PHYSIOLOGICAL AND MANAGEMENT STUDIES ON Alternaria alternata (Fr.) Keissler-THE INCITANT OF LEAF BLIGHT OF GROUNDNUT (Arachis hypogaea L.)
THESIS
SUBMITTED TO THE RAJASTHAN AGRICULTURAL UNIVERSITY, BIKANER IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
IN
AGRICULTURE (PLANT PATHOLOGY)
BY
SHANKER LAL KANTWA
2006
CONTENTS
S. No. Particulars Page No.
1. INTRODUCTION ......
2. REVIEW OF LITERATURE ......
3. MATERIALS AND METHODS ......
4. EXPERIMENTAL RESULTS ……..
5 DISCUSSION ......
6. SUMMARY ......
LITERATURE CITED ......
ABSTRACT (ENGLISH) ......
ABSTRACT (HINDI) ......
RAJASTHAN AGRICULTURAL UNIVERSITY, BIKANER S.K.N. COLLEGE OF AGRICULTURE, JOBNER
CERTIFICATE - I
Dated : ...... /2006
This is to certify that Mr. SHANKER LAL KANTWA had successfully completed the Comprehensive Examination held on 28th June,
2006 as required under the regulation of Master of Science.
(R.P. MAHARSHI)
Head Department of Plant Pathology S.K.N. College of Agriculture, Jobner
RAJASTHAN AGRICULTURAL UNIVERSITY, BIKANER S.K.N. COLLEGE OF AGRICULTURE, JOBNER
CERTIFICATE - II
Dated : ...... /2006
This is to certify that this thesis entitled “Physiological and management studies on Alternaria alternata (Fr.) Keissler-the incitant of leaf blight of groundnut (Arachis hypogaea L.)” submitted for the degree of Master of Science in the subject of Plant Pathology of the Rajasthan Agricultural University, Bikaner is a bonafide research work carried out by Mr. SHANKER LAL KANTWA under my guidance and supervision and that no part of this thesis has been submitted for any other degree. The assistance and help received during the course of investigation have been fully acknowledged. The draft of the thesis was also approved by the advisory committee on …………..
(R.P. MAHARSHI) (K.S. SHEKHAWAT) Head Major Advisor Department of Plant Pathology
DEAN S.K.N. College of Agriculture, Jobner
RAJASTHAN AGRICULTURAL UNIVERSITY, BIKANER S.K.N. COLLEGE OF AGRICULTURE, JOBNER
CERTIFICATE – III
Dated : ...... /2006
This is to certify that this thesis entitled “Physiological and management studies on Alternaria alternata (Fr.) Keissler-the incitant of leaf blight of groundnut (Arachis hypogaea L.)” submitted by Mr. SHANKER LAL KANTWA to the Rajasthan Agricultural University, Bikaner in partial fulfilment of the requirements for the degree of Master of Science in the subject of Plant Pathology, after recommendation by the external examiner was defended by the candidate before the following members of the examination committee. The performance of the candidate in the oral examination on his thesis has been found satisfactory, we therefore, recommend that the thesis be approved. (R.P. MAHARSHI) (K.S. SHEKHAWAT) Head Major Advisor Department of Plant Pathology
(S.C. JAIN) Advisor
APPROVED (M.L. JAKHAR) Advisor
(M.L. SHARMA) DEAN, Dean, PGS Nominee Post Graduate Studies RAJASTHAN AGRICULTURAL UNIVERSITY, BIKANER S.K.N. COLLEGE OF AGRICULTURE, JOBNER
CERTIFICATE - IV
Dated : ………../2006
This is to certify that Mr. SHANKER LAL KANTWA of the Department of Plant Pathology, S.K.N. College of Agriculture, Jobner has made all corrections/modifications in the thesis entitled “Physiological and management studies on Alternaria alternata (Fr.) Keissler-the incitant of leaf blight of groundnut (Arachis hypogaea L.)” which were suggested by the external examiner and the advisory committee in the oral examination held on ………/2006. The final copies of the thesis duly bound and corrected were submitted on ………/2006, are enclosed herewith for approval.
(K.S. SHEKHAWAT) Major Advisor
Enclosed one original and two bound copies of the thesis. Forward to the Dean, Post Graduate Studies, RAU, Bikaner, through the Dean, S.K.N. College of Agriculture, Jobner.
(R.P. MAHARSHI)
DEAN Head S.K.N. College of Agriculture Department of Plant Pathology Jobner
LIST OF TABLES
Table Particulars Page No. No. 4.1 Mycelial growth and sporulation of Alternaria alternata on …… different solid media (after 7 days of incubation at 25+ 20C) 4.2 Mycelial growth and sporulation of Alternaria alternata on …… different liquid media (after 7 days of incubation at 25+20C) 4.3 Effect of temperature on the mycelial growth and …… sporulation of Alternaria alternata in vitro (after 7 days of incubation) 4.4 Effect of relative humidity on the mycelial growth and …… sporulation of Alternaria alternata (after 7 days of incubation at 25 + 20C) 4.5 Mycelial growth and sporulation of Alternaria alternata at …… different hydrogen-ion concentration (after 7 days of incubation at 25 + 20C) 4.6 In vitro efficacy of bio-control agents against Alternaria …… alternata in dual culture plate method 4.7 In vitro efficacy of phytoextracts against mycelial growth …… of Alternaria alternata (after 7 days of incubation at 25+20C) 4.8 In vitro efficacy of phytoextracts against sporulation of …… Alternaria alternata (after 7 days of incubation at 25+2 0C) 4.9 In vitro efficacy of fungicides against mycelial growth of …… Alternaria alternata (after 7 days of incubation at 25 +2 0C) 4.10 In vitro efficacy of fungicides against sporulation of …… Alternaria alternata (after 7 days of incubation at 25 +20C) LIST OF FIGURES
Figure Particulars Page No. No. 4.1 Mycelial growth and sporulation of Alternaria alternata on …… different solid media (after 7 days of incubation at 25+2 0C)
4.2 Mycelial growth and sporulation of Alternaria alternata on …… different liquid media (after 7 days of incubation at 25+2 0C)
4.3 Effect of temperature on the mycelial growth and …… sporulation of Alternaria alternata in vitro (after 7 days of incubation)
4.4 Effect of relative humidity on the mycelial growth and …… sporulation of Alternaria alternata (after 7 days of incubation at 25+2 0C)
4.5 Mycelial growth and sporulation of Alternaria alternata at …… different hydrogen-ion concentration (after 7 days of incubation at 25+2 0C)
4.6 In vitro efficacy of phytoextracts against mycelial growth of …… Alternaria alternata (after 7 days of incubation at 25+2 0C)
4.7 In vitro efficacy of phytoextracts against sporulation of …… Alternaria alternata (after 7 days of incubation at 25+2 0C)
4.8 In vitro efficacy of fungicides against mycelial growth of …… Alternaria alternata (after 7 days of incubation at 25+2 0C)
4.9 In vitro efficacy of fungicides against sporulation of …… Alternaria alternata (after 7 days of incubation at 25+2 0C)
LIST OF PLATES
Plate PLATE Page No. No. 1a. Conidia of Alternaria alternata ……… 1b. Conidia with chain ……… 2a. Pathogenicity test ……… 2b Symptomology ……… 3a. Mycelial growth and sporulation of Alternaria alternata ……… on different solid media (after 7 days of incubation at 25+ 20C) 3b. Mycelial growth and sporulation of Alternaria alternata ……… on different liquid media (after 7 days of incubation at 25+20C) 4a. Effect of temperature on the mycelial growth and ……… sporulation of Alternaria alternata in vitro (after 7 days of incubation) 4b. Effect of relative humidity on the mycelial growth and ……… sporulation of Alternaria alternata (after 7 days of incubation at 25 + 20C) 5. In vitro efficacy of bio-control agents against Alternaria ……… alternata in dual culture plate method 6a In vitro efficacy of fungicides against mycelial growth of ……… Alternaria alternata at 50 ppm concentration (after 7 days of incubation at 25 + 20C) 6b In vitro efficacy of fungicides against mycelial growth of ……… Alternaria alternata at 100 ppm concentration (after 7 days of incubation at 25 + 20C) 6c In vitro efficacy of fungicides against mycelial growth of ……… Alternaria alternata at 200 ppm concentration (after 7 days of incubation at 25 + 20C) 6d In vitro efficacy of fungicides against mycelial growth of ……… Alternaria alternata at 500 ppm concentration (after 7 days of incubation at 25 + 20C) 6e In vitro efficacy of fungicides against mycelial growth of ……… Alternaria alternata at 1000 ppm concentration (after 7 days of incubation at 25 + 20C)
ACKNOWLEDGEMENT
I take this privilege to express my deep sense of gratitude and indebtedness to Dr. K.S. Shekhawat, Assistant Professor, Department of Plant Pathology, S.K.N. College of Agriculture, Jobner (Jaipur), for his valuable guidance, constant help, keen interest and ever inspiring encouragement during the period of course work, investigation and preparation of this comprehensive work. With full regard and honour I wise to place my sincere and heartfelt gratitude to members of my advisory committee Dr. S.C. Jain, Asstt. Prof., Deptt. of Plant Pathology, Dr. M.L. Jakhar, Asstt. Prof., Deptt. of Plant Breeding and Genetics and Dr. M.L. Sharma (Dean PGS), Assoc. Prof., Deptt. of Biochemistry, whose incessant help, constant advices and engrossing guidance made this task turn to success. I am highly grateful to Dr. R.P. Maharshi, Head, Department of Plant Pathology, S.K.N. College of Agriculture, Jobner for constructive suggestion, generous assistance and for rendering help as and when needed. I owe my sincere reverence to Dr. R.A. Singania, Dean, S.K.N. College of Agriculture, Jobner for sublime encouragement and providing necessary facilities for conducting the present investigation. My warm and sincere thanks to Dr. R.G. Jat and Sh. R.R. Ahiar, Asstt. Prof., Deptt. of Plant Pathology, other staff members for their everwilling help and valuable suggestions received during the course of investigation. I offer my sincere thanks to Dr. B.L. Jakhar, M.L. Tetarwal, B.R. Choudhary, Tara Chand, Gopal, Ashok, Jaipal, Rajendra, Kailash, Satyanarayan, Bhagwan Sahay, A.K. Bochalya and Jhabar who boosted my morale and extended unreserved help of varied nature. Word fail to convey my sense of profuse regards and affection to my respected mother Smt. Prabhu Devi and father Sh. Kana Ram Kantwa their blessing helped me to accomplish every such step of my academic career. I deeply owe my sense of regards to my dear elder brother Dr. S.R. Kantwa (Scientist), L.R. Kantwa, R.D. Kantwa and my sister Santosh whose ever inspiring thoughts boosted up my moral, throughout my studies. Word cannot express my heartiest feeling of gratitude to my spouse dear Mrs. Kokila Kantwa who has provided me mental support and inspiration not only in the course of study but also in various movements of troubles in my life. I fell pleasure in expressing souly love to my son Bajrang and Mahesh, whose smiles paved a way of roses for me and will go and go an motivating me for a delightful future I am also grateful to Sh. R.K. Bana, Shivam Computer's & Training Centre, Jobner, who typed this manuscript neatly and efficiently within a very short period. Place : Jobner Dated : ……….. (SHANKER LAL KANTWA) Physiological and management studies on Alternaria alternata (Fr.) Keissler-the incitant of leaf blight of groundnut (Arachis hypogaea L.)
SHANKER LAL KANTWA* Dr. K.S. SHEKHAWAT** (Research Scholar) (Major Advisor)
ABSTRACT
Leaf blight caused by Alternaria alternata was observed on leaves of groundnut (Arachis hypogaea L.) plants collected from farmer field around vicinity of Jobner. The fungus was isolated from infected leaves and found pathogenic under artificial inoculation conditions. Disease symptom appeared as small, isolated scattered pale yellow and orange-brown spots on the leaves. The lowest leaves were affected first and the disease progress upward. There were concentric rings with narrow chlorotic zone around the spots on the older leaves. Out of six different solid and liquid media tested for mycelial growth and sporulation of Alternaria alternata, potato dextrose agar and broth media was found best. Maximum growth and sporulation was observed at 25 0C temperature, 100 per cent relative humidity and pH 6.5. Four antagonist agents were tested against Alternaria alternata in vitro, Trichoderma polysporum was found most effective antagonist against Alternaria alternata in dual culture plate method. Among plants extracts, garlic clove extract was found most effective in inhibiting the mycelial growth and sporulation of Alternaria alternata followed by neem and datura leaf extracts. Babul leaf extract was found least effective in inhibiting the mycelial growth and sporulation of fungus in food poison technique. In general, as the concentration of plant extracts increased the inhibition of mycelial growth also increased and sporulation decreased. Among seven fungicides tested in vitro, mancozeb gave complete inhibition of mycelial growth and sporulation at 1000 ppm concentration followed by copper oxychloride and captafol. Propiconazole was least effective followed by captan and chlorothalonil. In general, as the concentration of fungicide increased the inhibition of mycelial growth also increased and sporulation decreased.
* Post graduate student, M.Sc. (Ag.), Deptt.of Plant Pathology, S.K.N. College of Agriculture, Jobner. ** Thesis submitted in partial fulfilment of the requirement for M.Sc. (Ag.), degree in Plant
Pathology under supervision of Dr. K.S. Shekhawat, Assistant Professor, Deptt. of Plant
Pathology, S.K.N. College of Agriculture, Jobner, Rajasthan Agricultural University,
Bikaner.
1. INTRODUCTION
Groundnut (Arachis hypogaea L.) is mainly grown as an oilseed kharif crop in India. Groundnut is also known as peanut, earthnut, monkeynut, goobernut, pinda and manillanut. De Candolle (1986) stated its origin to be in
Brazil (south America). The genus Arachis comes under sub family
Papillionaceae of the family Leguminaceae. The crop is principally cultivated as an oilseed but considerable quantities are used directly for human consumption like other pulses. The kernels are widely acknowledged as a rich and cheap source of vegetable protein. The chemical composition of the kernels, indicating their value as food as : protein 25.33, carbohydrates 10.20, fat 40.50, fibre 3.4 and ash 1.9 per cent. However, as source of oil, the groundnut finds its largest use thus deriving the reputation as “King of oil seed crops” (Reddy, 1976).
The groundnut is presently grown on a commercial scale in about 82 different countries in the world. In India, the total coverage under this crop is about 6.7 million hectare with an annual seed production 7.0 million tonnes (Anonymous, 2004-05). Major growing states of India are
Gujarat, Andhra Pradesh and Tamil Nadu. Among the different states in India,
Gujarat states first in its production while Andhra Pradesh in area. Rajasthan state seventh in term of area and production. The cultivation of groundnut is well adopted to be condition prevailing in Rajasthan and is cultivated in about
212040 hectares with an annual production 338118 tonnes (Anonymous,
2003-04) Jaipur, Bikaner, Sikar, Churu and Dausa are the major groundnut, producing district of Rajasthan.
All parts of the groundnut plants affected by disease causing losses of different magnitudes. The diseases, which affect the foliage cause extensive damage to tissues involved in photosynthesis which may because serious limiting factors in groundnut production.
Leaf blight of groundnut caused by Alternaria alternata is newly reported by Vyas et al. (1985) which is an important disease in the major groundnut growing areas of the state. The fungus causes yellowish green speaks of small size on the leaf lamina. The shape of the yellowish speck may be round, oval or irregular, could be seen clearly by holding leaf against light. As the disease advances dark-brown spots begin to appear in the centre of speck of which under favourable conditions begin to expand and term‟s dark-brown having characteristic concentric rings. When the disease is in severe form several such dark-brown spots appear on the leaf which later on coalesce to form big patches covering the coming partially or wholly. In such cases the leaf let become chloratic and generally drops.
The disease widely occurs in the crops grown in sandy soil of
Rajasthan where climatic conditions are dry and temperature remains high.
Literature search revealed that no much work has been carried out on incitant of this disease. When the conditions for the disease development are favourable the spread of disease is very quick involving all the plants in a field and such blighted plants can be recognized from a disease.
The objectives taken in the present study will add an important information in the available literature. This will be ultimately useful in the management of the disease.
The objectives of the presented study were as follows :
(i) Collection, isolation, purification, identification and pathogenicity
of disease causing fungus.
(ii) Physiological studies viz. effect of different solid and liquid media,
temperature, relative humidity and pH on the growth and
sporulation of A. alternata.
(iii) In vitro efficacy of some antagonists against A. alternata.
(iv) In vitro efficacy of some phyto extracts against A. alternata.
(v) In vitro efficacy of systemic and non-systemic fungicides against
A. alternata.
2. REVIEW OF LITERATURE
Groundnut (Arachis hypogaea L.) suffers from various diseases that are mainly caused by fungi. Following are some important fungal disease of groundnut.
S. Name of disease Causal organism Reference No. 1. Pre-emergence rot Rhizopus sp. Purss (1960) 2. Cercospora leaf spot Cercospora personata Sulaiman (1964) (Tikka disease) C. arachidicola 3. Afla root Aspergillus flavus Chohan and Gupta (1968) 4. Grey mould Botrytis sp. Figueiredo and Cardoso Rosa (1968) 5. Leaf blight Alternaria alternata Balasubramanian (1979) and Vyas et al. (1985) 6. Leaf blight Alternaria tennuissina Patil et al. (1990) 7. Leaf spot Alternaria arachidis Kulkarni (1974) 8. Eye spot - Bansal and Sobti (1988) 9. Crown rot Aspergillus niger Lashin et al. (1989) 10. Seedling or sclerotinia Sclerotinia minor Porter et al. (1989) blight 11. Seedling rot Aspergillus niger, A. flavus Subrahmanyam Rhizoctonia solani, (1991) Macrophomina phaseoli Rhizopus sp., Fusariam spp. Pythium spp. 12. Seed rot Aspergillus flavus, A. niger, Uumechuruba Macrophomina phaseoli et al. (1992)
2.1 Physiological studies
2.1.1 Media
Nutritional requirement of various fungi differ and there is no one medium which can be universally suited to all the fungi. Therefore, studies on the nutritional requirement of a pathogen is an important aspects as this help in better understanding of host pathogen relationship and disease management (Lilly and Bernett, 1951).
The growth of Alternaria tenuis was better on Richards‟s medium (Ashour and Kadi, 1959). Rangaswani and Sambandam (1960) observed maximum growth of Alternaria species on PDA. Gemawat (1969) studied that Alternaria brunsii grew better on PDA followed by oatmeal agar.
Singh and Prasada (1973) isolated the fungus in vitro using
PDA from diseased leaves of clusterbean after surface sterilization with 0.1% mercuric chloride (HgCl2) solution. They purified the pathogen by single spore isolation and maintained the culture on PDA. They also tried different solid and liquid media to know growth requirement of Alternaria cyamopsidis and found the PDA and Richard‟s medium suproted maximum growth of fungus.
Growth and sporulations of A. alternata was best on PDA followed by oatmeal agar (Ionnsidis and Main, 1973). Mathur and Sarbhoy
(1977) also observed that Richard‟s medium supported better growth of A. alternata isolated from sugarbeet followed by PDA, cornmeal and oat meal agar. Gemawat and Ghosh (1979) reported PDA and Richard‟s medium best for growth in case of A. solani. In vitro potato dextrose agar support optimum growth and sporulation of Alternaria porri have been reported by earlier workers (Saad and Hagedron, 1970; Raju and Mehta, 1982).
The fungus, Alternaria alternata grew maximum on
Sabouraud‟s agar medium followed by Richards‟s synthetic medium, corn‟s agar, PDA and Oatmeal agar resulted in luxuriant growth while Borwn‟s synthetic and glucose asparagines agar media were poor for growth (Shah,
1980). Mohanty et al. (1981) found maximum growth of A. carthemi causing lead blight of safflower on Czapek-Dox agar, followed by Sabouraud‟s
Richard‟s, PDA, Glucose, Peptone and host extract age media.
Gupta et al. (1987) observed that Czapeck‟s medium was best for growth of A. porri among isolates tested in the study. They observed that isolates could be differentiated more clearly on the basis of colony colour on
Sabouraud‟s medium and colour of culture filtrate on Brown‟s medium. Kaul and Saxena (1988) reported that the PDA found to be the best medium for growth of five isolates of Alternaria solani causing early blight of potato.
Andreu and Cupull (1991) found the growth rate of A. solani was higher on the new culture media test than on PDA, tomato glucose agar or Czapek malt extract agar. At 22-26 0C, the growth rate of A. solani on the new media was similar to that on PDA. Saeed et al. (1995) reported maximum colony growth of A. alternata of Richards agar medium.
Physiological studies conducted by Ayub et al. (1995) revealed that the PDA was best medium for growth and sporulation of A. cyamopsdis were temperature was best on 30+2 0C with pH.6.0. Pandey and Vishwakarma
(1998) studied the growth, sporulation and colony characters of A. alternata on different vegetables-based media and they were obtained maximum colony growth on capsicum dextrose agar medium. Maheshwari et al. (2001) tested eleven nutrient media for the growth and sporulation of A. alternata, maximum growth of the fungus was obtained in both liquid and solid media form on potato dextrose followed by oat meal which were statistically similar. Richard‟s and malt extract media supported excellent sporulation of the pathogen, whereas, minimum growth and poor sporulation were found on Asthana and Hawker‟s medium.
2.1.2 Temperature and pH
Generally, all the fungal species prefer a temperature range of
20-30 0C with neutral pH (7.0). Fungus, can grow over a wide range of pH but the optimum pH for mycelial growth and sporulation may differ for each species or even forms within species, most fungi growth best at neutral or slightly on acidic medium (Lilly and Barnett, 1951).
Optimum temperature is one of the important factor for the growth and sporulation of an organism for some fungi which also influences the occurance and development of disease. It may be as high as 50 0C (Lilly and Barnett, 1951) but most of the organisms grow between 0 to 42 0C (Wolf and Wolf, 1947).
Hydrogen ion concentration is one of the important factor and remarkably influences the physiology of growing organism both in nature and articial cultures (Lilly and Bernett, 1951).
Neergaard (1945) reported a good growth and sporulation of various species of Alternaria in the range of 23-28 0C. Ozcelik and Ozcelik (1990) studied affect of temperature and pH on biomass production by
Alternaria spp. and reported that biomass production was greatest at 250C and pH 8.0.
Hasija (1970) reported that optimum pH and temperature for growth and sporulation of A. alternata as 6.6 and 25 0C. Chandrashekhar and
Ball (1980) observed that Alternaria alternata causing leaf blight of grey manges grew from 15 to 35 0C with the optimum temperature being 30 0C for growth and sporulation. Herrera et al. (1989) reported that modified that
PDA, constant light and 26 0C temperature were the most favourable conditions for A. porri mycelial growth sporulation and chlamydospore production in vitro.
Choulwar and Datar (1991) observed that A. solani causing early blight of tomato, required the optimum temperature for growth on PDA was 28 0C. Exposure to sunlight produced maximum growth and distinct zonation but not sporulation. Hossain et al. (1997) studied the effect of culture media and temperature on the growth and sporulation of A. porri. Potato dextrose agar media supported the growth and sporulation of the fungus.
Luxuriant mycelial growth was obtained within a temperature ranged of 20-30
0C and having maximum growth at 25 0C.
Singh et al. (2001) observed that the optimum temperature for growth and sporulation of Alternaria alternata lies form 25 to 30 + 2 0C. Saharan et al. (2003) studied the conidial germination of A. cucumerina at different temperature viz., 20, 24, 28 and 32 0C.
Rangaswami and Sambandam (1960) observed that various species of Alternaria isolated from solanaceous hosts are capable of growing over a wide range of hydrogen-ion concentrations. The optimum pH for
Alternaria solani from potato found to be at 6.0. Saad and Hegedorn (1970) observed good growth of Alternaria alternata causing leaf spot of bean at pH
6.5. Verma (1970) noted that A. solani and A. tenuis could grow over a wide range of pH 6.5 however, pH was 6.6 for both the species. Reddy (1973) found that a pH range of 5.0 to 6.5 was food for the growth and sporulation of
A. alternata (Fr.) Kesis causing leaf spot of mungbean. Mathur and Sarbhoy
(1977) reported the best growth and sporulation of A. alternata of sugarbeet at pH 4.5. Wang and Dong (1991) reported the favourable temperature and pH for in vitro growth of A. alternata and it was found to be 20-25 0C and pH 5.5 to 5.7. Khatun (1996) reported good growth and sporulation of A. brassicae at pH 6 to 8 with maximum growth at pH 7.0.
The optimum temperature for growth and conidial germination of Alternaria solani isolated from tomato plants in the range of 23-28 0C
(Tong Yuntlui et al., 1994). Datar (1995) temperature range from 20 to 30 0C are reported to be optimum for the rapid development of fruit rot of chillies caused by A. alternata. The optimum temperature for growth and spore germination of Alternaria alternata, causing leaf blight of sunflower was favourable at 250C (Smita et al., 1998).
Akhtar et al. (1999) observed that A. alternata grew best at 30
0C while very negligible growth recorded at 100C and no growth at 50C.
Maheshwari et al. (2000) studied growth and sporulation of Alternaria alternata at 11 different temperatures and 16 pH levels ranging from 5 to
400C and 3.0 to 10.5 pH. They observed that the optimum temperature and pH for fungus were 280C and 6.5, respectively. Minimum fungus growth was recorded at 50C and pH 10.5. Excellent sporulation was observed at 25-30 0C and pH 5.5-6.5. The optimum pH for growth of A. alternata is pH 6.5
(Maheshwari et al., 2000). Growth and sporulation of A. alternata causing blight of chickpea was most favoured by 250C temperature (Singh et al.,
2001).
8.1.4 Relative humidity
Relative humidity is the most important factor on spore germination and causing disease. Dickinson and Bottomely (1980) suggested that conidia of A. alternata germinated quickly at the relative humidity. Khare and Nema (1981) studied that maximum sporulation of Alternaria porria on onion was recorded at 22 0C and 90 % relative humidity. These authors also reported that spore germination in vitro was 100% within 4 hours at 22 0C while on onion a maximum spore was recorded within 6 hours at 25 0C.
Hundred per cent relative humidity was found optimum for germination in vitro Khare and Nema (1982). Gupta et al. (1985) reported that conidial germination was maximum when 100% relative humidity prevailed for 6 hours or more at 25 0C.
Bhargava and Khare (1988) observed growth and sporulation of
Alternaria of chickpea at 25-27 0C and R.H. 80%. Everts and Lacy (1990) reported that conidia were formed at all relative humidity tested at 75-100 %, number of lesions were very low at 75-85% relative humidity but increased with increasing relative humidity. Conidia formed on lesions on senescent leaves incubated in dew at 25 0C. Rodriguez et al. (1991) observed that the intensity and dynamics of A. porri conidial germination were studied at 5, 10,
15, 20, 25, 30, 35, 37.5 and 40 0C and relative humidity 76-100%. Conidia developed at 5-37.5 0C, with an optimum temperature of 30 0C. Germination started within 1 hour of incubation at 20-25 0C and in 4 hours, 50% of conidia had germinated.
Kumar (1992) reported that infection by A. alternata was favoured by moderately high temperature, normal relative humidity and dry, rainless days. Out of 6 cultivars observed under natural infection at Pali
(Rajasthan), two cultivars i.e. RD 31 and BL 32 remained free from disease while the others exhibited varying reactions. Singh et al. (2001) tested the effect of different relative humidity on germination and development of
Alternaria tenuissima.
2.2 Biological control Biocontrol of plant pathogens is getting moment in recent years due to an increasing awareness of pesticide hazards, subsequent environmental pollutions, the failure of current fungicides due to resistant due to resistant races/pathotypes of specific diseases of crop plants, higher cost of development, greater difficulties associated with findings new fungicides and many disease posed to give more attention on alterative methods, like biocontrol. Therefore the search is for plant protection strategies which are sustainable, eco-friendly and non hazardous. One of such promising strategy is biological control, which is primarily based on microbial antagonism.
Strashnov et al. (1985) reported that effectiveness or
Trichoderma harizianum against fruit rot of tomato caused by Alternaria alternata.
Antagonistic strains of Trichoderma from garlic leaf showed
45.7 per cent and 87.7 per cent control of Alternaria solani by dual culture method and toxic filtrate, respectively (Sastrachidayat, 1995). Sas-Piotrowska and Dorszewski (1996) reported that T. koningii was found to be most effective antagonist followed by Trichoderma viride, Trichoderma harzianium against A. alternata.
Best control of Alternaria disease in tomato was achieved by T. harzianum when it was applied as a powder to seeds (Gromovikh et al., 1998)
Salgado et al. (1998) found that the Cuban strain of Trichoderma was effective against Alternaria solani affecting both mycelial growth and the metabolism of A. solani. Babu et al. (2000) reported that Trichoderma harzianium and
Trichoderma viride were significantly effective in inhibiting the mycelial growth of Alternaria solani, the causal agent of the leaf blight disease in tomato. Abdul et al. (2001) that Trichoderma harzianum and Nimokil 60 EC
(neem oil product) effective in vitro in retarding the mycelial growth of
Alternaria solani.
Lal and Upadhyay (2002) evaluated Trichoderma viride and T. harzianum against A. tenuissima (causing leaf blight of pigeonpea) in vitro. In dual plate culture experiment T. viride inhibited the growth of A. tenumissmia within 75 hr. incubation followed by T. harzianum.
Mandhare and Suyawanshi (2003) reported inhibited conidial germination and mycelial growth of two fungi (A. alternata and A. porri) by
Bacillus stubtilis at 2.2 x 109 and 2.2 x 108 CFU per ml respectively, this effect was generated by a theromo-liable metabolic toxic to A. porri and A. alternata.
2.3 Botanicals
Singh et al. (1990) reported that Ajoene a compound derived from garlic inhibited spore germination of some fungi including Alternaria solani, A. teniuissima, A. triticini Collectrichum curvularia and Fusarium which cause serious discusses in some important crop plant India.
Garlic extract has been reported effective against A. alternata, in vitro by several workers (Mistry, 1992; Barros et al., 1995; Shivpuri et al.,
1997; Karade and Sawant, 1999; Rashmi and Yadav, 1999 and Chaudhary et al., 2003). Extracts of other plants like neem, onion, tulsi and datura were also found effective against diseases caused by A. alternata (Mistry, 1992;
Tarkusay and Onogur, 1998; Singh and Majumdar, 2001 and Chaudhary et al., 2003).
Barros et al. (1995) tested effect of garlic extract aganists germination and mycelial growth of Alternaria alternata and A. longipes at
250, 500, 1000, 2000, 5000 and 10000 ppm concentrations and reported that garlic extract effectively inhibit the growth of both the species at 250 ppm and reduced colonies diameter.
Srivastava and Lal (1997) studied in vitro fungicidal properties in aqueous leaf extract of Calotropis procera, Azadirachta indica, Lantaana camara and Ocimum basilicum against Alternaria alternata. The leaf extract of Ocimum basilicum has inhibited germination of Alternaria alternata.
Singh and Majumdar (2001) tested the efficacy of plant extracts against Alternaria alternata, the incitant of fruit rot of pomegranate.
8.4 Fungicides
Application of fungicides plays a major role in the management of plant disease. In order to find out an effective fungicide with suitable concentration, it needs a lot of laboratory studies.
Management of plant diseases caused by Alternaria alternata has been attempted through various chemicals. Copper oxychloride and
Mancozeb has been found effective against A. alternata in vitro (Kalra and Sohi, 1984; Fadl et al., 1985; Mathur and Shekhawat, 1986; Kumar and
Pandey, 1988; Yadav et al., 1993; Navale et al., 1998; Rashmi et al., 1998;
Kamble et al., 2000; Johnson and Subramanyan, 2000; Gupta et al., 2001;
Ghosh et al., 2002; Singh et al., 2003 and Prasad and Naik, 2003).
Among the systemic fungicides, Carbendazim and Thiophanate methyl have been found effective in controlling the diseases caused by A. alternata (Singh and Shukla, 1984; Kumar and Pandey, 1988; Navale et al.,
1998; Rashmi et al., 1998; Smita et al., 1999; Johnson and Subramanyam,
2000; Gupta et al., 2001; Ghosh et al., 2002 and Singh et al., 2003).
Kalra and Sohi (1984) showed that Thiram (0.05-0.2%),
Dithane M-45 (Mancozeb 0.1-0.2%) and Difolatan (Captafol, 0.2%) completely inhibited the growth of Alternaria fruit root of tomato pathogen
(A. alternata). They also observed that systemic fungicides found ineffective except calixin (tridemorph) against the same fungus.
Choulwar and Datar (1992) studied that of 9 fungicides (Copper oxychloride, Zineb, Ziram, Mancozeb, Carbendazim, Dithanon, Thiophanate- methyl, Iprodine and Captafol) against early blight (Alternaria solani) tomato,
Carbendazim also reduced disease incidence. Ramaswamy et al. (1994) found isolate of A. solani sensitive when grown on PDA containing Carbendazim,
Thiophanate (Thiophanate-Methyl), Bliotox (Copper oxychloride), Daconil
(Chlorothalonil), Dexon (Fenaminosulf) or Captan. Spore germination of sensitive isolates was inhibited in the presence of fungicides. Choulwar and Datar (1994) studied the tolerance of A. solani, the causal agent of early blight of tomato of Mancozeb, Captafol, Thiophanate-methyl and Carbendazim at
1000, 1500, 2000 and 2500 ppm concentrations in vitro A. solani could tolerate 2500 ppm of all the fungicides.
Khan et al. (1995) reported that mycelial growth of A. alternata the causal agent of tomato fruit rot, completely inhibited by Propiconazole
(Tilt) and Tridemorph (Calixin-M) at 30 ppm whereas, in descending order of effectiveness, Mancozeb + Metalaxyl (Ridowil), Chlorothalonil (Daconil),
Carbendazim (Bavistin), Benomyl (Benlate), Propineb (Antracol), Zinc ammoniate ethylene bis (Dithiocarbamatepoly-ethylenethiram-disalfide)
Polyramcombi and Thiophanate methyl (Topsin M) significantly reduced mycelial growth at 10, 20 and 30 ppm concentrations.
Kumar and Singh (1997) studied that Carbendazim, Mancozeb and Captan gave good control of A. alternata and A. linicola the incitant of seedling blight of linseed. Mahashwari and Singh (1997) selected nine fungicides by bioassay against A. alternata causal organism of leaf blight of
Dolicus bean. Thiram and Carbendazim gave complete control followed by
Benimyl and Thiophanate-methyl as Indofofil M-45. Mayee (1997) found
Carbendazim, Carboxin or Ziram as highly effective in reducing Alternaria blight of sunflower. Srinivas et al. (1997) found that the Carbendazim gave most effective control of Alternaria blight of sunflower followed by Iprodione and Propiconazole. Smita Rajan et al. (1998) showed that Carbendazim and
Captafol (0.05%) completely inhibited spore germination of Alternaria blight of sunflower. Singh et al. (2001) revealed that six fungicides varied greatly in their efficiency to inhibited the growth of Alternaria cyamposisdis however, the Carbendazim, Pridemefon, Kitazin and Captan were less effective.
3. Materials and Methods
3.1 COLLECTION, ISOLATION AND PURIFICATION
OF THE PATHOGEN
Groundnut leaves showing yellowish green speacies with concentric rings were collected from farmer field around vicinity of Jobner.
Diseased specimens were brought to laboratory and examined under microscope for preliminary examination and were put in to humid chamber for observing the sporulation on naturally infected groundnut leaves. For isolation of pathogen, small pieces of the leaves were cut from the diseased portion along with some healthy tissues and surface sterilized with 0.1 per cent mercuric chloride solution for 1 minute followed by three washing with sterilized distilled water. The surface sterilized pieces were transferred to 2 per cent potato dextrose agar (PDA) slants and incubated at 25+2°C. After seven days of incubation, the fungal growth was transferred aseptically to
PDA slants and purified following single spore technique.
Purification was done by single spore method for purification, sub cutting of single spore of the fungus was done form the uncontaminated peripheral growth on 20 ml PDA poured in sterilized petridishes inoculated at
25+2 0C. After 3 days of incubation the single growing spore and hyphal tips were located under the microscope, hyphal tip and single spore was marked and cut with a dummy objectives and transferred separately to PDA slants.
Resulting cultures showed no variability in morphological characters. The culture were maintained by transferring on fresh PDA slants at an interval of
7 days.
3.2 IDENTIFICATION OF THE PATHOGEN
THE PATHOGEN WAS TENTATIVELY IDENTIFIED
THROUGH MICROSCOPIC STUDY OF CULTURAL AND
MORPHOLOGICAL CHARACTERS OF FUNGUS WERE
OBSERVED UNDER MICROSCOPE FOLLOWING STAINING THE
CULTURE IN LACTOPHENOL. MEASUREMENT OF FUNGAL
HYPHAE AND CONIDIAL SIZE WERE MADE WITH THE HELP OF
STAGE AND OCULAR MICROMETER. OBSERVATION ON SHAPE,
COLOUR, LENGTH, BREADTH AND SEPTATION OF CONIDIA
WERE RECORDED. THE CULTURE WAS IDENTIFIED BY
MYCOLOGIST, DEPARTMENT OF PLANT PATHOLOGY, S.K.N.
COLLEGE OF aGRICULTURE, JOBNER (JAIPUR) RAJASTHAN.
THE IDENTITY WAS FURTHER CONFIRMED FROM AGHARKAR
RESEARCH INSTITUTE, PUNE (MAHARASHTRA).
3.3 PATHOGENICITY
TO TEST THE PATHOGENICITY, GROUNDNUT
PLANTS WERE INOCULATED WITH THE SPORE SUSPENSION
OF ALTERNARIA ALTERNATA. THE SPORE SUSPENSION WAS
PREPARED IN STERILIZED DISTILLED WATER BY BLENDING 7
DAYS OLD CULTURE OF THE FUNGUS IN PESTLE AND
MORTAR. THE BLENDED CULTURE WAS FILTERED THROUGH DOUBLE LAYERED STERILIZED CHEESE CLOTH. DESIRED
SPORE CONCENTRATION (1 X 103 SPORES / ML) WAS OBTAINED
BY ADDING STERILIZED DISTILLED WATER AND COUNTING
THE NUMBER OF SPORES/ML. SIX WEEKS OLD GROUNDNUT
PLANTS WERE INOCULATED BY SPRAYING THE SPORE
SUSPENSION WITH THE HELP OF AN AUTOMIZER. THE CHECK
WAS MAINTAINED BY SPRAYING THE STERILIZED DISTILLED
WATER ONLY. INOCULATED AND CONTROL PLANTS WERE
EXPOSED TO IDENTICAL CONDITIONS UNDER MOIST
CHAMBER FOR 48 HRS. SYMPTOMATOLOGY OF THE DISEASE
WAS STUDIED THROUGH VISUAL OBSERVATION FROM
INITIATION TO FULL DEVELOPMENT OF THE DISEASE AND
WAS COMPARED WITH CONTROL.
PATHOGEN WAS REISOLATED FROM
ARTIFICIALLY INOCULATED PLANT AND RESULTING
CULTURE WAS COMPARED WITH ORIGINAL ONE TO CONFIRM
THE IDENTITY OF THE PATHOGEN.
3.4 PHYSIOLOGICAL STUDIES
All the glasswares were thoroughly cleaned and rinsed with distilled water. Chemicals of analar grade were used. Different synthetic and semi-synthetic media were prepared by weighing the different constituents of each medium and then adding the distilled water to make up the volume 1000 ml and autoclaved at 1.045 kg/cm2 for 20 minutes. In solid media studies agar-agar was added whereas, in liquid media studies no agar-agar was added.
In solid media 20 ml agar medium was poured in each petridishes whereas in liquid media 20 ml of the medium was dispensed in each of 100 ml conical flask.
Inoculation was done with 2 mm disc of mycelial mat taken from 7 days old fungal culture and incubated at 25+2 0C for 7 days. In all experiments the treatment were replicated four times.
3.4.1 Growth and sporulation on solid media
Growth on solid media was determined by measuring the colony diameter along the two diagonals passing through the centre of colony and sporulation was recorded by using Haemocytometer. The measurements were recorded at an interval of 7 days.
The composition of variance soil is media used in the present study are given below : (i) Czapeck’s dox agar Agar 15.0 g Dipotassium phosphate 1.00 g Distilled water 1000 ml Ferrous sulphate 0.01 g Magnesium sulphate 0.50 g Potassium chloride 0.50 g Sodium nitrate 2.00 g Sucrose 30.00 g (ii) Richard's medium Agar 15.00 g Distilled water 1000 ml Ferric chloride 0.02 g Magnesium sulphate 2.50 g Potassium dihydrogen phosphate 5.00 g Potassium nitrate 10.00 g Sucrose 50.00 g (iii) Potato dextrose Agar (PDA) Agar-agar 20.00 g Dextrose 20.0 g Distilled water 1000 ml Peeled potato 250 g (iv) Asthana media Agar-agar 20.00 g Distilled water 1000 ml Glucose 5.00 g Magnesium sulphate 0.75 g Potassium dihydrogen phosphate 1.75 g Potassium nitrate 3.50 g (v) Oat meal agar Agar-agar 20.00 g Distilled water 1000 ml Glucose 20.00 g Oat meal 20.00 g (vi) Corn meal agar Agar-agar 20.00 g Corn meal 20.00 g Distilled water 1000 ml Glucose 20.00 g 3.4.2 Growth and sporulation on liquid media
In case of liquid media the mycelial growth was harvested by filtration through whatman‟s filter paper No. 42, washed repeatedly, dried at 60 0C till it had constant weight after cooling in desicators. Observation on radial growth and sporulation were recorded after 7 days of incubation. The liquid media used in the present study were :
Synthetic and semi-synthetic media
(i) Asthana media
(ii) Corn meal agar
(iii) Czapeck‟s dox agar
(iv) Oat meal agar
(v) Potato dextrose Agar (PDA)
(vi) Richard‟s medium 3.4.3 Effect of temperature on growth and sporulation
Effect of temperature on growth and sporulation of Alternaria alternata was studied in vitro. 20 ml of sterilized potato dextrose agar medium was poured in each in each sterilized petridish. Inoculation was made with 2 mm disc from 7 days old fungal culture and incubated at 7 different temperatures viz., 20, 25, 30, 35, 40, 45 and 50 0C. Observation on radial growth and sporulation were recorded after 7 days of inoculation.
3.4.4 Effect of relative humidity on mycelial growth and sporulation
To study the effect of relative humidity on mycelial growth and sporulation of Alternaria alternata, six different levels of relative humidity i.e. 50, 60, 70, 80, 90 and 100 per cent was maintained by using the concentrate sulphuric acid and sterilized distilled water in different proportions. The different relative humidity levels were maintained by the method suggested by Buxton and Mellanby (1934). Composition of the acid solution used were as follows :
Relative humidity (%) Stock solution (ml)* Distilled water (ml) 50 514.0 420.0 60 374.0 396.0 70 348.0 510.3 80 294.0 640.0 90 161.0 712.0 100 0.00 Only distilled water * 50% v/v solution of concentrate sulphuric acid Humidity levels were maintained in 90 mm diameter petriplates.
For this sixty petriplates were sterilized, out of which 30 were for preparing culture plates by pouring 20 ml of PDA medium in each of them and 30 for holding humidity solution. In each case 20 ml aliquate of required humidity solution were poured in bottoms of the sterilized petriplates. These bottoms, containing humidity solutions were then covered aseptically with the inverted bottom plates holding inoculated medium plates. In this way inoculated surface was directly exposed to the humidity condition created by the solution.
Incubation was done at 25+2 for 7 days, radial growth and sporulation was recorded at 2 diagonals. Experiment was replicated six different levels of relative humidity viz., 50, 60, 70, 80, 90 and 100 per cent were produced by mixing concentrated sulphuric acid and distilled water in different proportions in glass desiccators.
Petriplates containing PDA medium were inoculated with 2 mm disc of 7 days culture of test fungus. Inoculated petriplates were immediately accommodated in glass desiccators containing mixture of sulphuric acid and distilled water in required proportions and inoculation was done at 25+2 0C for 7 days. Observations on radial growth and sporulation were recorded after
7 days of incubation. Influence of weather parameters and disease development in natural condition was recorded quarterly and correlated.
3.4.5 Effect of hydrogen ion concentrations on mycelial growth and
sporulation The effect of hydrogen ion concentration on the growth and sporulation of Alternaria alternata was determined by adjusting the pH of
PDA medium from at 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5 by using citrate phosphate buffer before sterilization with the help of pH meter. Aliquate of 20 ml medium were dispensed in 100 ml conical flask and autoclaved at 1.045 kg/cm2 for at 15 minutes. Inoculations were made with 2 mm disc of mycelial mat obtained from 7 days old culture of Alternaria alternata. Inoculated flasks were incubated 25+2 0C for 7 days. The mycelial mats were harvested, dried in incubator at 60 0C for 24 hours and weighed. Observation on radial growth and sporulation were recorded after 7 days of incubation.
Stock solutions
Solution A. : 0.1 m solution of citric acid (192.1 mol) in one litre of distilled water 19.21 gm of citric was dissolved.
Solution B. : 0.2 m solution of diabasic sodium hydrogen phosphate (293.9 mol wt) in one litre of distilled water 58.78 gm of diabasic sodium hydrogen phosphate was dissolved.
Solution A (cm) Solution B (cm) pH 84.0 116.0 5.5 73.7 126.3 6.0 54.7 145.5 6.5 35.3 164.7 7.0 12.7 187.3 7.5 5.5 194.5 8.0 8.5
3.5 In vitro efficacy of biocontrol agent against Alternaria alternata
through dual culture plate method
For antagonistic test in PDA inoculation techniques was employed under laboratory conditions following dual culture method. PDA was used as the basal medium. 20 ml of sterilized melted PDA was poured in each petridish and allowed to solidify. After 3 hours of pouring, these plates were inoculated with 2 mm disc taken from 7 days old culture of Alternaria alternata and antagonistic agents namely Bacillus spp., Trichoderma harzianum, T. viride and T. polysporum. Separately opposite to each other in the periphery of the petridishes. Four replications were maintained for each treatment. Petridishes both with antagonistic agents Alternaria alternata and only pathogen (control) were incubated at 25+1 0C for 7 days, zone of inhibition, if developed any was measured and recorded for these antagonistic agents.
3.6. Effect of plant extracts on growth and sporulation of Alternaria
alternata
The effect of each plant extract was tested at five concentrations viz., 50, 100, 200, 500 and 1000 ppm. Plant parts were thoroughly washed with sterilized distilled water and were ground separately in electric grinder using equal amount of sterilized water to get stock solution. The mixture was squeezed with of double layered sterilized cheese cloth. The extract thus obtained was considered as of 100 per cent concentration. Required amount of stock solution was added to PDA to get desired concentration. The effect of against mycelial growth and sporulation was tested using food poisoned technique (FPT). Required quantity of each plant extract was mixed thoroughly in melted PDA to get desired concentration just before pouring in sterilized petridishes and was allowed to solidify for 3 hours. Each plate was inoculated with 2 mm disc of mycelial bit taken from the periphery of 7 days of old culture of Alternaria alternata growing on PDA. The inoculated petridishes were then incubated at 25+2 0C. Four petridishes were used for each treatment serving as four replication, petridishes without plant extract severed as control. The experiment was conducted in Completely
Randomized Block Design (CRD). Colony diameter (two diagonals) was measured after 7 days of incubation. Per cent growth inhibition was calculated by Vincent‟s (1947) formula follows :
C - T Per cent growth inhibition = ------x 100 C Where,
C = Diameter of the colony in check (average of both diagonals)
T = Diameter of colony in treatment (average of both diagonals)
Following plant extracts were used to test their efficacy against
Alternaria alternata in vitro.
Table 3.1 Plant extracts tested against A. alternata
S.No. Name of plant Botanical name Plant part used 1 Babul Acacia nilotica Leaves 2 Dhatura Datura stramonium L. Leaves 3 Garlic Allium sativum Clove 4 Ginger Zingiber officinale Roscoe Rhizome 5 Jangly chualia Amaranthus viridis Leaves 6 Neem Azardirachta indica A. Juss Kernel and leaves 7 Tulsi Ocimum sactum Leaves
3.5 In vitro efficacy of systemic and non systemic fungicides against, A.
alternata
Efficacy of seven systemic and non systemic fungicides against mycelial growth and sporulation of Alternaria alternata was tested by using food poisoned technique. Five different concentrations viz., 50, 100, 200, 500 and 1000 ppm of each fungicide was tested required quantity weighed of each fungicide was added separately in autoclaved PDA and mixed thoroughly.
The poisoned medium was poured in sterilized petriplates and allowed to solidity. Each plate was inoculated with 2 mm disc of the fungal culture and incubated at 25+2 0C for 7 days. The linear growth of the test fungus was recorded per cent growth inhibition was calculated by Vincent‟s (1947) formula referred under 3.4.
Following fungicides were tested against Alternaria alternata.
S.No. Common name Trade name Chemical name 1 Captafol Difoltan N (1, 12, 2, tetrachloroenc- 1-2-dicarboximide 2 Captan Captan 50 WP N-trichloromethl thiomethoxam 4 cyclo 3 Carbendazim Bavistin Methyl –1, 2, benzimidazole carbamate 4 Chlorothalonil Kavach 75 WP Tetrachloraiso- phthalonitrile 5 Copper oxychlooride Blitox 50 Dicoper chloride trihy droxide 6 Mancozeb Dithane M-45 Hexane-1, 2 dicarboximide 7 Propiconazole Tilt -
4. EXPERIMENTAL RESULTS
4.1 PATHOLOGICAL INVESTIGATION
4.4.1 Collection leaf blight infected samples, isolation and maintenance
of the pathogen
Leaf blight infected leaves were collected from the vicinity of
Jobner (Jaipur). The fungus Alternaria alternata was isolated from the lighted leaves of groundnut in potato dextrose agar medium. The pure culture was maintained on potato dextrose agar slants. The periodical subculturing and multiplication were done on the same medium to keep the entire fresh and was used throughout studies.
4.1.2 Morphology and identification of the pathogen
Morphological characters of the fungus were studied by observing slides, stained with cotton blue under a microscope.
Photomicrographs of mycelium and conidia were taken.
Colony
On third day after inoculation on the potato dextrose agar, fungus colony was seed as olivaceous in colour surrounded by whitish fluffy growth of the fungus. It changed gradually in fourth day from light olivaceous to dark olivaceous in colour upto 12 days of incubation and after 15-20 days to inoculation, whole fungal growth were converted into dark brown to black in colour. Average growth rate of the colony of the fungus was 8 to 10 mm per day which completely covered the petridish within 10 days when incubated at 25 + 1 0C.
Mycelium
The fungus produced profuse mycelial growth on PDA which was circular olivaceous in colour when young, later on turned dark brown to black colonies. The microscopic observations of fungus on PDA showed that mycelium was irregularly branched at acute angle, septate and light brown in colour.
Conidiophores
The conidiophores of the fungus were simple, septate and light brown in colour.
Conidia
Conidia were light brown to dark brown, muriform with 1-5 transverse septa and 0-3 longitudinal septa, variable in size and shape. They were obclavate to oval in shape with short cylindrical beak which measured
14.00-63.50 x 7.50 – 14.00 µm (Av. 37.72 x 12.60 µm).
The cultural and morphological characters observed under laboratory conditions were compared with those described in literature. Thus, it was identified as Alternaria alternata (Fr.) Keissler. The pure culture was also sent for identification to Department of Plant Pathology, S.K.N. College of agriculture, Jobner (Jaipur) Rajasthan. The identity was further confirmed from Agharkar Research Institute, Pune (Maharashtra) and identified as
Alternaria alternata.
4.1.3 Pathogenicity
Pathogenicity test of leaf blight pathogen of groundnut was carious out by spraying the spore suspension (105 spore/ml) on groundnut leaves.
The leaf blight pathogen was able to infect the inoculated groundnut leaves and developed the spot on leaves by spraying the spore suspension. Spraying of sterile distilled water (control) showed no symptoms on groundnut leaves.
The first typical symptoms were observed after five days of inoculation as small, isolated, scattered, pale yellow, brown spots on the leaves. The lowest leaves were affected first and the disease progress upward.
Latter on, after seven to eight days to inoculation the disease symptoms appeared as necrotic spots which were covered with a deep greenish blue growth of the fungus. There were concentric rings with narrow chlorotic zone around the spots on the older leaves. After nine days of inoculation, the symptoms appeared as light brown, irregular necrotic specks increased in size and because angular to irregular in shape and developed dark brown spots with yellow halo after 10 to 12 days. After 15 to 16 days, the spots enlarged and coalesced to cover large areas of the leaves which lead to leaf blight. As a result, the infected leaves started drying on re-isolation form diseased leaves of groundnut, identical culture of Alternaria alternata (Fr.) Keissler to that of original culture was obtained. Hence, pathogenicity was proved.
4.2 PHYSIOLOGICAL STUDIES ON FUNGAL GROWTH IN
VITRO
4.2.1 Growth and sporulation of A. alternata on different solid media
The pathogen i.e. Alternaria alternata were grown on six synthetic and semi synthetic media. All the media were autoclaved, distributed in petri-dishes, inoculation with 2 mm disc of mycelial mats obtained from 7 days old culture and incubated at 25 + 2 0C for 7 days.
Observation recorded for growth and sporulation are presented in table 4.1.
Among the six different solid media understudy, the best medium was found to be potato dextrose agar with maximum radial growth
(88 mm) and sporulation (35.00 x 106/ml). It was followed by Richard‟s,
Oatmeal, Czapeck‟s, Cornmeal and Asthana medium whereas minimum growth and sporulation of this fungus was observed on Asthana medium
(14.25 mm and 20.25 x 106 /ml). It can be concluded that potato dextrose agar medium was best supporter of growth and sporulation of the fungus. The
PDA was selective medium for further studies. 4.2.2 Growth and sporulation of A. alternata on different liquid media
To find out a suitable liquid medium for the mycelial growth and sporulation of Alternaria alternata six different media (without agar, agar) were tested in vitro. Inoculated flask were incubated at 25 + 2 0C for 7 days and mycelial mats were harvested, dried and weighed. Observations were recorded on growth and sporulation of Alternaria alternata and are presented in table 4.2. On perusal of data presented in table 4.2, it is evident that are media, under study, supported the growth and sporulation of
Alternaria alternata. Maximum growth and sporulation was recorded on potato dextrose agar medium (54.75 mg and 13.75 x 106 /ml) followed by
Richard‟s, Oatmeal, Czapeck‟s, Cornmean and Asthana medium. Richard‟s and Oatmeal media were moderate for mycelial growth and sporulation whereas cornmeal and Asthana media were found to be poor supporter for mycelial growth and sporulation. It can be concluded that potato dextrose agar medium was best supporter for growth and sporulation of fungus.
4.2.3 Growth and sporulation of A. alternata on different temperature
The temperature ranges for the growth vary for all micro- organisms as well as for host pathogen interactions. It is evident from the data presented in table 4.3 that the fungus grew at all the temperature (20 to 40 0C) under study. Maximum mycelial growth (88.00 mm) and sporulation (35.50 x
106 /ml) was observed at 25 0C. A sudden fall in mycelial growth and sporulation was observed at 30 0C, 35 0C and 40 0C. However, 20 0C and 30 0C also favoured good growth and sporulation of Alternaria alternata but differ significantly for growth at 25 0C. No growth and sporulation observed at 45 0C and 50 0C temperature. IT can be concluded that 25 0C is the optimum temperature for mycelial growth and sporulation of Alternaria alternata (Table 4.3).
4.2.4 Effect of relative humidity on growth and sporulation of A. alternata
To evaluate the effect of atmospheric moisture, the fungus was exposed directly to different level of relative humidity. It was observed (table
4.4) that all the six humidity levels (50 to 100 per cent) induced the growth and sporulation of Alternaria alternata. Significantly best mycelial growth
(88.25 mm) and sporulation (37.25 x 106/ml) was recorded at 100 per cent relative humidity followed by growth and sporulation at 90 per cent (86.00 mm and 35.00 x 106 /ml) relative humidity level. A significantly decrease in mycelium growth and sporulation was observed at 80, 70 and 60 per cent humidity level. Minimum mycelium growth and sporulation was observed relative humidity level. It can be concluded that high humidity favoured the growth and sporulation of Alternaria alternata.
4.2.5 Effect of different hydrogen-ion concentration
To determine the optimum pH requirement for growth and sporulation of Alternaria alternata. Seven different pH levels ranging from
5.5 to 8.5 with a difference of 0.5 unit in each case were studied in liquid potato dextrose agar medium. The results presented in Table 4.5 showed that the fungus grew on wide range of pH from 5.5 to 8.5. Maximum dry mycelium weight and sporulation was observed at pH 6.5 (95.00 mg and
30.50 106/ml). Thus was followed by pH 7.0, 7.5, 8.0, 5.5 and 8.5. The weight of dry mycelium weight and sporulation harvested from the liquid media with pH 6.5 and 7.0 were significantly more than any other pH range tested. Least fungal growth and sporulation (62.00 mg and 19.75 x 106 /ml) was recorded at ph 8.5.
4.2.6 In vitro efficacy of biocontrol agent against Alternaria alternata
through dual culture plate method
Three different species of Trichoderma viz., Trichoderma viride, Trichoderma harzianum and Trichoderma polysporum and one bacterial, antagonist i.e. Bacillus spp. were evaluated for their antagonism against Alternaria alternata by dual culture plate method.
The result presented in Table 4.6 revealed that all the antagonists significantly inhibited the growth and Alternaria alternata. All species of Trichoderma inhibited the growth of Alternaria alternata, however
Trichoderma polysporum showed significantly maximum zone of inhibition
(7.50 mm) and grew over the Alternaria alternata. Colonies followed
Trichoderma harzianum, Trichoderma viride and Bacillus spp.
6. SUMMARY
Leaf blight disease of groundnut (Arachis hypogaea L.) caused by Alternaria alternata was observed at farmers field near by Jobner (Jaipur).
The isolated fungus Alternaria alternata proved to be pathogenic on groundnut. Initially symptoms developed as a small isolated scattered pale yellow orange-brown spots on the leaves. These spots become irregular in shape with increase in size and appear brown to grey in colour covered with a deep greenish blue growth of fungus. There were concentric ring with narrow chlorotic zones around the spot.
Out of six different solid media, potato dextrose agar (PDA) supported best mycelial growth and sporulation of the fungus followed by
Richard‟s and Oatmeal medium. While in case of broth media best growth and sporulation was observed on potato dextrose broth medium followed by
Richard‟s and Oatmeal broth medium. Poor mycelial growth and sporulation was observed on Asthana broth media.
Studies on effect of temperature, relative humidity and pH in vitro conditions, maximum mycelial growth and sporulation was observed at
250C temperature, 100 per cent relative humidity and pH 6.5.
The relative efficacy of biocontrol agent showed that
Trichoderma polysporum was found most effective antagonist against Alternaria alternata followed by Trichoderma viride and T. harzianum.
Bacillus sp. was found least effective against the pathogen.
Studies on the relative efficacy of plant extracts at different concentration in in vitro. Garlic clove extract was found most effective in reducing mycelial growth and sporulation of the fungus followed by neem, datura and tulsi. Zinger, jungly choulia and babul were found least effective.
The relative efficacy of fungicides at different concentration in vitro, showed mancozeb at 1000 ppm concentration was found most effective followed by copper oxychloride, captafol and carbendazim. Propiconazole and captan was least effective in reducing mycelial growth and sporulation of
Alternaria alternata.
Table 4.1 Mycelial growth and sporulation of Alternaria alternata on different
solid media (after 7 days of incubation at 25 + 2 0C)
S.No. Medium Colony diameter (mm)* No. of spore/ml (x106)* 1. Asthana media 44.25 20.25
(41.69) (26.74)
2. Corn meal media 50.00 22.50
(40.00) (28.31)
3. Czapeck‟s media 57.50 25.25
(49.31) (30.16)
4. Oat meal media 60.25 26.00
(50.91) (30.65)
5. Potato dextrose agar 88.00 35.00
(69.73) (36.27)
6. Richard‟s medium 74.75 30.25
(59.83) (33.36)
SEm+ 0.73 0.26
CD at 5% 2.24 0.79
* Average of four replications Figures given in parenthesis are angular transformed values
Table 4.2 Mycelial growth and sporulation of Alternaria alternata on different
liquid media (after 7 days of incubation at 25 + 20C)
S.No. Medium Dry mycelial weight No. of spore/ml (mg)* (x106)* 1. Asthana media 20.25 5.75
(26.74) (13.87)
2. Corn meal media 26.75 7.25
(31.14) (15.62)
3. Czapeck‟s media 42.00 9.75
(40.39) (18.19)
4. Oat meal media 45.75 10.75
(42.56) (19.13)
5. Potato dextrose agar 54.75 13.75
(46.57) (21.76)
6. Richard‟s medium 49.25 12.50
(44.57) (20.70)
SEm+ 0.49 0.18
CD at 5% 1.50 0.54
* Average of four replications Figures given in parenthesis are angular transformed values
Table 4.3 Effect of temperature on the mycelial growth and sporulation of
Alternaria alternata in vitro (after 7 days of incubation at 25 + 2 0C)
S.No. Temperature (0C) Mycelial growth No. of spore/ml (mm)* (X 106)* 1. 20 41.50 15.75 (40.10) (23.38) 2. 25 88.00 35.50 (69.73) (36.57) 3. 30 66.00 23.75 (54.33) (29.16) 4. 35 49.75 18.25 (44.85) (25.29) 5. 40 10.25 3.25 (18.67) (10.38) 6. 45 0.00 0.00 (0.00) (0.00) 7. 50 0.00 0.00 (0.00) (0.00) SEm+ 0.30 0.15 CD at 5% 0.92 0.47 * Average of four replications Figures given in parenthesis are angular transformed values
Table 4.4 Effect of relative humidity on the growth and sporulation of
Alternaria alternata (after 7 days of incubation at 25 + 2 0C)
S.No. Relative humidity (%) Mycelial growth No. of spore/ml (mm)* (X 106)* 1. 50 42.00 11.25 (42.39) (19.59) 2. 60 49.25 14.75 (44.57) (22.58) 3. 70 67.50 25.25 (55.24) (30.16) 4. 80 80.50 32.50 (63.79) (34.75) 5. 90 86.00 35.00 (68.02) (36.27) 6. 100 88.25 37.25 (69.95) (37.90) SEm+ 0.70 0.37 CD at 5% 2.14 1.12 * Average of four replications Figures given in parenthesis are angular transformed values
Table 4.5 Mycelial growth and sporulation of Alternaria alternata at different
hydrogen-ion concentration (after 7 days of incubation at 25 + 2 0C)
S.No. pH level Dry mycelial weight No. of spore/ml (mg)* (X 106)* 1. 5.5 67.50 21.00 (55.24) (27.27) 2. 6.0 72.00 23.00 (58.05) (28.65) 3. 6.5 95.00 30.50 (77.07) (33.52) 4. 7.0 91.00 28.75 (72.54) (32.42) 5. 7.5 80.50 26.00 (63.79) (30.65) 6. 8.0 76.50 25.25 (61.00) (30.16) 7. 8.5 62.00 19.75 (51.94) (26.38) SEm+ 0.66 0.29 CD at 5% 2.03 0.88 * Average of four replications Figures given in parenthesis are angular transformed values
Table 4.6 : In vitro efficacy of bio-control agents against Alternaria alternata in dual culture plate method
S.No. Bioagents Zone of inhibition (mm)*
1. Bacillus spp. 3.00
(9.97)
2. Trichoderma harzianum 6.50
(14.77)
3. Trichoderma polysporum 7.50 (15.89)
4. Trichoderma viride 5.0
(12.92)
5. Check 0.0
(0.00)
* Average of four replications Figures given in parenthesis are angular transformed values
Table 4.9 In vitro efficacy of various fungicides against mycelial growth of Alternaria alternata (after 7 days of incubation at 25 +2 0C)
S. Fungicides Per cent inhibition mycelial growth* No. concentration (ppm) Mean 50 100 200 500 1000 1. Captan 15.50 20.25 23.70 32.80 38.25 26.10
(23.18) (26.74) (29.13) (34.93) (38.26) (30.72)
2. Carbendazim 42.80 47.70 61.25 75.00 78.50 61.05 (53.98) (43.68) (51.50) (60.00) (62.37) (51.38)
3. Chlorothalonil 35.50 42.50 54.25 68.10 76.50 55.37
(36.57) (40.68) (47.43) (55.61) (61.00) (48.08)
4. Copper oxchloride 50.30 58.30 70.20 85.75 95.20 71.95 (45.17) (49.77) (56.91) (67.82) (77.34) (58.02)
5. Difoltan 44.80 50.80 65.70 80.30 84.40 65.20 (42.01) (45.45) (54.15) (63.65) (66.34) (53.84)
6. Mancozeb 50.70 60.80 75.30 90.50 100.00 75.46
(45.40) (51.23) (60.19) (72.04) (90.00) (60.30)
7. Tilt 8.42 11.40 16.20 28.40 32.75 19.43
(Propiconazole)
(16.86) (19.73) (23.73) (32.20) (34.90) (26.15)
8. Check 0.00 0.00 0.00 0.00 0.00 0.00
(0.00) (0.00) (0.00) (0.00) (0.00) (0.00)
Mean 31.00 36.46 45.82 57.60 63.19
(33.83) (37.14) (42.60) (49.37) (52.64)
SEm+ C.D. at 5% Fungicide (F) 0.50 1.41 Concentration (C) 0.38 1.05 F x C 1.13 3.16
* Average of four replications Figures given in parenthesis are angular transformed values
Table 4.10 In vitro efficacy of various fungicides against sporulation of Alternaria alternata (after 7 days of incubation at 25 +2 0C)
S. Fungicides No. of spore/ml (x106)* Mean No. 50 100 200 500 1000 1. Captan 17.00 15.25 14.25 11.25 7.50 13.05
(24.35) (22.98) (22.17) (19.59) (15.89) (21.1 7) 2. Carbendazim 15.75 14.00 11.25 7.50 5.00 10.70 (23.38) (21.91) (19.59) (15.89) (12.92) (19.0 9) 3. Chlorothalonil 16.50 14.25 13.75 11.00 7.00 12.50
(23.96) (22.17) (21.76) (19.36) (15.34) (20.7 0) 4. Copper oxchloride 11.50 7.75 7.00 5.00 4.00 7.05 (19.82) (16.16) (15.34) (12.92) (11.53) (15.3 9) 5. Difoltan 13.50 8.25 7.75 5.50 4.50 7.87 (21.55) (16.69) (16.16) (13.56) (12.58) (16.2 9) 6. Mancozeb 9.00 7.25 6.50 4.25 0.00 5.39
(17.45) (15.62) (14.77) (11.82) (0.00) (13.4 2) 7. Tilt 18.00 16.50 14.25 12.75 8.50 14.20
(Propiconazole)
(25.10) (23.96) (22.17) (20.72) (16.95) (22.1 3) 8. Check 0.00 0.00 0.00 0.00 0.00 0.00
(0.00) (0.00) (0.00) (0.00) (0.00) (0.00)
Mean 12.65 10.40 9.47 7.15 4.54
(20.83) (18.81) (17.92) (15.50) (11.30)
SEm+ C.D. at 5% Fungicide (F) 0.20 0.55 Concentration (C) 0.15 0.41 F x C 0.44 1.22
* Average of four replications Figures given in parenthesis are angular transformed values
Table 4.7 In vitro efficacy of phytoextracts against mycelial growth of Alternaria alternata (after 7 days of incubation at 25 +2 0C)
S. Name of plant Per cent inhibition of mycelial growth Mean No. concentration in ppm* 50 100 200 500 1000 1. Babul 21.75 25.00 27.25 30.50 35.75 28.05
(27.79) (30.00) (31.46) (33.52) (36.72) (31.97)
2. 27.25 32.75 35.50 39.00 44.75 35.85 Datura (31.46) (34.90) (36.57) (38.64) (41.98) (36.78)
3. Jangly Choulai 25.50 27.75 31.25 35.50 39.25 31.85
(30.32) (31.78) (33.98) (36.57) (38.79) (34.35)
4. Garlic 36.25 39.75 42.50 49.75 64.75 46.60 (37.01) (39.08) (40.68) (44.85) (53.57) (43.05)
5. Ginger 26.75 31.00 34.40 38.00 42.25 34.48 (31.14) (33.83) (35.91) (38.05) (40.54) (35.95)
6. Neem 28.00 35.25 38.75 42.25 57.50 40.35
(31.94) (36.42) (38.49) (40.54) (49.31) (39.43)
7. Tulsi 29.75 36.50 41.00 46.50 62.75 43.30
(33.05) (37.16) (39.81) (42.99) (52.38) (41.14)
8. Dithane M-45 50.70 60.80 75.30 90.50 100.00 75.46
(45.40) (51.23) (60.19) (42.04) (90.00) (60.30)
9. Check 0.00 0.00 0.00 0.00 0.00 0.00
(0.00) (0.00) (0.00) (0.00) (0.00) (0.00)
Mean 27.32 32.08 36.21 41.33 49.66
(31.51) (34.49) (36.99) (40.00) (44.80)
SEm+ C.D. at 5% Fungicide (F) 0.43 1.21 Concentration (C) 0.32 0.90 F x C 0.96 2.70
* Average of four replications Figures given in parenthesis are angular transformed values
Table 4.8 In vitro efficacy of phytoextracts against sporulation of Alternaria alternata (after 7 days of incubation at 25 +2 0C)
S. Name of plant No. of spore/ml (x106)* Mean No. 50 100 200 500 1000 1. Babul 21.75 19.00 18.25 16.00 13.50 17.70
(27.79) (25.84) (25.29) (23.57) (21.55) (24.87)
2. 17.25 15.50 14.25 11.50 8.00 13.30 Datura (24.54) (23.18) (22.17) (19.82) (16.42) (21.38)
3. Jangly Choulai 14.25 17.50 16.25 14.00 11.50 15.70
(26.02) (24.72) (23.77) (21.97) (19.32) (23.34)
4. Garlic 13.25 8.00 7.50 5.25 4.00 7.60 (21.34) (16.42) (15.89) (13.24) (11.53) (16.00)
5. Ginger 18.50 16.75 15.00 12.75 9.00 14.40 (25.47) (24.15) (22.78) (20.92) (17.45) (22.30)
6. Neem 16.50 14.00 13.00 11.25 7.00 12.35
(23.96) (21.97) (21.13) (19.59) (15.35) (20.57)
7. Tulsi 15.00 13.50 11.25 7.50 5.25 10.50
(22.78) (21.55) (19.59) (15.89) (13.24) (18.90)
8. Mencozeb 9.00 7.25 6.50 4.25 0.00 5.39
(17.45) (15.62) (14.77) (11.82) (0.00) (13.42)
9. Check 0.00 0.00 0.00 0.00 0.00 0.00
(0.00) (0.00) (0.00) (0.00) (0.00) (0.00)
Mean 14.50 12.38 11.33 9.16 6.47
(22.38) (20.60) (19.66) (17.61) (14.73)
SEm+ C.D. at 5% Fungicide (F) 0.23 0.65 Concentration (C) 0.17 0.48 F x C 0.52 1.45
* Average of four replications Figures given in parenthesis are angular transformed values
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