhibition. All of these treatments minimize the colony growth (Table 3.1, 3.2). Gemstar super 325 SC Azoxystrobin+Difenoconazole and Nativo 75 WG both were more effective fungicides and completely inhibit the growth. Allete 80%WP was at second numeral effective and reduces colony growth effectively when compared to control.

Sr. No Scale Disease reaction

1 1 Highly resistant

2 3 Resistant

3 5 Moderately resistant

4 7 Susceptible

5 9 Highly susceptible


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Table 3.1 In-vitro evaluation of fungicides against Ascochyta rabiei

Sr. no Treatments Colony Diameter (cm)

Percentage of Inhibition (%)

1 Control 2.36 a 0%

2 Nativo 75 WG (130ppm) 0.0 b 100%

3 Nativo 75 WG (250ppm) 0.0 b 100%

4 Allete 80%WP (130ppm) 0.86 c 63.55%

5 Allete 80%WP (250ppm) 0.73 c 69.06%

6 Gemstar super 325 SC Azoxystrobin+Difenoconazole (130ppm)

0.0 b 100%

7 Gemstar super 325 SC Azoxystrobin+Difenoconazole (250ppm)

0.0 b 100%

Mean values were average of three values Mean values were followed by same letters were not significantly different at (P < 0.05). Table 3.2 Analysis of Variance for evaluation of fungicides

ANOVA

Source DF SS MS F P

Treatment 6 24.5114 4.08524 672.86 0.0000

Date 3 1.8467 0.61556 101.39 0.0000

Treatment*Date 18 3.8800 0.21556 35.50 0.0000

Error 56 0.3400 0.00607

Total 83 30.5781

Significance at P<0.05

Fig 3.2 in vitro efficiency of fungicides against Ascochyta rabiei


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3.3 Screening of gram varieties against ascochyta blight For the evaluation of resistance of gram cultivar against blight four varieties were sown under Randomized Complete Block Design (RCBD) and were screened out with the help of disease rating scale. The results of screening trial revealed that no variety was resistant to disease while one line 07005 was moderately resistant to disease (Table 3.3, 3.4). Table 3.3 Gram varieties evaluated against blight

Table 3.4 Response of gram varieties against Ascochyta blight

Variety Disease Incidence (%) Grading Response

1 88.41 7 S

2 63.66 5 MR

3 96.23 9 HS

4 80 7 S

3.4 Development of Ascochyta rabiei and environmental models The objective of investigation is to understand and explain the relationship among different environmental variables. In statistical relationship, the relation between variable is not known exactly and we have to approximate the relationship and develop model that characterize their main features. Regression analysis is concerned with developing such "approximating" models.In the present study data of environmental conditions including disease severity was collected and was evaluated for the development of predictive models for Ascochyta blight. The influence of these environmental conditions on Ascochyta blight was determined by correlation. 3.5 Evaluation of environmental variables and environmental conditions for the development of ascochyta blight The important environmental factors in the disease development were temperature (maximum and minimum), relative humidity and rainfall. In the present study these environmental variables were recorded in three different time intervals and the corresponding value for the disease severity of Ascochyta blight. The corresponding value for disease incidence was found to its peak of 59.01% at maximum Temperature of 20.680C similar pattern of disease development was observed during the temperature of 28.610C the disease severity was decreased to 49.27%. The lowest value for disease severity was recorded during high maximum temperature of 32.710C. Maximum temperature has influence on the disease as it increase the disease severity decreases. Similarly minimum temperature was recorded at three intervals and the corresponding value for disease incidence was on its peak at lower temperature. The disease was 59.01% at the temperature of 9.590C, 49.27% at 14.730C and the lowest value of diseases was observed at 18.340C where disease severity was 40.95%. The relative humidity conductive for disease development was recorded and the disease was 59.01%, 49.27% and 40.95% at the relative humidity of 60.29%, 50.71% and 40.29%. Relative humidity also contributed towards the disease development at high level of humidity there is increase in disease but as the humidity decrease there is less disease incidence. Rainfall also play a significant role in disease development high rainfall is conductive to disease spread. There was decrease in disease when there is no rainfall the disease incidence was 45.11% at 0mm rainfall and 59.01% at average rainfall of 2.07mm. So rainfall also contributed towards the disease development as it increases the chances of disease spread also increases. 3.6 Correlation of environmental conditions with ascochyta blight The correlation of environmental variables i.e maximum temperature, minimum temperate, relative humidity and rainfall with Asochyta blight was observed (Table 3.5). A significant (P<0.05) but negative correlation was observed

Sr. No. Scale Disease reaction Varieties

1 1 Highly resistant

2 3 Resistant

3 5 Moderately resistant 07005

4 7 Susceptible Noor-2013 V.K. 15011

5 9 Highly susceptible V.N. 15014


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between maximum temperature and disease severity of Ascochyta blight. The value of coefficient of correlation observed was r=-0.70. As the temperature increases the disease severity decreases and the level of significance is < 0.05. There is also negative correlation between minimum temperature and disease severity, as the minimum temperature increases, disease severity decreases. The value of coefficient of correlation coefficient observed was r=-0.71. The relationship was significant at P<0.05 (Table 3.5). There is positive correlation between relative humidity and disease severity as the humidity increases disease severity also increases. The relationship was found significant at P<0.05 and the value of correlation coefficient was r=0.72. There is also positive correlation between rainfall and disease severity, as there is increase in rainfall the disease severity also increases and the level of significance for rainfall is significant at P<0.05 and the value of correlation coefficient is r=0.59 (Table 3.5). Table 3.5 Correlation: variety, disease severity, Temperature (maximum and minimum),

relative humidity and rainfall

Variety Disease severity

Temp. (max.)

Temp (min.)

Relative humidity

Disease severity 0.066 0.702

Temperature (maximum)

0.000 1.000

-0.704 0.000

Temperature (minimum)

0.000 1.000

-0.718 0.000

0.997 0.000

Relative humidity 0.000 1.000

0.729 0.000

-0.979 0.000

-0.992 0.000

Rainfall

0.000 1.000

0.598 0.000

-0.942 0.000

-0.912 0.000

0.854 0.000

Fig 3.3 Effects of environmental variables on disease incidence


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3.7 Evaluation of fungicides against Ascochyta rabiei The disease management is by chemical control. Three fungicidal treatments were used for in-vivo evaluation of fungicides. The chemicals used were Gemstar super 325 SC Azoxystrobin+Difenoconazole, Nativo 75 WG, Allete 80%WP for the management of disease. These chemical significantly reduces the disease. For the management of Ascochyta rabiei on gram these fungicides were used at three time intervals and data was recorded after each spray. 3.7.1 Evaluation of fungicides after third spray against Ascochyta rabiei The three same fungicides Gemstar super 325 SC Azoxystrobin+Difenoconazole, Nativo 75 WG and Allete 80%WP were again applied after the interval of one week to check the disease incidence and disease severity and data was analyzed statistically as shown in Table. All the fungicides reduces the disease effectively with Gemstar super 325 SC Azoxystrobin+Difenoconazole was most effective fungicide for controlling gram blight, followed by Nativo 75 WG was found to be second best treatment an then Allete 80%WP (Figure 4.16, 4.17). The best variety was 07005. Statistical data also show significant results at P<0.05 shown in (Table 4.22, 4.23). Table 3.6 Analysis of variance for disease incidence of 3rd spray

ANOVA

Source DF SS MS F P

Variety 3 1316.7 438.89 52.67 0.0000

Tretment 3 19850.0 6616.67 794.00 0.0000

Variety*Tretment 9 933.3 103.70 12.44 0.0000

Error 32 266.7 8.33

Total 47 22366.7

Significant at P<0.05 Table 3.7 Analysis of variance for disease severity of 3rd spray

ANOVA

Source DF SS MS F P

Variety 3 946.9 315.62 20.95 0.0000

Tretment 3 18580.9 6193.62 411.16 0.0000

Variety*Tretment 9 953.3 105.92 7.03 0.0000

Error 32 482.0 15.06

Total 47 20963.0

Significant at P<0.05

Fig 3.4 Disease incidence after 3rd spray


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Fig 3.5 Disease severity after 3rd spray DISCUSSION Gram (Cicer arietinum L.) usually known as chickpea is thought to be the third most essential grain vegetable on the world after dry beans and pea, being broadly developed in numerous subtropical and warm-mild places. Pakistan contributed 9.5% of total gram production in world. Gram is a important source of proteins and other mineral nutrition. Unfortunately, the crop was attacked by more than 50 pathogens but ascochyta blight was most destructive disease of gram (Singh et al., 1984). The current study was conducted to discover the solution of this problem. Different fungicides were used to control the disease. According to strategy three fungicides were used at two different concentrations in in-vitro experiment against Ascochyta rabiei. Three replicates of all treatments were made under Complete Randomized Design (CRD) and this experiment was directed in lab of Department of Plant Pathology, University of Agriculture, Faisalabad. Three fungicides were used are Gemstar super 325 SC Azoxystrobin+Difenoconazole, Nativo 75 WG and Allete 80%WP at two different concentrations at 130 ppm and 250 ppm and the best of them were applied in in-vivo experiment. Among these fungicides Azoxystrobin+Difenoconazole and Nativo 75 WG were completely inhibit the colony growth of Ascochyta rabiei at both concentrations and the percentage of inhibition was 100% followed by Allete 80%WP show inhibition 63.55% at 130 ppm and 69.06% at 250 ppm. In 2007 Chang et al., conducted a trial had resemblance to present study to observe mycelia growth in the presence of fungicides pyraclostrobinat and chlorothalonil at different concentrations (0.1, 0.12, 0.5, 0.15, 0.25, 0.3 ppm). The results show 50% reduction in the spore germination and radial growth when compare with control. Another trial was conducted by kosIada et al., 2013 uses commercially available fungicides for the in-vitro suppression of blight at the concentrations of 1, 10, 100, 1000 mg dm-3 by using PDA. Tebuconazole, spiroxamine and triadimenol results complete inhibition of growth and benomyl, azoxystrobin, thiophanate and chlorotalonil least significantly reduce the mycelial growth. Screening of gram varieties was done to find out the better one which tolerates the disease and show better growth responses. Four varieties V.K 15011, 07005, V.N. 15014 and Noor-2013 were evaluated on the basis of disease incidence. The result tells that no variety was found to be resistant against blight but one variety was found to be moderately resistant against Ascochyta blight was 07005 other two varieties Noor-2013 and V.K. 15011 was susceptible and V.N. 15014 was found to be highly susceptible to Ascochyta blight. Rehman et al., 2013 uses twenty varieties BALKASSAR-2000, C-2000, DG-92, KARAK-3, KARAK-2, KARAK-2, PUNJAB-91, VANHAR2000, PB-2008, Noor-2009, BITAL-


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98, C-98, C-88, DASHT, KARAK-1, KARAK-98, LAWAGHAR-2000, NOOR-91, PAIDAR-91, PARBAT, PUNJAB-2000, SHEENGHAR-2000, CM-2000, THAL-2006 and CM-2008 for evaluating varieties that show resistance against blight. None of them was highly resistant to disease. Environmental factors i.e., temperature (maximum, minimum), relative humidity and rainfall play a main role in the development of disease. Correlation of these factors shows that temperature (maximum, minimum) was negative towards disease development as decrease in temperature increase the chances of disease and rainfall and humidity was positively correlated as increase in both of them also increases the chances of disease. In the present study Ascochyta blight model was developed to check out the contribution of these factors on disease. The model was statistically justified (R2 =49.56) at P<0.05 that developed to predict probable attack of Asochyta rabiei under maximum temperature given as under: Y=81.42-122 X1. R2=49.56 Where Y= Ascochyta blight, X1= Maximum temperature. It indicated that 1 unit increase in maximum temperature there would be probable 1.22 unit decreases in disease. Similarly, minimum temperature predictive model is statistically justified (R2 =51.49) at P<0.05 was as under: Y = 73.38 - 1.589 X2. R2=51.49 Where Y= Ascochyta blight, X2= Minimum temperature. The model predicts that one unit increase in temperature there is 1.589 times decrease in disease severity. Relative humidity also play role in disease development and model was developed (R2=53.16) at P<0.05 and the model summary was as under Y = 14.98 + 0.710 X3.

R2=53.16 Where Y= Ascochyta blight, X3= Relative humidity. The model predicts that on unit increase in relative humidity there is 0.710 times increase in disease. Also, rainfall model is statistically justified (R2=35.72) at P<0.05 was as under: Y = 47.42 + 4.87X4.

R2=35.72 Where Y= Ascochyta blight, X4= Rainfall. It is evident from the model that factor responsible for the attack of Ascochyta raiei includes rainfall. The model predicts that on unit increase in relative rainfall there is 4.87 times increase in disease severity. Ketelaer et al., 1988 states monthly high rainfall and at minimum 80C temperature was conductive to the disease epidemic. This model prediction uses to restrict the spread of Ascochyta blight. Trapero-casas, (1992) develop a model for environmental variables responsible for gram blight. The results show that low temperature and humidity significantly influence on the spread of disease. Regression model predicts that at the temperature of 210C with humid period of 7 hours causes significantly influence on infection (Y=25%). The results of their study reveals that the temperature and humidity influence on the blight disease and helps to predicts the chances of Ascochyta blight on different areas. Ali, (2011) finds considerable influence of environmental variables on cotton and the model helps to predict the CLCuVD considering influence of environmental variables (Y=145+4.47X1-0.151X2-0.490X3) Where Y= CLCuVD, X1= minimum temperature, X2= rainfall, X3= relative humidity) and uses step by step regression analysis to develop model for prediction of disease. All environmental variables show significant correlation. Fungicides were more likely to be used for the control of Ascochyta blight. Three fungicides were used in in-vivo experiment that found good in in-vitro trial are Gemstar super 325 SC Azoxystrobin+Difenoconazole, Nativo 75 WG, Allete 80%WP. Three sprays were used with different time intervals. In-vivo experimental was directed in research area of Department of Plant Pathology, University of Agriculture, Faisalabad following the Randomized Complete Block Design (RCBD) with three replications. For this purpose four varieties and three treatments were used. The effect of these FUNGICIDES was observed in disease severity and disease incidence. Gemstar super 325 SC


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Azoxystrobin+Difenoconazole was proved most effective fungicides on all varieties followed by Allete 80%WP and then Allete 80%WP in regulating disease over control. Demirci et al., 2003 works on in-vitro evaluation of fungicides by using difenoconazole, diniconazole, Diniconazole and tebuconazole. Among these tebuconazole, difenoconazole and diniconazole were most effective while others are not very effective. In 2013 Rehman et al., use five fungicides mancozeb, thiophenate methyl, difenaconazole, chlorothalonil and metalxyl + mancozeb for the varieties Vanhar-2000, Noor2009, CM- 2008, Thal-2006 and PB-2008 was used in the field. All fungicides were reducing the disease after two sprays with different time intervals. CONFLICT OF INTEREST: Nothing to declare FUNDING: Nothing to declare 1. Ahmad, S., M.A. Khan, S.T. Sahi and R. Ahmad. 2014.

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