Incidence and management of Alternaria sp. associated with blond psyllium (Plantago ovata L.) seeds bZlcxksy ¼IykUVsxks vksosVk fy-½ ds chtksa ls lg&lacaf/kr vkWYVjusfj;k iztkfr;ksa dk izdksi ,oa izcU/ku
Sajjan Choudhary
Thesis
Master of Science in Agriculture (Plant Pathology)
2015
Department of Plant Pathology S.K.N. COLLEGE OF AGRICULTURE, JOBNER-303 329 SRI KARAN NARENDRA AGRICULTURE UNIVERSITY, JOBNER Incidence and management of Alternaria sp. associated with blond psyllium (Plantago ovate L.) seeds bZlcxksy ¼IykUVsxks vksosVk fy-½ ds chtksa ls lg&lacaf/kr vkWYVjusfj;k iztkfr;ksa dk izdksi ,oa izcU/ku
Thesis
Submitted to the Sri Karan Narendra Agriculture University, Jobner
In partial fulfillment of the requirement for the degree of
Master of Science In the Faculty of Agriculture (Plant Pathology)
By
Sajjan Choudhary
2015
Sri Karan Narendra Agriculture University, Jobner S.K.N. College of Agriculture, Jobner
CERTIFICATE-I
Dated :______2015
This is to certify that Mr. Sajjan Choudhary has successfully completed the comprehensive examination held on ------2014 as required under the regulation for Master’s degree.
(K.S. SHEKHAWAT) Professor & HEAD Department of Plant Pathology S.K.N. College of Agriculture, Jobner
Sri Karan Narendra Agriculture University, Jobner S.K.N. College of Agriculture, Jobner
CERTIFICATE-II
Dated :______2015
This is to certify that the thesis entitled “Incidence and management of Alternaria sp. associated with blond psyllium (Plantago ovata) seeds” submitted for the degree of Master of Science in the subject of Plant Pathology embodies bonafide research work carried out by Mr. Sajjan Choudhary 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 13.12.2014.
(K.S. Shekhawat) Professor & HEAD (R.P. Ghasolia) Department of Plant Pathology Major Advisor S.K.N. College of Agriculture, Jobner
(G.L. KESHWA) DEAN S.K.N. College of Agriculture, Jobner
Sri Karan Narendra Agriculture University, Jobner S.K.N. College of Agriculture, Jobner
CERTIFICATE-III Dated :______2015 This is to certify that the thesis entitled “Incidence and management of Alternaria sp. associated with blond psyllium (Plantago ovata) seeds” submitted by Mr Sajjan Choudhary to Sri Karan Narendra Agriculture University, Jobner, in partial fulfillment 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. Ghasolia) (R.R. Ahir) Major Advisor Advisor
(B.L. Jat) (B.L. Kakraliya) Advisor Director Education Nominee
(K.S. Shekhawat) Professor & HEAD (G.L. KESHWA) Department of Plant Pathology Dean S.K.N. College of Agriculture, S.K.N. College of Agriculture, Jobner Jobner
Approved
DIRECTOR EDUCATION S.K.N. Agriculture University, Jobner
Sri Karan Narendra Agriculture University, Jobner S.K.N. College of Agriculture, Jobner
CERTIFICATE-IV
Dated : _____ 2015
This is to certify that Mr Sajjan Choudhary of the Department of Plant Pathology, S.K.N., College of Agriculture, Jobner has made all corrections/ modifications in the thesis entitled “Incidence and management of Alternaria sp. associated with blond psyllium (Plantago ovata) seeds” which were suggested by the external examiner and the advisory committee in the oral examination held on ______2014. The final copies of the thesis duly bound and corrected were submitted on ______2014 and forwarded herewith for approval.
(R.P. Ghasolia) Major Advisor (K.S. Shekhawat) PROFESSOR & HEAD Department of Plant Pathology S.K.N. College of Agriculture, Jobner
(G.L. KESHWA) DEAN S.K.N. College of Agriculture, Jobner
APPROVED
DIRECTOR EDUCATION SKNAU, Jobner
CONTENTS
CHAPTER PARTICULARS PAGE NO. NO.
1. INTRODUCTION ………..
2. REVIEW OF LITERATURE ………..
3. MATERIALS AND METHODS ………..
4. RESULTS ………..
5. DISCUSSION ………..
6. SUMMARY ………..
LITERATURE CITED ………..
ABSTRACT (English) ………..
ABSTRACT (Hindi) ………..
LIST OF TABLES Table Particulars Page No. No. 3.1 Districts where from seed samples collected and their …… code number 3.2 Phytoextracts used against Alternaria alternata …… 4.1 Details of seed samples collected …… 4.2 Seed abnormalities and impurities in different isabgol …… seed samples 4.3 Incidence of mycoflora associated with isabgol seeds …… analysed by Standard Blotter Method 4.4 Incidence of mycoflora associated with isabgol seeds …… analysed by Standard Agar Plate Method 4.5 Pathogenicity of seed borne Alternaria alternata of …… isabgol by Seed Inoculation Technique (in Petri plates) 4.6 Pathogenicity of seed borne Alternaria alternata of …… isabgol by Seed Inoculation Technique (in pots) 4.7 Pathogenicity of Alternaria alternata by foliar …… inoculation technique (in pots) 4.8 Effect of different levels of temperature on mycelial …… growth of Alternaria alternata at 7th day of incubation in vitro 4.9 Effect of pH on mycelial growth of Alternaria alternata …… on PD broth at 10th day of incubation at 25 + 10C in vitro 4.10 Effect of solid media on mycelial growth of Alternaria …… alternata at 25+1°C 4.11 Effect of relative humidity on the mycelial growth of …… Alternaria alternata at 10th day of incubation at 25 + 10C in vitro
Table Particulars Page No. No. 4.12 Effect of fungicidal foliar spray on disease intensity …… 4.13 Comparative efficacy of different fungicides on …… mycelial growth of Alternaria alternata in vitro after 7 days of incubation at 25 + 1 0C 4.14 In vitro fungitoxicity of plant extracts against Alternaria …… alternata by poisoned food technique after 7 days of incubation at 25 + 1 0C
LIST OF FIGURES
Figure Particulars Page No. No. 4.1 Incidence of mycoflora associated with isabgol seeds …… analysed by Standard Blotter Method 4.2 Incidence of mycoflora associated with isabgol seeds …… analysed by Standard Agar Plate Method 4.3 Effect of different levels of temperature on mycelial growth of Alternaria alternata at 10th day of incubation …… in vitro 4.4 Effect of pH on mycelial growth of Alternaria alternata on PD broth at 10th day of incubation at 25 + 10C in …… vitro 4.5 Effect of different levels of relative humidity on the mycelial growth of Alternaria alternata at 10th day of ……. incubation in vitro 4.6 Effect of fungicidal foliar spray on disease intensity (in ……. vivo) 4.7 Comparative efficacy of different fungicides on mycelial growth of Alternaria alternata in vitro after 7 ……. days of incubation at 25 + 1 0C
LIST OF PLATES
Plate Plate Page No. No. 1 Categorization of isabgol seeds ……
2 Pathogenicity test and culture of Alternaria alternata ……
3a Effect of different pH levels on growth of Alternaria ……
alternata on PD broth media
3b Effect of different levels of temperature on growth of ……
Alternaria alternata on PDA
3c Effect of different levels of relative humidity on growth ……
of Alternaria alternata on PDA
4 Efficacy of different fungicides against A. alternata (in ……
vitro)
5. Fungitoxicity of different plant extracts against A. ……
alternata (in vitro)
1. INTRODUCTION
Blond psyllium (Plantago ovata Forsk.) commonly known as isabgol, is an annual herb with narrow linear rosette like leaves belonging to the family Plantaginaceae. Out of about 200 species of psyllium available in various parts of the world, only 10 are commonly found in India and among these blond psyllium, Plantago ovata Forsk. known for superior quality of husk is preferred over others. It is indigenous to the Mediterranean and West Asia extending upto Sutlej and Sindh in Pakistan. The name isabgol or asphgol is derived from two Persian words "Isab" or “Asph” means horse and "Ghol" means ear, combinedly "horse ear". The Persian word Aspha is said to have originated from Sanskrit word "Ashwa" means horse and the appearance of seeds is just like boat shaped. Isabgol seed and husk is used in medicines especially for relieving constipation.
Isabgol is an important cash crop cultivated for its export and being of important medicinal value is reported to have larger demands and is traded in major medicinal drug markets of the world. Isabgol has pharmaceutical importance to treat dysentery, chronic constipation and chronic diarrhoea and as laxative demulcents, emollients and diuretics. India commands nearly monopoly in the production and export of the seed and husk to the world market. India is earning about Rs. 1600 million as foreign exchange from the export of blond psyllium products to countries like USA, Germany, France, England, Spain and Belgium (Maiti, 2000).
In India, the isabgol crop is mainly grown as commercial crop in Gujarat, Rajasthan and Madhya Pradesh. However, the crop is spreading to other non-traditional parts of the country such as Haryana, Uttar Pradesh and Karnataka. In Rajasthan, it is being cultivated in 190081 hectare area with a total production of 99950 tonnes of seeds with an average productivity of 525 kg/ha (Anonymous, 2012-13). In Rajasthan, lsabgol mainly cultivated in Barmer, Jalore, Nagaur, Jodhpur and Jaisalmer districts. Presently, Rajasthan is on the top in productivity in India.
The seeds of isabgol have neither odour nor taste, but consist of colloidal mucilaginous rosy white husk, which is composed of reserved carbohydrates particularly pantosans like xylose, arabinose, rhamnose, galactose besides galacturonic acid. The husk constitutes about 30 per cent part of seeds and is mainly used as bulk laxative. In addition to mucilage, the seeds also contain semi-drying fatty oils (5 per cent), small amount of acubin and tannin, an active ingredient exhibiting acetyl-choline like action. It has a high water absorbing capacity. The very high water retentive capacity of blond psyllium helps in controlling diarrhoea, chronic dysenteries of amoebic and bacilliary origin and ulcerated surface of intestinal mucosa. It is also used for treating constipation, cervical dilater for pregnancy termination (Anonymous, 1979), to cure diuretic, alleviate kidney and bladder disorders, gonorrhoea, urethrites, hemorrhoids, burning sensation in feet, polyurea, difficult mucturitions and piles. It is also reported to reduce blood cholesterol (Taneja et al., 1989). Other than medicinal uses, it is also used in dyeing, calico-printing, ice-cream stabilizer, confectionery and cosmetic industry etc. The seed after removing husk contain 17 to 19 per cent protein and is used as poultry and cattle feed.
One of the major factors, reducing isabgol yield is onslaught of diseses like leaf blight (Alternaria alternata); wilt (Fusarium sp.); damping off (Pythium ultimum) and downy mildew (Peronospora plantaginis) which were reported by Patel et al. (1984); Russel (1975); Kapoor and Choudhary (1978) and Richardson (1990), respectively.
Out of these diseases, leaf blight or blight caused by Alternaria alternata, generally affects the crop at all stages of growth. Initially, the infected plants show minute spots with loss of chlorophyll. The spots enlarge gradually covering larger area and the colour of affected portions change from light brown to dark brown and black. Necrosis of the affected area also occurs (Patel et al., 1984, Maharshi, 1992, Meena and Maharshi, 2013b).
Alternaria alternata (Fr.) Keissler, is a ubiquitous and omnivorous plant pathogen, inciting several diseases in many plant species. The genus Alternaria belongs to the Hyphomycetous Anamorphic (earlier deuteromycetes) group of Kingdom Fungi, which comprises different saprophytic as well as endophytic species and is well known for its notoriously destructive plant pathogen member. The necrotrophic nature of Alternaria species typically leads to extensive damage of the plant and harvest product, with seedlings seldom surviving an attack (Humpherson- Jones, 1985, Rimmer and Buchwaldt, 1995 and Mamgain et al., 2013). The genus is characterized by its transversely and longitudinally septate (muriform) conidia. The multicelluar pigmented (dark coloured) conidia are produced in chains. The conidia are broadest near the base and taper gradually to an elongate beak (Dube, 2013).
Doshi and Sharma (2002) reported 30-40 per cent incidence of leaf spot of Aloe vera caused by A. alternata. Similar incidence of the disease was reported in Ruhelkhand division of Uttar Pradesh (Gupta and Masood, 2003). Besides Aloe vera, the fungus A. altemata also causes heavy losses in several other crops (Tak et al., 1985; lvanovic et al., 1994 and Kumari, 2001). Meena and Maharshi (2013a) recovered ten fungi including Alternaria alternata from the seeds of isabgol.
However, not much work has been conducted on incidence and management of Alternaria spp. associated with seeds of isabgol in Rajasthan, which produces almost exclusively this grain for medicinal use. The complains made by farmers of western Rajasthan and discussions held with extension personelles of Department of Agriculture, Government of Rajasthan, about occurrence of blight disease of isabgol, it has been decided to assess the incidence of Alternaria spp. with seeds, effect of physio-pathological factors and its management by plant extracts and fungicides.
Therefore, keeping in view of the above facts and the recognized role of seed borne fungi, endeavours have been made to fill up some of the several gaps existing in literature. Investigation entitled “Incidence and management of Alternaria alternata associated with blond psyllium (Plantago ovata) seeds” was therefore undertaken to retain the monopoly of India and especially of Rajasthan in production of isabgol with the following objectives:
(i) To record the incidence of Alternaria spp. and other fungi associated with blond psyllium seeds.
(ii) To observe the effect of Alternaria spp. on seed germination and seedling vigour
(iii) To know the effect of physiopathological factors on Alternaria spp.
(iv) To manage the Alternaria spp. by various antifungal agents. 2. REVIEW OF LITERATURE
Isabgol is one of the prime medicinal crop, which is being exported to developed countries and middle east. It is one of the major foreign exchange earner and having the highest contribution in exports of medicinal plants. Rajasthan is now on the top to cultivate this crop. However, cultivation of the crop has been threatened by many biotic and abiotic factors which affect its quality and production adversely. Among the major biotic stress downy mildew (Kapoor and Choudhary, 1976 and Rathore and Rathore, 1996), damping off (Chastagner et al., 1978), Fusarium wilt (Russel, 1975), leaf blight (Patel et al., 1984) and oozing (Rathore, 2003) are reported on Isabgol.
The Alternaria blight of Isabgol was reported after confirming its pathogenic nature through Koch’s postulates (Patel et al., 1984). Some of the reports of Alternaria blight where, pathogenicity was proved are being quoted to support its pathogenic nature. Gaddanakeri and Kulkarni (1998) isolated Alternaria alternata using tissue isolation from infected leaves of turmeric (Curcuma longa). Akhtar et al. (2004) isolated A. alternata causing leaf blight of tomato and also proved its pathogenicity.
Maiti et al. (2007) found that leaf blight disease of Gloriosa superb (Liliaceous), an endangered, herbaceous, perennial, climbing lily that produces colchicine, in West Bengal (India) showed brownish spots on leaves, developed in concentric rings, which eventually darkened and coalesced to blight the entire leaf. The causal fungus was morphologically identified as Alternaria alternata (Fr.) Keissler.
Ramjegathesh and Ebenezar (2012) collected ten isolates of A. alternata causing leaf blight of onion from conventional onion growing areas of Tamil Nadu, India and their pathogenicity was established. 2.1 Incidence of Alternaria alternata and other fungi associated with blond psyllium seeds
Literature revealed that only a few attempts have been made as to know about the seed mycoflora of isabgol. Patel et al. (1984) first reported leaf disease of isabgol caused by Alternaria alternata in Gujarat. Elwakil and Ghoneem (1999) reported 41 species of fungi belonging to 21 genera from seed samples of blond psyllium from Egypt. They proved the pathogenic nature of Fusarium oxysporum, Fusarium solani and Fusarium moniliforme with isabgol seeds and seedlings.
Singh and Khan (1999) reported the presence of Aspergillus niger, Penicillium citrinum, Rhizopus stolonifer, Cladosporium cladosporoides and Trichoderma spp. on isabgol seeds collected from local market of Dehradun (Uttarakhand, India).
The common fungal diseases recorded on isabgol crop are listed as follow :
S.No. Name of Causal organism Reference disease
1 Plantago wilt Fusarium Russel (1975) oxysporum
2 Plantago wilt Fusarium solani Mehta et al. (1985)
3 Leaf disease Alternaria alternata Patel et al. (1984)
4 Downy mildew Peronospora Richardson (1990) plantaginis
5 Damping off Pythium ultimum Richardson (1990)
Godika et al. (2000) reported that one hundred and eighty eight sunflower seed samples collected in Rajasthan, India were subjected to dry seed examination and the blotter test to determine the incidence of Alternaria alternata. Symptomatic seeds with grayish brown and brown to black discolouration yielded A. altternata on incubation on moistened blotters. Samples from Jhalawar (17.0-95.0%), Jaipur (2.0-94.0%), Kota (10.0-91.0%), Baran (1.0-72.0%), Bhilwara (3.0-62.0%), Alwar (1.0-51.0%) and Sriganganagar (4.0-52.0%) districts revealed high infection percentage.
Anjli et al. (2005) observed that young mature and ripe fruits of C. papaya collected from farmer’s fields in Abdulapur, Gokulpur and markets in Meerut and Mawana, Uttar Pradesh, India were analysed for associated mycoflora. Isolation on potato dextrose agar and Czapek’s Dox agar media showed that ripe fruits harboured more fungi. Steritized or unsterilized seeds (100 to 400 seeds per sample), collected from different localities and cultivars were examined by blotter and agar plate methods. The percentage of occurrence and incidence varied between methods, places and cultivars. The fungi isolated from fruits and seeds consisted of 61 species belonging to 32 genera. Of these, Aspergillus, Alternaria (15 spp.), Colletotrichum and Fusarium (3 spp. each) were dominant.
Sumanth et al. (2010) observed that the seed-borne pathogens are one of the major causes of serious diseases in growing crops because of poor health and quality of seeds. To realize this aspect, the study has been undertaken and it is observed that among the tested spices, Alternaria alternata, Aspergillus flavus, Aspergillus niger, Aspergillus ustus, Cladosporium cladosporoides, Curvularia lunata, Fusarium oxysporum, Fusarium roseum, Helminthosporium tetramera, Trichoderma viride showed maximum incidence on agar plates and A. alternata, Aspergillus flavus, A. niger, A. ustus and H. tetramera have maximum incidence on blotter plates. Meena and Maharshi (2013a) observed that seed samples of isabgol collected from Jalore, exhibited more presence of deformed (shrivelled+inflated) and discolored (black colored) seeds. They recovered ten fungi viz. Alternaria alternata, Aspergillus niger, Chaetomium murorum, Curvularia clavata, C. fallax, Emericella nidulans, E. rugulosa, F. semitectum, Fusidium spp. and Penicillium spp.. Out of which, Alternaria alternata, A. niger and F. semitectum dominated and Chaetomium murorum, Curvularia clavata, C. fallax, Emericella nidulans, E. rugulosa, Fusidium spp., Penicillium sp. were present in the variable frequency based on spore load.
Meena and Maharshi (2013b) studied environmental factors and reported that 8 per cent seed moisture and 40 0C temperature showed minimum incidence at 6 month of storage. Therefore, 8 per cent moisture is suitable to store seed of isabgol for maximum period. In case of 90 per cent relative humidity, seeds showed minimum incidence of Alternaria alternata and Fusarium semitectum after six months of storage.
2.2 Effect of Alternaria alternata on seed germination and seedling vigour
Seed fungi cause various disorders including blackening of fruit (Raicu et al., 1964), seedling rot (Anahosur et al., 1972), wilt (Singh et al., 1972), blight (Gemawat and Prasad,1972), blossom blight (Bandopadhyay et al., 1980), seed rot (Dwivedi et al., 1982), inhibition of seed germination (Prasad, 1984) and seedling death (Srivastava, 1984).
Similarly, Dhingra and Muchovej (1979) have found F. semitectum to be seed borne on snap bean causing pod rot, seed rot and root rot symptoms.
Costa et al. (2005) found F. semitectum to be the major seed colonizing fungus in the commercial acid delinted cotton seed lots. The internally seed borne inoculum besides reducing seedling emergence incited an array of symptoms finally leading to dry root rot and seedling mortality.
Erpelding and Prom (2006) studied seed mycoflora of sorghum and found that Fusarium semitectum was the most frequently occurring fungal species leading to reduction in seed germination followed by Curvularia lunata.
Among seed mycoflora of carrot, Alternaria alternata, A. dauci, A. radicina, Aspergillus spp., Fusarium spp., Penicillium sp. and Phoma spp. were reported to be associated with carrot seeds and affect germination (Kim and Mathur, 2006).
Meena and Maharshi (2013b) reported that seed samples having association of Alternaria spp. and other fungi reduced seed germination and seedling vigour of isabgol.
2.3 Physiopathological studies
2.3.1 Effect of different media
Effect of various culture media on the growth, sporulation and morphological variations of A. alternata, causing leaf spot disease in several crops have been reported in various crops.
Among the different liquid media, Richard's medium supported maximum mycelial growth of Alternaria alternata (Kulkarni, 1998). The poorest growth with the less sporulation was observed in Conn's medium (Gopinath et al., 2002).
Potato dextrose agar, Richards agar, maize meal agar, Czapex agar, oat meal agar, host leaf extracts agar, malt extracts agar, Kirchoff's medium, Coon's agar and Asthana and Hawker's media were used against A. alternata by many workers Pandey et. al., 2006, Mishra and Mishra, 2012 and Joshi et. al., 2012. Among these, potato dextrose agar medium was found most effective for growth and sporulation of A. alternata.
2.3.2 Effect of temperature
Maheshwari et al. (2000) reported maximum growth and sporulation of A. alternata (leaf spot of bean) at 28 0C temperature. Singh et al. (2001) recorded maximum growth and sporulation of Alternaria alternata between 25 to 30 ± 2 ºC. Prashanthi and Kulkarni (2003) tried different temperatures from 5-45 ºC (5.0, 10, 15, 20, 25, 30, 35, 40, and 45 ºC) for A. alternata, Colletotrichum gloeosporoides and Aureobasidium pullulans. They found 25 ºC as optimum temperature for A. alternata, while, the optimum temperature for both Colletotrichum gloeosporoides and Aureobasidium pullulans was 30 º C. Singh and Majumdar (2004) studied post harvest rot of pomegranate incited by Alternaria alternata and reported that 25 0C temperature was favourable for disease development. Ma-Guillong et al. (2006) reported that 24 ºC temperatures was most suitable for sporulation of Alternaria alternata causing leaf spot of tobacco. Israram et al. (2007) observed maximum growth and sporulation of Alternaria alternata causing Alternaria fruit rot of Ber at 25 to 30 ºC temperature. Bochalya (2010) evaluated effect of temperature and relative humidity on Alternaria alternata causing fruit rot of brinjal in vitro revealed that maximum mycelial growth and sporulation was observed at 25 0C and 90 per cent RH whereas, minimum mycelial growth and sporulation at 15 0C and 60 per cent RH. Prasad and Ahir (2013) also observed maximum mycelium growth of A. alternata at 25 °C temperature.
2.3.3 Effect of hydrogen ions concentration (pH)
Maheshwari et al. (2000) evaluated 16 different pH levels on Alternaria alternata ranging from 3.5 to 10.5 and optimum pH for growth of fungus was found to be 6.5. Singh et al. (2001) reported the growth and sporulation of A. alternata on 11 different media at 10 different pH values. The optimum pH for growth and sporulation was 5.5. Prashanti and Kulkarni (2003) studied the effect of different pH levels (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5) on growth of Chromolana odorata, Alternaria alternata, Colletotrichum gloesporiodes and Aureobasidium pullulans. It was inferred that A. alternata, Colletotrichum gloesporiodes and Aureobasidium pullulans exhibited optimum growth at pH 5.5, 6.0 and 6.5, respectively. However, in most of the studies conducted on different pH levels, it was found that Alternaria alternata favours pH 6.5 (slightly acidic).
Hubballi et al. (2010) evaluated 11 different pH levels on Alternaria alternata ranging from 4.0 to 9.0 and pH 6.5 was found to be ideal and produced the maximum mycelial growth (87.50 mm). Ramjegathesh and Ebenezar (2012) determined growth of Alternaria alternata at 11 different pH levels ranging from 4.0 to 9.0 with a narrow fraction of 0.5. All of the eleven pH levels, pH 4.5 was favoured to be ideal and produced the maximum mycelial growth (8.87 cm) followed by pH 4.0 (8.66 cm) and pH 5.0 (8.46 cm). 2.3.4 Effect of relative humidity
A range of 70 to 80 per cent relative humidity was found most favourable for development of sunflower leaf spot caused by A. alternata (Kumar and Singh, 1996).
Prasad and Ahir (2013) also observed maximum mycelium growth of Alternaria spp. at 100 per cent relative humidity.
2.4 Management of Alternaria alternata by various antifungal agents
2.4.1 Evaluation of fungicides
Various fungicides and biocides have been tested for their efficacy against fungal pathogens causing different disease in crop plants. In bioassay, studies on different fungicides have been evaluated in vitro. Noon et al. (2001) estimated the effectiveness of chlorothalonil and mancozeb @ 2.5 kg/ha as spray against Alternaria blight which reduces the disease severity by 14.2% and 73.6%, respectively as compared to untreated plots (90.9%). Rao and Rao (2002) tested captan, carbendazim, mancozeb and copper oxychloride against stem blight of sesame caused by Alternaria alternata and observed that mancozeb was most effective in reducing disease incidence followed by copper oxychloride and carbendazim.
Ghosh et al. (2002) found that mancozeb (0.25%) and carbendazim (0.1%) were most effective against mycelial growth and sporulation of Alternaria alternata in vitro causing leaf spot of gerbera. Singh et al. (2003) conducted an experiment in vitro to determine the effect of Indofil M-45, Indofil Z-78, Vitavax, Benlate, Thiram, Kavach, Calixin, Ridomil, Blue Copper-50, Miltox, Plantvax and Captan on the growth of Alternaria alternata causing leaf spot of brinjal by poisoned food technique. Indofil M- 45, Indofil Z-78, Vitavax and Kavach caused complete inhibition of growth of Alternaria alternata. Mancozeb and copper oxychloride were found effective against A. alternata both in vitro and in vivo (Singh and Rai, 2003). Nasreen (2003) reported that the growth of Alternaria alternata, Cladosporium cladosporoides, Macrophomina phaseolina, Drechslera specifera, Fusarium oxysporum and Rhizoctonia solani were significantly reduced on PDA amended with 0.1 per cent of either Captan, Vitavax, Dithane M-45, Thiram and Benomyl. Benomyl was found to be most effective.
Mahalinga et al. (2003) investigated the chemical control of Alternaria blight (Alternaria helianthi) in sunflower cv. Modern Sunflower. The treatments were: T1, seed treatment (0.2%) with thiram (ST); T2, ST + one spray of mancozeb at (0.2%) at disease appearance stage (SDA); T3, ST + 2 sprays of the same chemical (mancozeb) at SDA; T4, ST + one spray at 30 days after sowing (DAS); T5, ST + one spray at 45 DAS; T6, ST + 2 sprays at 30 and 40 DAS; T7, untreated seeds with one spray at SDA; T8, untreated seeds + 2 sprays at intervals of 15 days starting from SDA; T9, untreated seeds + one spray at 30 DAS; T10, untreated seeds + one spray at 45 DAS; T11, untreated seeds + 2 sprays at 30 and 45 DAS; and T12, control. The best yield was obtained with mancozeb applied at SDA with and without seed treatment (578 and 825 kg/ha, respectively). T3 and T8 were recorded with the lowest disease incidence in both years (2.53 and 3.79%), followed by T1 and T7. Seed treatment alone was not effective in controlling the disease.
Efficacy of fungicides viz. mancozeb, copper oxychloride, carbendazim, dinocap and IBP (Iprobenphos) in controlling A. alternata causing leaf spot in French bean (Phaseolus vulgaris) cv. PDR-14 was evaluated in field experiments. Mancozeb treatment resulted in maximum control of the disease (Singh et. al. 2003). Shukla et. al. (2005) managed blight disease of Eucalyptus caused by Alternaria alternata using seven fungicides, viz. Dithane M-45, Difolatan, Zineb, Bavistin (0.2%), Blue Copper-50 (0.3%), Captan (0.2%) and Brestan-60 (0.1%). The results indicated that all the fungicides significantly reduced the disease incidence as compared to control and they were equally good for controlling the disease.
Chatta (2005) evaluated six fungicides to control Alternaria blight of castor under field trials for two years. Maximum control of disease was recorded with mancozeb (0.2%) which reduced the disease and produced higher seed yield (15 to 17 q/ha). Efficacy of fungicides viz. carbendazim, captan, mancozeb, copper oxychloride, chlorothalonil and iprodione were tested and found effective against Alternaria alternata causing Alternaria blight of periwinkle (Kumari et.al., 2006).
In bioassay studies of fungicides, Akbari and Parakhia (2007) found difenoconazole and propiconazole to be equally effective and completely inhibited the growth of A. alternata causing blight of sesame even at minimum concentration of 50 ppm. Kumar et al. (2013) tested twelve fungicides namely, Indofil M-45 (0.2%), Indofil Z- 78 (0.2%), Chlorothalonil (0.2%), Bavistin (0.1%), Blitox- 50 (0.2%), Captafol (0.2%), Kitazin (0.2%), Vitavax (0.1%), Thiram (0.2%), Captan (0.2%), Ziram (0.2%) and Topsin-M (0.2%) against leaf spot disease of chilli caused by Alternaria alternata. Out of 12 fungicides, only 5 fungicides viz., Bavistin, Indofil M-45, Chlorothalonil, Vitavax and Thiram proved to be effective fungicides as they inhibited the growth of the pathogen completely. All the fungicides employed in vitro were further evaluated in vivo during two consecutive years (2007 and 2008), in order to assess their efficacy in controlling the disease. All the fungicides gave significantly better control of the disease. Indofil M-45 proved to be most effective in checking the disease followed by Indofil Z-78 and Chlorothalonil. Meena and Maharishi (2013b) tested six fungicides and observed that seed treatment with Bavistin and Topsin M (0.2%) gave effective control of seed borne pathogenic Alternaria alternata causing leaf blight of isabgol.
2.4.2 Evaluation of phyto-extracts
Barros et al., (1995) tested the effectiveness of garlic extract against spore germination and mycelial growth of Alternaria alternata and A. longipes at 250, 500, 1000, 2000, 5000 and 10000 ppm and concluded that garlic extract effectively inhibited the growth of both the species at 250 ppm.
Singh and Majumdar (2001) tested the efficacy of plant extracts viz. Allium sativum, Allium cepa, Curcuma longa, Zingiber officinale, Azadirachta indica, Datura stramonium and Ociumum sanctum against Alternaria alternata causing fruit rot of pomegranate and concluded that extract of Allium sativum, showed minimum disease severity followed by Ocimum sanctum and Zingiber officinale while others were highly effective. Choudhary et al. (2003) evaluated the efficacy of leaf extracts of Eucalyptus globolus, Datura stramonium, Solanum xanthocarpum, Azadirachta indica, Lantana camara, Ricinus communis and Lawsonia inermix, bulb extracts of Allium sativum, Allium cepa and rhizome extracts of Zingiber officinale in controlling early blight of potato caused by Alternaria alternata in vitro.The bulb extracts of Allium sativum recorded the highest inhibition of the pathogen (56.19%) followed by the bulb extracts of Allium cepa (54.27%) at 10 per cent concentration. Panchal and Patil (2009) tested the efficacy of extracts of garlic, turmeric and neem at 10 per cent concentration against Alternaria alternata and reported that garlic clove extract proved highly effective in reducing Alternaria fruit rot of tomato followed by turmeric and neem extract.
The antifungal activities of extracts from Allium sativum, Azadirachta indica, Cassia occidentalis, Clerodendron inerme, Duranta repens, Ferula asafoetida, Lantana camara, Ocimum sanctum, Parthenium hysterophorus Tridex procumbens and Vinca rosea were evaluated at 5 or 10 per cent against A. alternata causing leaf blight of turmeric and found effective against the pathogen (Gorawar and Hedge, 2008).
Fawzi et al. (2009) carried out in vitro studies to test the antifungal activity of 5 plant extracts, Cinnamomum zeylanicum (cinnamon), Cymbopogon proximus (halfa barr), Laurus nobilis (laurel), Perrsea americana (avocado) and Zingiber officinale (ginger) against two pathogenic fungi, Alternaria alternata and Fusarium oxysporum. The results revealed that plants extracts especially those performed with CDW (cold distilled water) had a strong antifungal activity with significant inhibition on the growth of the two tested fungi. Extracts of halfa barr and ginger were found most effective in inhibiting the growth of the tested fungi followed by avocado, cinnamon and laurel. Different plant extracts viz. Eucalyptus globulus, Datura stramonium, Solanum xanthocarpum, Azadirachta indica, Lantana camara, Ricinis communis, Lawsonia inermis, Alliurn sativum and Allium cepa were tested and found effective against, A. alternata in vitro (Karade and Sawant, 1999, Choudhary et.al., 2003 and Bochalya et al., 2012).
3 MATERIALS AND METHODS
3.1 Collection of seed samples
To study the seed borne fungi, seed samples were collected from Barmer, Nagaur, Pali, Jodhpur and Jalore districts of Rajasthan. These samples were collected from farmers, where isabgol is generally cultivated. From each district, 5 samples were collected from farmer’s houses belonging to different villages situated 5-10 km away from district headquarter in different geographical directions where isabgol is generally cultivated and stored by using old traditional practices, under variable environmental conditions. At each district headquarter; all collected seed samples were mixed to represent a composite sample of that particular district. Samples were collected 4-6 weeks before sowing in cloth bags, brought to the laboratory and then stored at 10 + 1 ͦC temperature for further studies.
Table 3.1. Districts where from seed samples collected and their code number
S.No. District Code number 1. Barmer IG-1 2. Jodhpur IG-2 3. Pali IG-3 4. Nagaur IG-4 5. Jalore IG-5
In all, 5 working samples were analyzed by different procedures suggested by ISTA (1985), Agarwal and Sinclair (1987) and Jha (1995).
3.2 Examination of dry seeds
The method suggested by Agarwal and Sinclair (1987) was followed. For examination, 400 g seeds per sample were drawn and divided into four fractions of 100 g each. Each fraction was spread on bottom of the Petri dish and examined with the help of a hand lens or if needed under stereo binocular microscope. The inspected seed materials were categorized as follows:
(A) Deformed seeds (shrivelled or inflated )
(B) Discoloured seeds (black or brown)
(C) Damaged seeds (insect or mechanically)
(D) Impurities
(i) Inert material (stone and sand )
(ii) Plant debries
(iii) Seeds of other crop /weed
(E) Apparently healthy seeds
Seeds and impurities of each category were pooled separately and weighted on electronic balance and per cent content by weight was calculated.
3.3 Isolation of mycoflora associated with isabgol seeds
Five seed samples i.e. IG-1, IG-2, IG-3, IG-4 and IG-5 were used separately for the isolation of mycoflora from the isabgol seeds. Two incubation methods viz., (a) Standard Blotter Method and (b) Agar Plate Method (ISTA, 1976) were employed for this purpose.
3.3.1 Standard Blotter Method
Four hundred seeds from each sample were analyzed after surface sterilization and unsterilization. Blotter papers were cut into 9 cm diameter circle and sterilized at 1.045 kg sq cm for 20 minutes. Three circles of blotter paper were placed at the bottom of each sterilized Petri plate aseptically and moistened by sterile distilled water. Twenty five seeds were placed at equal distance in each Petri plate. For sterilized condition, seeds were treated with 0.1% mercuric chloride (HgCl2) solution for one minute and rotated continuously to avoid clumping followed by three washing with sterile distilled water, were used. These Petri plates were incubated at 25 + 1 0C for 12 h of light alternating period. The seeds were examined on 8th day of incubation for the presence of seed borne fungi.
3.3.2 Standard Agar Plate Method
Two hundred seeds of each of the 5 samples were analyzed after surface sterilization with 0.1 per cent mercuric chloride solution for one minute, followed by 3 washings with sterile distilled water. Twenty five seeds were equispaced aseptically per Petri plate (9 cm diameter) containing 20 ml of PDA (250 ppm streptomycin was added to PDA just before pouring into the Petri plates to check bacterial contamination). The plates were incubated at 25 + 1 0C for 12 h of light alternating with 12 h of dark period. The fungal colonies emanating from seeds were examined from 4th to 7th days of incubation.
Isolation of mycoflora from isabgol seeds were carried out and maintained on 2 per cent PDA medium. Observations on per cent incidence of seed mycoflora were recorded in both Blotter and Agar Plate Methods. Per cent incidence of fungus (PIF) was calculated as follows:
Number of seeds sowing fungal growth PIF = ------x100 Total number of seeds incubated
3.4 Purification and identification of seed mycoflora
Isolated seed borne fungi were purified by two methods viz. (a) single spore technique (b) hyphal tip method
3.4.1 Single Spore Technique
In case of single spore technique, serial dilution of the spore suspension from 7 days old culture was made in sterile distilled water until a solution containing 10-15 spores per ml was achieved. One ml of diluted spore suspension was poured in Petri plate containing 2 per cent plain agar (20 g agar-agar in 1000 ml distilled water, autoclaved at 1.045 kg/sq. cm) under aseptic conditions. Spore suspension was evenly distributed by tilting the Petri plate in clock and anticlockwise directions. After few minutes, excess suspension was removed from the Petri plates. Inoculated Petri plates were incubated at 25 + 1 0C for 24 h. Germinating single spore was located and marked under the microscope with the help of dummy objective and transferred on 2 per cent PDA slants aseptically. Fungal growth on aseptically inoculated slants was subsequently allowed to grow and sporulate.
3.4.2 Hyphal Tip Method
This method is same as described earlier (as under 3.4.1) except that instead of single spore, hyphal tip was marked and transferred on 2 per cent PDA slants.
The different fungal isolates( Aspergillus niger,Curvularia clavata, Curvularia fallax,Fusarium, Alternari alternata and Semitectum) obtained were tentatively identified based on the standard literature. For confirmation of identiy, the pure cultures were sent to Indian Type Culture Collection (ITCC), Division of Plant Pathology, IARI, New Delhi-110012. The letter was received from ITCC and the results are mentioned in chapter of experimental results. Incidence in different seed samples was calculated and further studies were carried out only on Alternaria alternata.
3.5 Effect of Alternaria alternata on seed germination and seedling vigour (Pathogenicity Test)
For this, two tests were performed as follows:
3.5.1. Seed Inoculation Technique
One hundred apparently healthy surface sterilized seeds were taken. The seeds were then rolled on 10 days old sporulating culture of Alternaria alternata, thriving on PDA contained in Petri dishes. Inoculated seeds were plated on sterilized moist blotter in Petri dishes and same number of inoculated seeds were also sown in 30 cm earthen pots (Pre- sterilized and having autoclaved soil) at the rate of 5 seeds per pot x 20. The uninoculated surface sterilized apparently healthy seeds served as control. These Petri dishes and pots were kept in incubator (24+1 0C) and cage house (22-25 0C), respectively. The pots were watered as and when required. Observations on seed germination and pre emergence mortality were recorded on 10th day of sowing whereas, post emergence mortality and root and shoot length were recorded at 30 DAS. Seedlings showing symptoms were recorded on 60th day of sowing.
Seedling vigour was also calculated by following formula of Abdul- Baki and Anderson (1973).
Seeding Vigour Index = Germination % x (Root length + Shoot length)
3.5.2 Spray Inoculation Technique
The method suggested by Mathur et al. (1973) was followed to observe the symptoms on seedlings and growing on plants. Thirty days old seedlings, raised from disinfected seeds in 30 cm earthen pots having autoclaved soil (soil : FYM = 3 : 1) were inoculated by spraying with spore suspension of 7 days old culture of Alternaria alternata, having concentration @ 106 spores/ml. The seedlings were sprayed to run off. Earthen pots having inoculated seedlings were kept in a humid chamber for 48 hours. Check plants were sprayed with distilled water only. Each treatment was comprised of 100 seedlings (5 seedlings/ pots x 20). The sprayed seedlings were accommodated in a cage house where temperature ranged from 20 to 25 0C. Plants were inspected on 15th day of incubation. Isolations were carried out from inoculated plants parts showing typical disease symptoms.
3.6 Physio-pathological 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.
Inoculation was done with 5 mm disc of mycelial mat taken from 7 days old fungal culture and incubated at 25 + 10C (except temperature study) for 10 days. The each experiment under physio-pathological studies was arranged in completely randomized design (CRD) with three replications.
3.6.1 Effect of temperature on mycelial growth
Effect of temperature on mycelial growth of Alternaria alternata was studied in vitro. Twenty ml of sterilized PDA medium was poured in each sterilized Petriplates. Inoculation was made with 5 mm disc of 7 days old culture of Alternaria alternata with the help of sterilized cork borer and incubated at 5 different levels of temperature viz., 15, 20, 25, 30 and 35 0C for 10 days. Observations on mycelial growth was recorded after 10 days of incubation.
3.6.2 Effect of relative humidity on mycelial growth
To study the effect of relative humidity on mycelial growth of Alternaria alternata, five different levels of relative humidity i.e. 60, 70, 80, 90 and 100 per cent were maintained by using the concentrate sulphuric acid and sterilized distilled water in different proportions in glass desiccators by the method suggested by Buxton and Mellanby (1934). The composition of the acid solution was used as followed.
Relative humidity (%) Stock solution (ml)* Distilled water (ml)
60 374.0 396.0
70 348.0 510.3
80 294.0 640.0
90 161.0 712.0
* 50% v/v solution of concentrate sulphuric acid.
Petriplates containing PDA medium were inoculated with 5 mm disc of 7 days old culture of Alternaria alternata with the help of sterilized cork borer. Inoculated Petriplates were immediately accommodated in glass desiccators containing mixture of sulphuric acid and distilled water in required proportion and incubated at 25 + 10C for 10 days. Observations on mycelial growth was recorded after 10 days of incubation.
3.6.3 Mycelial growth on different solid media
Growth on solid media was determined by measuring the colony diameter along with the two diagonals passing through the center of colony by excluding initial diameter (5 mm) of bit was recorded.
Five solid media whose compositions given below were taken for in vitro studies. Petriplates having sterilized medium were inoculated with 5 mm disc of mycelial growth with the help of sterilized cork borer and incubated at 25 + 10C in incubator for 10 days. Observations on mycelial growth (radial growth) was taken after 10 days of incubation.
Medium giving best readial growth of mycelia was used for further studies.
(1) Potato Dextrose Agar (PDA) medium
Agar- Agar 20.00 g
Dextrose 20.00 g
Peeled potato 250.00 g
Distilled water 1000 ml
(2) Czapeck’s Dox Agar medium
Agar-Agar 15.00 g
Sucrose 30.00 g
Distilled water 1000 ml
Dipotassium phosphate 1.00 g
Magnesium 0.50 g
Potassium chloride 0.50 g
Sodium nitrate 2.00 g
Ferrous sulphate 0.01g
(3). Oat Meal medium
Agar- Agar 20.00 g
Glucose 20.00 g
Oat meal 20.00 g
Distilled water 1000 ml
(4). Corn Meal medium
Agar-Agar 20.00 g
Corn meal 20.00g
Glucose 20.00g Distilled water 1000ml
(5). Martin’s medium
Agar-Agar 15.00g
Dextrose 10.00g
Peptone 5.00g
Distilled water 1000ml
Rose Bengal 1 part in 3000 part of media
Potassium dihydrogen phosphate 1.00g
Magnesium sulphate 0.50g
Streptomycin 30.00mg
3.6.4 Effect of pH
To study the effect of different levels of pH on mycelial growth, the pH of medium (broth) was adjusted at 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and
8.0 using citrate phosphate buffer before sterilization with the help of pH meter. Flasks having liquid medium of each pH level were inoculated with
5 mm disc of seven days old fungus culture. Flasks were incubated at 25 +
1°C and the observation on mycelial growth (on dry weight basis) were recorded after 7th day of inoculation.
3.7. Management studies
Efficacy of different fungicides and plant extracts were evaluated against Alternaria alternata.
3.7.1 Efficacy of fungicides against Alternaria alternata (in vitro)
Efficacy of five systemic and non-systemic fungicides against mycelial growth of Alternaria alternata was tested by Poisoned Food Technique (PFT) suggested by Nene and Thapliyal (1979). Four different concentrations viz., 100, 300, 500 and 700 ppm of each fungicide was tested. Required quantity of each fungicide was added separately to sterilized medium mixed thoroughly and poured in sterilized Petriplates and allowed to solidify for 12 hours. Each plate was inoculated with 5 mm disc of 7 days old culture of Alternaria alternata with the help of sterilized cork borer and incubated at 25 + 10C for 10 days. A control was also maintained where medium was not supplemented with any fungicides. The mycelial growth of the test fungus was recorded and per cent growth inhibition was calculated by Vincent’s (1947) formula given below. The experiment was conducted in completely randomized design with three replications.
C-T Per cent growth inhibition = ------x 100 C Where, C = Diameter of colony in check (Average of both diagonals) T = Diameter of colony in treatment (Average of both diagonals) Following fungicides were tested in vitro against Alternaria alternata by Poisoned Food Technique.
Common name Trade name Chemical name Concentration (ppm)
Carbendazim Bavistin Methyl-1H benzimidazole-2-yl 100, 300, 500,700 carbamate
Mancozeb Indofil M-45 Manganese ethylene 100, 300, 500,700 bisdithiocarbamate plus zinc
Carbendazim (12%) Companion Methyl-1H benzimidazole-2-yl 100, 300, 500,700 + Mancozeb (63%) carbamate + Manganese ethylene bisdithiocarbamate plus zinc
Thiophanate methyl Topsin M 100, 300, 500,700
Difenoconazole Score 3-chloro-4-(2RS, 4RS; 2RS, 100, 300, 500,700 4SR)-4-methyl-2-(1H-1,2,4- triazol-1-ylmethyl)-1,3- dioxolan-2-yl] phenyl 4- chlorophenyl ether
3.7.2 Efficacy of fungicides against A. alternata (in vivo) and disease assessment
The field experiments were conducted during 2013-14 in cage house of Department of Plant Pathology, SKN COA, Jobner, Jaipur in RBD manner with four replication in 1 x 1m plots, using seeds from sample ‘IG-5’. All the recommended agronomic practices were followed to raise the crop. The fungicides viz., thiophanate methyl (0.2%), carbendazim *0.1%), mancozeb (0.25%), carbendazim + mancozeb (0.2%) and Difenoconazole (0.2%) were tested by applying as foliar spray (40 DAS). The middle two rows of each plot were rated for disease incidence (the presence or absence of one or more lesions) at 60 DAS by examining five leaves from five adjacent plants at 5 random sites within each plot for a total of 25 leaves per plot. The per cent disease intensity (PDI) was assessed for each plot on a disease rating scale of 1-9 (Gugino et al., 2007) based on the percentage of leaf area symptomatic (1= 0% tissue symptomatic, 2= up to 1%, 3= 2-5%, 4= 6-10%, 5= 11-20%, 6= 21-30%, 7= 31-40%, 8= 41-50%, and 9= over 50%). The intensity was calculated by using the formula of Wheeler (1969).
Sum of individual ratings PDI = x 100 Number of leaves observed x Maximum disease rating
The per cent disease control was calculated by using the following formula:
Disease in control – Disease in treatment PDC = x 100 Disease in control
3.7.3 Efficacy of plant extracts against Alternaria alternata (in vitro)
Following plant extracts were tested in vitro against Alternaria alternata by Poisoned Food Technique.
Table 3.2 Efficacy of plant extracts on mycelial growth of Alternaria alternata (in vitro)
Name of Plant Botanical Part used Concentration (%) name
Neem Azadirachta Leaves 5, 10, 15 indica
Garlic Allium sativum Cloves 5, 10, 15
Lantana Lantana Leaves 5, 10, 15 camara
Turmeric Curcuma longa Rhizomes 5, 10, 15
Ashwaganda Withania Leaves 5, 10, 15 somnifera
The effect of each plant extract was tested at three different concentrations i.e. 5, 10 and 15 per cent. To get these, the required plant parts were thoroughly washed with sterilized water and grinded separately in electric grinder using equal amount of sterilized distilled water. The mixture was squeezed with double layered sterilized cheese cloth. The extracts thus obtained were considered as of 100% concentration. Required amount of stock solution was added to PDA to get desired concentration.
The effect of plant extracts against mycelial growth of Alternaria alternata were tested by PFT. Required quantity of each plant extracts were mixed thoroughly in melted PDA, to get desired concentration, just before pouring in sterilized Petriplates and was allowed to solidify for 12 hours. Each plate was inoculated with 5 mm disc of 7 days old culture of Alternaria alternata with the help of sterilized cork borer. The inoculated Petriplates were incubated at 25 + 10C for 10 days. A control was also maintained where medium was not supplemented with any plant extracts. The experiment was conducted in completely randomized design with four replications. Colony diameter (two diagonals) was measured after 10 days of incubation. The per cent growth inhibition was calculated by Vincent’s (1947) formula.
4. EXPERIMENTAL RESULTS
All the experiments related to the “Incidence and management of Alternaria sp. associated with blond psyllium (Plantago ovata) seeds” were conducted in the Department of Plant Pathology, S.K.N. College of Agriculture, Jobner except seed samples. The results and observations are recorded here as under:-
4.1 Collection of seed samples
Five seed samples were collected from Barmer, Jalore, Jodhpur, Nagaur and Pali duing 2013-14. Details of collected samples with their code number are given in Table 4.1.
4.2 Inspection of dry seeds
Seeds from each sample were categorized in five groups (Table 4.2 and Plate 1). Seed deformity in the form of shriveling was maximum in sample IG-5 (10.6%). Discolouration and damaged seeds were observed in all the samples. Maximum black (10.9%), brown (3.6%) discoloration and damaged seeds (4.8%) were observed in sample IG-5 while minimum black (3.1%), brown (2.2%) discolouration and damaged seeds (1.9%) in IG-2.
All five seed samples contained plant debries, seeds of other crops and sand, stones and other particles as impurities. Maximum healthy seeds were observed in sample IG-2 (88.8%) whereas, minimum was observed in IG-5 (66.5%) seed sample (Table 4.2)
4.3 Isolation of mycoflora associated with isabgol seeds
4.3.1 Standard Blotter Method
Five fungal species belong to four genera in addition to un-identified mycoflora were isolated from sterilized and un-sterilized isabgol seeds (Table 4.3 and Fig. 1). Fungi and their respective per cent incidence from sterilized and un-sterilized seeds were Alternaria alternata (1.00-11.25), Aspergillus niger (1.50-8.25), Curvularia clavata (0.25-3.75), C. fallax (0.00-2.50), Fusarium semitectum (1.50-7.25) and un-identified mycoflora (0.00-6.25).
Per cent incidence of Alternaria alternata, out of total per cent mycoflora was recorded maximum in un-sterilized seeds of Jalore sample (29.41) followed by Barmer (27.02), Jodhpur (25.80) and Pali (22.54) while it was minimum in Nagaur sample (21.50). Per cent incidence of Alternaria alternata including other fungi was less in sterilized seeds as compared to un- sterilized seeds.
The sterilized and un-sterilized seeds were plated on moist blotter paper in sterilized Petri plates for detection of fungal flora. The total frequency of mycoflora was observed maximum in unsterilized seeds of sample of Jalore (38.50%) followed by Pali (25.70%), Barmer (18.50%) and Jodhpur (15.50%) and it was minimum in Nagaur (12.75%), whereas, in surface sterilized seeds, the incidence of mycoflora was maximum in the sample of Jalore (11.25%) followed by Pali (9.25%) and Barmer (7.25%) and it was minimum in samples of Jodhpur (6.00%) and Nagaur (6.00%).
4.3.2 Standard Agar Plate Method
Five fungal species belong to four genera in addition to un-identified mycoflora were isolated from isabgol seeds (Table 4.4 and Fig. 2)). Fungi and their per cent occurrence recorded were Alternaria alternata (1.50-5.00), Aspergillus niger (2.00-4.50), Curvularia clavata (0.50-2.50), C. fallax (0.00- 2.50) and Fusarium semitectum (2.00-3.50). Total per cent mycoflora was maximum in Jalore sample (19.00%) followed by Pali (13.00%), Nagaur (12.00%), Barmer (12.00%) and Jodhpur (9.00%). Per cent incidence of Alternaria alternata, out of total per cent mycoflora was highest in Jalore (27.77%) followed by Barmer (25.00%), Pali (21.15%), Nagaur (20.83%) and Jodhpur (16.86%).
4.4. Pathogenicity Test Among various fungi isolated from seeds. Results on the effect of seed borne fungi on germination and seedling vigour obtained by seed and foliar inoculation methods are presented in the (Table 4.5, 4.6 and 4.7) Alternaria alternata was most predominantly associated with them. Hence, for further studies pathogenicity was tested for Alternaria alternata only. 4.4.1 Seed inoculation (in Petri plates) Inoculation of healthy seeds with Alternaria alternata (Table 4.5) caused both pre- and post-emergence mortality (11.42% and 3.07%, respectively), reduction in vigour index (292.5) in comparison to control (1003.3). Amongst the seedlings raised in the test, 74.60 per cent seedlings showed symptoms and they were yellowing and brown or black necrotic spots on the leaves. 4.4.2 Seed inoculation (in pots) In seed inoculation, Alternaria alternata caused higher pre- and post- emergence mortality i.e. 9.37% and 4.41%, respectively and reduction in vigour index (639.2) in comparison to control (850.5). Amongst the seedlings raised in the test, 83.07 per cent seedlings showed symptoms like seedling blight, leaf blight and yellowing of leaves (Table 4.6). 4.4.3 Foliar inoculation Out of 100 seedlings sprayed with conidial suspension of Alternaria alternata, 85.0 per cent of seedlings showed symptoms (Table 4.7). Symptoms observed at seedling stage were yellowing, tip burning, marginal blight and brown to black small spots on the leaves. Re-isolation from affected tissues revealed the presence of the pathogen. 4.5 Physiology of the pathogen
4.5.1 Effect of temperature on mycelial growth of Alternaria alternata (in vitro)
The effect of different temperature levels viz., 15, 20, 25, 30 and 35 0C was studied according to methods described, on PDA as basal medium with essentially pH 6.5. This was aimed to know the optimum temperature for growth of Alternaria alternata. Results depicted in Table 4.8, Fig. 3 and Plate 3 reveals that fungus could grow at all the temperature levels viz., 15, 20, 25, 30 and 35 ºC. Maximum mycelial growth (88.00 mm) was observed at 25 ºC. However, the temperature of 20 ºC, 30 ºC and 35 0C resulted in 75.00 mm, 71.00 mm and 65.00 mm mycelial growth of Alternaria alternata, respectively, but differ significantly from the growth at 25ºC.
4.5.2 Effect of different pH levels on growth of Alternaria alternata
In general, fungi are capable of growing within a wide range of hydrogen ion concentrations of the medium while, most of them grow best in neutral or slightly acidic medium. The pH preference of most of the foliar pathogens ranges between 5.0 to 6.5 which obviously favour the establishment of pathogen in their host. Hydrogen ion concentration governs several physiological and metabolic processes of microorganisms. The relationship of pH to the mycelial growth of Alternaria alternata was determined at different pH levels viz. 4.0 to 8.0 at 25 ± 1 ºC for 7 days.
Of all the eight pH levels, pH 6.5 was found to be idea and produced the maximum dry mycelial weight of 845 mg followed by pH 6.0 (820 mg) which was at par to each other. The dry mycelial weight was lowest at pH 9 which recorded 290 mg (Table 4.9, Fig.4 and plate 3). The pH below six and above 6.5 was noticed to be inhibitory to the growth.
4.5.3 Effect of solid media on mycelial growth of Alternaria alternata
To find out a suitable medium for the mycelial growth of Alternaria alternata, five different synthetic and semi synthetic media were tested in vitro.
On perusal of data (Table 4.10) revealed that among the five different solid media understudy, the potato dextrose agar was significantly superior in supporting maximum mycelial growth (85.55 mm) at 7th day of incubation followed by Richard’s media (76.70 mm) and oat meal agar (63.33 mm). Minimum growth of the fungus was observed on Martin’s medium (37.77 mm). The potato dextrose agar medium was best supporter of growth of the fungus. The PDA medium was selected for further studies. 4.5.4 Effect of relative humidity on mycelial growth of A. altermata To evaluate the effect of atmospheric moisture, the fungus was exposed directly to different levels of relative humidity viz., 50, 60, 70, 80, 90 and 100 per cent and incubated at 25 + 1 0C for 7 days. It was observed (Table 4.11, Fig.5 and Plate 3) that all the six humidity levels induced the growth of Alternaria alternata. Maximum mycelial growth (87.50 mm) was recorded at 100 per cent relative humidity which was at par with 90 per cent (85.00 mm) relative humidity. A significantly decrease in mycelial growth was observed at 80, 70 and 60 per cent humidity. Minimum mycelial growth (40.00 mm) was observed at 50 per cent relative humidity.
4.6 Studies on disease management
4.6.1 Effect of fungicides on mycelial growth of Alternaria alternata in vitro
In view of the literature available on management aspects of Alternaria alternata, five fungicides were selected for this experiment taking their different concentrations. Results recorded as per cent inhibition of radial mycelial growth are presented in Table 4.13. The data suggested that increase in concentrations of the fungicides caused increased inhibition of mycelial growth of the fungus. Among five fungicides tested, carbendazim+mancozeb (Companion) was found most effective which was showing 66.66, 79.99, 87.77 and 94.44 per cent inhibition of mycelial growth of Alternaria alternata at 100, 300, 500 and 700 ppm concentrations, respectively followed by mancozeb (48.88, 71.11, 83.33 and 87.77, respectively). Thiophanate methyl was found to be least effective in inhibiting mycelial growth. This experiment shows that carbendazim+mancozeb is highly effective in inhibiting the mycelial growth of Alternaria alternata in vitro.
4.6.2 Efficacy of fungicides in reducing disease intensity (in vivo) Data presented in Table 4.12 shows that all the fungicides tested differ significantly superior over control. Carbendazim +mancozeb was found most effective in reducing per cent disease intensity (31.09%) over control. It was found statistically at par with mancozeb. The least effective was thiophanate methyl with 11.95 per cent disease reduction over control. 4.6.3 Efficacy of plant extracts on mycelial growth of Alternaria alternata (in vitro)
To evaluate the efficacy of five plant extracts i.e. garlic cloves (Allium sativum), neem leaves (Azadirachta indica), turmeric rhizomes (Curcuma longa), lantana leaves (Lantana camara) and ashwagandha leaves (Withania somnifera) were tested at three different concentrations viz., 5, 10 and 15 per cent against Alternaria alternata on PDA by poisoned food technique. Data presented as per cent inhibition of radial mycelial growth are presented in Table 4.14. The data clearly shows that increase in concentrations of the plant extracts caused a decrease in mycelial growth of the fungus thereby, resulting in increased inhibition. Among five plant extracts, garlic was found most effective showing 78.88, 82.22 and 89.99 per cent inhibition of mycelial growth of Alternaria alternata at 5, 10 and 15 per cent concentrations, respectively followed by neem which caused 72.22, 80.00 and 91.11 per cent inhibition of linear growth of Alternaria alternata, respectively.
Leaf extract of Lantana resulted in 33.33, 38.88 and 42.22 per cent inhibition at 5, 10 and 15 per cent concentrations respectively and it was found to be least effective against Alternaria alternata.
In general, higher concentrations of plant extract were more effective in reducing the mycelial growth as compared to lower concentrations. Plant extract (P) x concentration (C) interaction was also found significant. This suggests that plant extracts offer a better alternative to fungicides as they are safer and effective too.
5. DISCUSSION
Every seed is a potential harbor of a wide variety of microfungi containing both pathogenic and saprophytic microorpanisms, both externally and internally (Utobo et al., 2011). These microfungi may reduce seed quality and impair seed germination resulting in the production of abnormal seedlings (Paul, 1989, Vijayan and Rehill, 1990, Bateman and Kwasna, 1999 and Khanzada et al., 2002). Seeds play a vital role in the production of healthy crops. They are carrier of many important seed borne pathogens inciting various diseases, which results in considerable yield loss. Seeds carry a number of fungi. Although, majority of them are saprophytes, a few are potential pathogens capable of ruining the crop. Weather during the harvesting period plays a decisive role in the infection of seeds by certain fungi. Storage of seeds result in dominance of storage fungi (Christensen, 1973).
The importance of seed as basic input in crop production programme has been fully recognized, which has to be high quality and pathogen free. Seed of most cultivated crops are known to transmit the dangerous plant pathogens from diseased area to disease free area ultimately resulting in production of disease(s) and causing lesser yield and poor quality and claimed to be an impediment in marketing. The product that we wish to keep out of our market, consequently losses its export value especially price because of the potential negative impact on plant health (or) produce.
Fungi, bacteria, viruses, and nematodes are seed borne, where they remain safe and transmitted to long distances. They are associated as intraembryonal, extarambryonal, concomitant, inert matter or associated with inert matter and lead to systemic, local or both the types of infections. The seed borne inoculums play a vital role in the disease cycle leading to epidemics based on the inoculum potential available and other factors. Micro- organisms present in the seed result in prolong seed dormancy, reduction in germination and adversely affect survival of seedling and seedling vigour. It is essential to know the exact role of micro organisms associated with seed. Hundreds of micro organisms have been reported associated with seeds without establishing their exact association and their role. Such reports are of no use but create confusion in seed trade. Hence, it is essential to prepare lists of seed borne pathogens and the diseases caused by them in specific agro climatic region, covering individual crop.
Blond psyllium or isabgol (Plantago ovata) is an annual herb with potential medicinal properties. This crop suffers from vagaries of fungal and other diseases. Among fungal diseases, blight caused by Alternaria alternata (Fr.) Keissler, earlier considered to be a minor disease, is now becoming increasingly destructive and widely damaging in recent years. In view of economic importance of the disease and limited information available on incidence and disease management, studies were carried out on seed mycoflora, effect on seed germination and seedling vigour, physio-pathology and effective management of the disease through botanicals and chemicals. The results on these aspects are discussed below.
During present investigation five seed samples of isabgol were collected from Barmer, Jalore, Jodhpur, Nagaur and Pali districts of Rajasthan during 2013-14. Examination of dry seed samples revealed the presence of deformed (shriveled), discoloured (black and brown coloured) and damaged (mechanically and insect ) seeds, in addition to impurities like plant debries (pieces of leaves and stem), seeds of other crops, inert material (stones and sand) and apparently healthy seeds. It is likely that different types of seed mycoflora during development of seeds and there storage might have caused such deformation and discoloration of seeds. Occurrence of such deformation and discoloration along with impurities have also been reported during examination of dry seeds of pearlmillet, barley and isabgol by Singh and Singh, 1983; Randhava and Aulakh, 1984 and Meena and Maharshi, 2013, respectively. For the isolation of mycoflora from isabgol seeds, two standard methods viz. Blotter and Agar Plate Methods were used to know the incidence of mycoflora. The Blotter Method displayed 5 fungi viz. Alternaria alternata, Aspergillus niger, Curvularia clavata, Curvularia fallax and Fusarium semitectum alongwith un-identified mycoflora. It also reveled that the seed sample from Jalore was infected and infested with maximum number of fungi and the sample from Jodhpur showed minimum incidence.
The incidence of Alternaria alternata has been noted following standard Blotter and Agar Plate Methods. In the present study, maximum counts of A. alternata and other fungi were observed by blotter (un-sterilized and sterilized seeds) method as compared to agar plate (sterilized seeds) method. Other fungi detected were Aspergillus niger, Curvularia clavata, C. fallax, Fusarium semitectum and un-identified fungi. This study is in conformity to earlier findings of Russell (1975), Patel et al. (1984), Mehta et al. (1985), Maharshi (1992), Elwakil and Ghoneem (1999) and Meena and Maharshi (2013) who isolated Alternaria sp., Aspergillus sp., Curvularia sp., Fusarium sp. and other fungi from seeds of isabgol.
In general, little variation was observed in blotter and agar plate methods for the presence of Alternaria alternata and other fungi recorded on seeds. Detection of A. alternata was higher in blotter method in comparison to agar plate method. This variation might be due to the reasons that some of the weak and slow growing fungi could not grow in agar culture in comparison to fast growing saprophytic fungi. Pre-surface sterilization of seeds and use of substratum in the method employed may be another reason (de Temple, 1961 and Neergaard and Saad, 1962). To have a complete spectrum of the mycoflora, it seems essential to deploy both the methods. Neergaard and Saad (1962) also observed that blotter and agar plate methods are equally valuable and supplementary to each other.
Seed germination and seedling growth are generally influenced by seed borne fungi (Christensen and Kaufmann, 1965; Rati and Ramalingam, 1974 and Prasad, 1980 a & b). In the present investigation, the effect of A. alternata as estimated by seed and foliar inoculation methods showed significant reduction in the seed germination and seedling vigour and proved to be pathogenic. It is likely that fungi which reduced seed germination are pathogenic to host seedlings at the pre-emergence stage (Jayaweera et al., 1988). Our findings of seed and foliar inoculation techniques revealed that Alternaria alternata reduced seed germination, root and shoot elongation by causing higher percentage of pre- and post- emergence mortality and subsequently it also reduced vigor index, whereas in control of both the test showed good germination, root and shoot elongation and higher vigour index.
Symptoms like chlorotic/ necrotic leaf spot and leaf blight were observed on young seedlings by inoculating them with conidial suspension of Alternaria alternata through Spray Inoculation Technique. Association of seed borne Alternaria alternata with infected plant parts has also been reported by Neergaard, (1977); Vala and Kapoor, (1985); Strandberg and White, (1989), Thind, (2005) and Meena and Maharshi (2013).
Every living being requires food for its growth and reproduction and fungi are not an exception to it (Kiryu, 1939). Fungi secure food and energy from the substrate upon which they live in the nature. In order to culture the fungi in the laboratory, it is necessary to furnish those essential elements and compounds in the medium which are required for their growth and other life process. Neither all media are equally good for all fungi nor there can a universal substrates or artificial medium on which all fungi grow well. So different media including synthetic and non-synthetic were tried for the growth of A. alternata. Physiological studies for this pathogen were carried out extensively covering media, temperature, pH and relative humidity. Out of the five media tested, PDA proved to be the best in terms of growth. Similar results were obtained by Kapoor and Hingorani (1958), Ionnidis and Main (1973), Pandey and Vishwakarma (1998), Maheshwari et al. (2001), Akhtar et al. (2004), Gorwar et al. (2006) and Israram et al. (2007). Waghunde and Patil (2010), Bochalya (2010) and Joshi et al. (2012) reported that Alternaria alternata fungus grew well on potato dextrose agar medium. Temperature is most important physical environmental factor for regulating the growth and reproduction of fungi. Ultra low or high temperature adversely affects the germination and growth. The results obtained in the present investigation showed that optimum temperature for growth is 25 ºC. Similar results were recorded by Gupta et al. (1985), Prashanthi and Kulkarni (2003), Ma-Guillong et al. (2006), Bochalya (2010) and Joshi et al. (2012). Garibaldi et al. (2007) and Balai and Ahir (2013) recorded optimum growth of A. alternata at 27 0C.
Fungi generally utilize substrates in the form of solution only if the reaction of solution conducive to fungal growth and metabolism (Kiryu, 1939). This brings importance of hydrogen ion concentration for better fungal growth. In this set of experiment taking different pH, the most suitable with maximum dry mycelial weight was 6.5. This shows that fungus prefers near neutral medium. The results obtained in the present study are in accordance with the results of Hasija (1970), Saad and Hegedorn (1970), Maheshwari et al. (2000), Hubballi et al. (2010), Waghunde and Patil (2010) and Meena et al. (2013).
In vitro studies on different levels of relative humidity revealed that 100 per cent relative humidity supported maximum mycelial growth of Alternaria alternata while minimum mycelial growth at 50 per cent relative humidity. The results are in close conformity with the observations of Prasad and Roy (1979) who observed maximum growth of Alternaria alternata at 90-100 per cent relative humidity. Ma-Guilong et al. (2006) and Balai and Ahir (2013) also reported that 90 to 100 per cent relative humidity was most suitable for mycelial growth of Alternaria alternata.
In vitro efficacy of five fungicides namely mancozeb, carbendazim, mancozeb + carbendazim, thiophanate methyl and difenoconazole were evaluated at 100, 300, 500 and 700 ppm concentration against Alternaria alternata. Among five tested fungicides, mancozeb + carbendazim was found most effective in inhibiting (94.44 %) mycelial growth of Alternaria alternata at 700 ppm concentration followed by mancozeb and carbendazim. Thiophanate methyl was found to be least effective to control Alternaria alternata. It has been observed that mancozeb+carbendazim is one of the most suitable and safer fungicide for foliar diseases like Alternaria blight. Similar observations were recorded by Kamble et al. (2000), Rao and Rao (2002), Singh et al. (2003), Kumari et al. (2006), Bochalya, (2010), Meena et al. (2013) and Meena and Ratnoo (2014).
On the basis of performance of fungicides in in vitro studies, all the five fungicides viz., mancozeb, carbendazim, mancozeb + carbendazim, thiophanate methyl and difenoconazole were evaluated by foliar spray in field against blight disease of isabgol. The results revealed that there was clear reduction in the severity of disease, when fungicides were applied as foliar spray. The severity reduction by fungicides at different concentrations had similar trend as in in vitro experiments. Present results are well supported by several workers like Singh et al. (2003), Shukla et al. (2005), Chatta (2005), Pal et al. (2008) and Kumar et al. (2013).
Fungicides can effectively control the disease but the residual problems are increasing and this is causing health hazards in human beings and animals. To find out possibilities of replacing fungicides with other eco - friendly products for management of disease, five plant leaf extracts i.e. garlic clove (Allium sativum), neem leaves (Azadirachta indica), turmeric rhizome (Curcuma longa), ashwagandha leaves (Withania somnifera) and lantana leaves (Lantana camara) were tested at three different concentrations viz. 5, 10 and 15 per cent against Alternaria alternata on PDA by poisoned food technique. Highly effective plant extract was garlic (Allium sativum) followed by neem leaves (Azadirachta indica). In general, higher concentrations of plant extracts were more effective in reducing the mycelial growth as compared to lower concentrations. The results obtained in the present study are in accordance with the results obtained by Mistry, (1992), Barros et al. (1995), Singh and Majumdar (2001), Jadeja and Pipliya (2008) and Panchal and Patil (2009). Looking to the increasing residues in crop, the extracts are the future to manage the disease and evaluation of some of the plant extracts in present studies have been carried out for the first time on this pathogen.
6. SUMMARY
The results of experiments conducted in regard to thesis entitled “Incidence and management of Alternaria sp. associated with blond psyllium (Plantago ovata) seeds“ are summarized below.
Out of five seed samples, collected from Barmer, Jalore, Jodhpur, Nagaur, Pali and Jalore seed sample depicted maximum discoloured and shrivelled seeds along with other impurities than other samples.
Five fungi viz. Alternaria alternata, Aspergillus niger, Curvularia clavata, Curvularia fallax, Fusarium semitectum alongwith un-identified fungi were isolated from all 5 samples by using standard blotter and agar plate methods. Blotter method showed more number of mycoflora than agar plate method. Among all seed samples tested, Jalore sample exhibited maximum incidence of mycoflora.
Out of 5 fungi isolated, Alternaria alternata, showed higher incidence among all the samples with less germination of seeds. Its identify was further confirmed from ITCC, IARI, New Delhi (no. 9336.14).
The effect of Alternaria alternata as estimated by seed and foliar spray inoculation methods showed significant reduction in the seed germination and seedling vigour. The disease symptoms on affected plants of isabgol initiated with yellowing and browning of the lower leaves and progressing upwards. Yellowing from the leaf tip along the margins of the leaf petiole were also observed. Under severe infections, lesions enlarged and coalesced causing blighting of entire leaf. Concentric circles with dark layers of conidia were observed on blighted leaf portions.
Out of five different solid media tested, potato dextrose agar (PDA) proved to be the best for mycelial growth of the fungus, followed by Richard’s medium. Minimum mycelial growth was observed on Martin’s medium. Out of six levels of temperature, maximum mycelial growth of A. alternata was recorded at 25 0C. Good mycelial growth of Alternaria alternata was observed at 100 per cent RH followed by 90 per cent.
The pathogen was able to grow in a wide range of pH from 4.0 to 8.0. However, the optimum pH for mycelial growth was 6.5.
Disease management is one of the remarkable event in reducing the amount of inoculum and further spread of the diseases. There cannot be any such occasion in nature that a disease can be permanently controlled. The scientists have endeavoured to develop many strategies for controlling plant pathogens, which includes use of chemicals, cultural practices, phyto- extracts agents. Of all the five fungicides evaluated in vitro, mancozeb + carbendazim (Companion) was found most effective in inhibiting mycelial growth of A. alternata followed by mancozeb. Among fungicides used as foliar sprays, carbendazim +mancozeb (Companion) proved to be most effective in reducing per cent disease intensity over control against Alternaria alternata followed by mancozeb and carbendazim.
In general, higher concentration of plant extracts were more effective in reducing mycelial growth as compared to lower concentrations. Plant extracts of garlic (Allium sativum), neem (Azadirachta indica), turmeric (Curcuma longa), lantana (Lantana camara) and Ashwaganda (Withania somnifera) were tested in vitro against mycelial growth of Alternaria alternata. Among these, garlic (Allium sativum) was found to be most effective in inhibiting mycelial growth of Alternaria alternata at 5, 10 and 15 per cent concentrations of the extract followed by Azadirachta indica. Lantana camara was found to be the least effective. BIBLIOGRAPHY
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Abstr. 70 (Suppl).:V-10. Singh, J. and Majumdar V.L. 2001. Efficacy of plant extract against Alternaria alternata the incitant of fruit rot of pomegranate (Punica granatum L.) J. Mycol. Pl. Pathol., 31:347-349. Singh, K., Rai, M. and Singh, K. 2003. Evaluation of chemicals against Alternaria leaf spot of brinjal. Ann. Pl. Protec. Sci., 11:394-395. Singh, P. and Khan, S.N. 1999. Assessment of seed borne fungi of some forest trees and their management. Indian J. of Fores., 22 : 281-284. Singh, R. S. 2005. Diseases of Vegetable Crops, 3rd Edition, Oxford and IBH Publishing Co. Pvt. Ltd., New Delhi. Singh, R.D., Choudhary, S.L. and K.G.Patel 1972. Seed transmission and control of Fusarium wilt of cumin Phytopathologic Mediterranea. 11 :19- 24. Singh, T. and Singh, D. 1983. Seed borne mycoflora of sesame with special reference to Rajasthan. Indian J. Mycol. Pl. Pathol. 13 : 32-42. Srivastava, R.N. 1984. 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Incidence and management of Alternaria sp. associated with blond psyllium (Plantago ovate L.) seeds
Sajjan Choudhary* Dr. R. P. Ghasolia ** (Research Scholar) (Major Advisor) ABSTRACT
Isabgol is an important medicinal crop of Rajasthan. The crop suffers severely from various seed borne diseases including Alternaria leaf blight which affects seed germination, seedling vigor, plant growth and isabgol yield.
Out of five seed samples, collected from Barmer, Jalore, Jodhpur, Nagaur and Pali, seed sample of Jalore showed maximum shriveled, discoloured and damaged seeds along with other impurities. Alternaria alternata, Aspergillus niger, Curvularia clavata, C. fallax and Fusarium senitectum were isolated from isabgol seeds.
Highest incidence of mycoflora was observed in sample ‘IG-5’ collected from Jalore, while it was lowest in sample ‘IG-2’ collected from Jodhpur. Incidence of blight causing fungus Alternaria alternata was also highest in sample ‘IG-5’, while it was lowest again in sample ‘IG-2’.
Alternaria alternata caused blight symptoms and observed highly pathogenic causing pre- and post- emergence mortality and reducing vigor index.
Maximum mycelial growth of Alternaria alternata was recorded at 25 0C temperature, 6.5 pH, 100 per cent relative humidity and on PDA medium.
All the fungicides, tested in vitro, inhibited the growth of fungus. Companion gave maximum inhibition of mycelial growth at 700 ppm concentration followed by mancozeb. Foliar application of companion was also observed to be most effective in reducing the per cent disease intensity.
Under in vitro conditions, garlic extract was found most effective in inhibiting mycelial growth followed by neem.
* Post graduate student, M.Sc. (Ag.), Department of Plant Pathology, S.K.N. College of Agriculture, Jobner. ** Thesis submitted in partial fulfillment of the requirement for M.Sc. (Ag.), degree in Plant Pathology under supervision of Dr. R.P. Ghasolia, Assistant Professor, Department of Plant Pathology, (S.K.N. Agriculture University, Jobner), S.K.N. College of Agriculture, Jobner, Jaipur.
Table 4.1: Major districts of Rajasthan where from seed samples
collected and their code number
S.No. District Code number
1 Barmer IG -1
2 Jodhpur IG-2
3 Pali IG-3
4 Nagaur IG-4
5 Jalore IG-5
Table 4.2: Seed abnormalities and impurities in different isabgol
seed samples
S. Categories of seed/ Per cent content (by weight basis / sample)* No. impurities IG-1 IG-2 IG-3 IG-4 IG-5 1. Deformed (i) Shrivelled 4.0 2.7 3.6 4.5 9.4 2. Discoloured (i) Black 6.5 3.1 7.6 8.5 10.9 (ii) Brown 2.8 2.2 2.7 3.1 3.6 3. Damaged seeds (i) Mechanical damage/ 1.9 1.9 3.4 3.9 4.8 insect damage 4. Impurities (i) Other crop seeds 1.4 0.2 1.6 1.0 1.2 (ii) Sand, stone and 1.5 0.5 1.1 1.5 2.3 other particles (iii) Plant debries 1.2 0.6 1.8 1.8 1.3 5. Apparently healthy 80.7 88.8 78.2 75.7 66.5 seeds *Average of 4 replications (100g seeds/ replication)
IG-1= Barmer, IG-2 = Jodhpur, IG-3 = Pali, IG-4 = Nagaur IG-5 = Jalore
Table: 4.3 Incidence of mycoflora associated with isabgol seeds analysed by Standard Blotter Method
Mycoflora Per cent incidence / sample* Barmer Nagaur Pali Jalore Jodhpur
US S US S US S US S US S Alternaria alternata 5.00 1.25 2.75 1.25 5.75 2.00 11.25 2.00 4.00 1.00
Aspergillus niger 4.00 1.75 2.25 1.50 5.50 2.25 8.25 2.25 3.25 1.50
Curvularia clavata 1.25 0.75 0.50 0.25 3.75 1.00 2.75 1.25 1.25 0.50
Curvularia fallax 2.25 1.50 2.00 1.00 0.75 0.00 2.50 1.50 1.50 1.00
Fusarium 4.25 1.50 3.25 2.00 6.00 2.75 7.25 2.00 2.50 1.50 semitectum
Unidentified 1.75 0.50 2.00 0.00 3.75 1.25 6.25 2.25 3.00 0.50 mycoflora
Total mycoflora 18.50 7.25 12.75 6.00 25.70 9.25 38.25 11.25 15.50 6.00
Alternaria alternata 27.02 17.24 21.56 20.83 22.54 21.62 29.41 17.77 25.80 16.66
* Seed tested 400/ sample
US- Un-sterilized, S- Sterilized
Table 4.4 Incidence of mycoflora associated with isabgol seeds analysed by standard Agar Plate Method
Mycoflora Per cent incidence / sample* Barmer Jodhpur Pali Nagaur Jalore Alternaria alternata 3.00 1.50 3.00 2.50 5.00
Aspergillus niger 2.50 2.00 3.50 3.50 4.50
1.50 1.00 1.00 0.50 2.50 Curvularia clavata
Curvularia fallax 2.50 1.50 0.00 1.00 2.00
Fusarium semitectum 2.00 2.50 2.50 2.50 3.50
Unidentified mycoflora 0.50 0.50 3.00 2.00 3.50
Total mycoflora 12.00 9.00 13.00 12.00 19.00
Alternaria alternata 25.00 16.66 21.15 20.83 27.77
* Seed tested 200/ sample
Table 4.8 Effect of different levels of temperature on mycelial growth of Alternaria alternata at 7th day of incubation in vitro
S.No. Temperature (0C) Mycelial growth (mm)* 1 15 61.00
2 20 75.00
3 25 88.00
4 30 71.00
5 35 65.00
SEm+ 1.082 CD (p=0.05) 3.052
* Average of three replications
Table 4.11 Effect of relative humidity on the mycelial growth of Alternaria alternata at 10 days of incubation at 25 + 10C in vitro
S.No. Relative humidity (%) Mycelial growth (mm)* 1 50 40.00
2 60 47.25
3 70 65.50
4 80 79.00
5 90 85.00
6 100 87.50
SEm+ 1.21
CD (p=0.05) 3.97
* Average of three replications
Table 4.9 Effect of pH on the mycelial growth of Alternaria alternata on PD broth at 10 days of incubation at 25 + 10C in vitro
S.No. pH Dry mycelial weight (mg)* 1 4.5 690
2 5.0 730
3 5.5 600
4 6.0 820
5 6.5 845
6 7.0 750
7 7.5 550
8 8.0 290
SEm+ 12.046
CD (p=0.05) 36.108
* Average of three replications
Table 4.10 Effect of solid media on mycelial growth of Alternaria alternata at 25+1°C
S.NO. Medium Mycelial growth (mm)* After 7 days 1. Czapeck’s medium 55.55
2. Martin’s medium 37.77
3. Oat meal medium 63.33
4. Potato dextrose agar medium 85.55
5. Richard’s medium 76.70 SEm+ 2.80
CD (p=0.05) 8.34
* Average of three replication
Table 4.13: Effect of fungicidal foliar spray on disease intensity
S. No. Fungicides Dose Per cent Per cent disease disease intensity reduction (%) intensity over control
1 Thiophanate 0.2 55.25 11.95
methyl (47.29)
2 Carbendazim 0.1 52.75 19.93
(46.58)
3 Mancozeb 0.25 49.75 20.71
(44.86)
4 Carbendazim+ 0.2 43.25 31.07
mancozeb (41.12)
5 Difenoconazole 0.2 54.25 13.94
(47.44)
6 Control - 62.75 -
(52.39)
SEm+ 1.44
CD (p=0.05) 4.21
Figures given in parentheses are angular transformed values
Table:4.12 Comparative efficiacy of different fungicides on mycelial growth of Alternaria alternata in vitro after 7 days of incubation at 25 + 1 0C
Fungicides Per cent growth inhibition at various Mean concentration (ppm)* 100 300 500 700 Mancozeb 48.88 71.11 83.33 87.77 73.33
(44.36) (57.49) (65.90) (69.53) (59.32)
Carbendazim 42.22 48.88 54.44 60.00 51.39
(40.52) (44.36) (47.55) (50.77) (45.80)
Thiophanate methyl 19.00 42.22 51.11 65.00 44.71
(25.84) (40.52) (45.64) (53.73) (41.43)
Carbendazim+mancozeb 66.66 79.99 87.77 94.44 82.21
(54.73) (63.43) (69.53) (76.36) (66.01)
Difenoconazole 40.00 44.44 51.11 70.00 49.72
(39.23) (41.81) (45.64) (56.79) (45.86)
Check 0.00 0.00 0.00 0.00 0.00
SEm+ CD (p=0.05)
F 0.60 1.72
C 0.49 1.40
FxC 1.21 3.44
* Average of 4 replications
Figures given in parentheses are angular transformed values
Table:4.14 In vitro fungitoxicity of plant extracts against Alternaria alternata by poisoned food technique after 7 days of incubation at 25 + 1 0C
Plant extract Part Per cent growth inhibition at Mean used different concentration (%) 5 10 15 Garlic Clove 78.88 82.22 89.99 83.69
(62.64) (65.06) (71.56)
Neem Leaves 72.22 80.00 91.11 81.11
(58.19) (63.43) (72.65)
Turmeric Rhizome 60.00 77.77 74.44 70.74
(50.77) (61.87) (59.63)
Ashwagandha Leaves 42.22 70.00 78.88 63.70
(40.52) (56.79) (62.64)
Lantana Leaves 33.33 38.88 42.22 38.14
(35.26) (38.57) (40.52)
Check - 00.00 00.00 00.00 -
SEm+ CD (p=0.05)
F 0.59 1.67
Con. 0.48 1.36
F x Con. 1.17 3.34
Table 4.5: Pathogenicity of seed borne Alternaria alternata of isabgol by Seed Inoculation Technique (in
Petri plates)
Fungus Per cent Per cent Per cent seedling Elongation Vigour Type of symptoms Germination seedlings mortality (cm) index showing Pre - Post- Root Shoot symptoms* emergence emergence
Alternaria 65.00 74.60 11.42 3.07 1.75 2.75 292.50 Yellowing, small isolated brown / black alternata necrotic spots on leaves
Control 79.00 3.89 4.76 2.53 5.70 7.00 1003.30 Yellowing and tip (uninoculated burning of leaves seed) No. of seed tested =100 * Based on emerged seedlings
Table 4.6: Pathogenicity of seed borne Alternaria alternata of isabgol by Seed Inoculation Technique (in
pots)
Fungus Per cent Per cent Per cent seedling Elongation Vigour Type of Germination seedlings mortality (cm) index symptoms showing Pre - Post- Root Shoot symptoms* emergence emergence
Alternaria alternata 68.00 83.07 9.37 4.41 4.10 5.30 639.20 Seedling blight, leaf blight and yellowing of leaves. Control 81.00 1.25 5.26 1.23 5.10 5.40 850.50 Yellowing, (uninoculated) burning of leaves. No. of seed tested =100 * Based on emerged seedlings
Table: 4.7: Pathogenicity of Alternaria alternata by foliar inoculation technique (in pots)
Fungus No. of seedlings sprayed Per cent seedlings Type of symptoms showing symptom Alternaria alternata 100 85.00 Yellowing, tip burning, marginal
blight and brown to black small
spots on the leaves.
Control 100 0.00 -
(uninoculated)
Average of 5 replications (5 seedlings/pot x 4/replication