EPIDEMIOLOGY AND MANAGEMENT OF LEAF BLIGHT OF MUNGBEAN [Vigna radiata (L.) Wilczek.] CAUSED BY Macrophomina phaseolina (Tassi) Goid.
THESIS SUBMITTED TO THE RAJASTHAN AGRICULTURAL UNIVERSITY, BIKANER IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY IN AGRICULTURE (PLANT PATHOLOGY) BY SURAJ MAL MEHTA
2004
RAJASTHAN AGRICULTURAL UNIVERSITY, BIKANER
S.K.N. COLLEGE OF AGRICULTURE, JOBNER
CERTIFICATE-I
Dated :------2004
This is to certify that Mr. SURAJ MAL MEHTA successfully completed the comprehensive examination held on 12th. May. 2001 as required under the regulation for Doctor of Philosophy degree.
(O.P. VERMA) Head & Professor 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 :------2004
This is to certify that this thesis entitled “Epidemiology and management of leaf blight of mungbean [Vigna radiata (L.) Wilczek.] caused by Macrophomina phaseolina (Tassi) Goid” submitted for the degree of Doctor of Philosophy in the subject of Plant Pathology embodies bonafied research work carried out by Mr. Suraj Mal Mehta 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 ------2004.
(O.P. VERMA) (J.P. GOYAL) Head & Professor Major Advisor Department of Plant Pathology S.K.N. College of Agriculture, Jobner
DEAN S.K.N. College of Agriculture, Jobner
RAJASTHAN AGRICULTURAL UNIVERSITY, BIKANER S.K.N. COLLEGE OF AGRICULTURE, JOBNER
CERTIFICATE-III
Dated :------2004
This is to certify that this thesis entitled “Epidemiology and management of leaf blight of mungbean [Vigna radiata (L.) Wilczek.] caused by Macrophomina phaseolina (Tassi) Goid” submitted by Mr. Suraj Mal
Mehta to the Rajasthan Agricultural University, Bikaner in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Plant Pathology after recommendation by the external examiner was defended by the candidate before the following members of the advisory 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.
(J.P. GOYAL) (O.P. VERMA) Major Advisor Head & Professor Department of Soil Science & Agricultural Chemistry (P.M. KANWAT) S.K.N. College of Agriculture, Jobner Advisor
(N.L. JAT) Adviso
External Examiner (R.K. BANSAL) Advisor
APPROVED DEAN, Post Graduate Studies (G.R. CHOUDHAR) Rajasthan Agricultural University, DEAN, PGS Nominee Bikaner
RAJASTHAN AGRICULTURAL UNIVERSITY, BIKANER S.K.N. COLLEGE OF AGRICULTURE, JOBNER
CERTIFICATE-IV
Dated :------2004
This is to certify that Mr. Suraj Mal Mehta of the Department of Plant Pathology, S.K.N. College of Agriculture, Jobner has made all corrections/modifications in the thesis entitled “Epidemiology and management of leaf blight of mungbean [Vigna radiata (L.) Wilczek.] caused by Macrophomina phaseolina (Tassi) Goid” which were suggested by the external examiner and the advisory committee in the oral examination held on ------2004. The final copies of the thesis duly bound and corrected were submitted on------2004, are enclosed herewith for approval. (J.P. GOYAL) Major Advisor
(O.P. VERMA)
Head & Professor
Department of Plant Pathology S.K.N. College of Agriculture, Jobner
DEAN S.K.N. College of Agriculture, Jobner
APPROVED DEAN, Post Graduate Studies Rajasthan Agricultural University
1. INTRODUCTION
Pulses are the main source of protein particularly for vegetarians and contribute about 14 percent of the total protein of an Indian average diet. Production of pulses in the country is far below the requirement to meet even the minimum level of per capita consumption. The per capita availability of pulses is dwindling fast from 74.9 gms in 1959 to 33 gms in 1998 as against the minimum requirement of 70 gms per day/capita prescribed by ICMR, which is causing malnutrition among the growing people. (Anonymous, 1998-99). Therefore, it is necessary that agricultural scientists should evolve the strategy of increasing the production of pulses to meet the protein requirement of increasing population of the country. Mungbean [Vigna radiata (L.) Wilczek] is primarily a rainy season crop but with the development of early maturing varieties, it has also proved to be an ideal crop for spring and summer season. Mungbean is an excellent source of protein (24.5%) with high quality of lysine (460 mg/g N) and tryptophane (60 mg/g N). It has also remarkable quantity of ascorbic acid when sprouted and also contains riboflavin (0.21 mg/ 100 gm) and minerals (3.84 g/100g) (Gopalan et. al., 1995). Mungbean, being a short duration crop, fits well in various multiple and inter-cropping systems. After picking of pods, mungbean plants may be used as green fodder or green manure. Beside these, the crop also enriches soil by fixing atmopheric nitrogen. In India, mungbean is mainly grown in the states of Maharashtra, Madhya Pradesh, Orissa, Andhra Pradesh, Tamil Nadu, Rajasthan and Uttar Pradesh. Total area sown under mungbean crop in the Rajasthan state is 7.02 lakh ha. with the grain production of 2.07 lakh tonnes and an average yield of 2.95 qt./ha (Anonymous, 2001-02). The major mungbean growing districts in Rajasthan are Nagaur, Jodhpur, Jalore, Churu, Jhunjhunu, Ajmer, Barmer, Tonk, Jaipur and Sikar. This crop is grown mainly as rainfed but some times cultivated under irrigated conditions specially in Srignaganagar district and to some extent in other districts also. Main limiting factor in profitable cultivation of this crop in Rajasthan is the attack of several diseases caused by fungi, bacteria and viruses which take heavy toll of the crop at all the stages of growth right from sowing to harvest and also during storage. The major fungal diseases which infect the crop are leaf blight [Macrophomina phaseolina (Tassi) Goid], powdery mildew [Erysiphe polygoni DC), web blight (Thanatephorus cucumeris (Fr.) Donk (=Rhizoctonia solani Kuhn) and Cercospora leaf spots (Cercospora canescens Ellis and Martin, C. cruenta Sacc., C. dolichi Ellis and Everlast, C. kikuchi Matsumoto & Tomoyasu and Anthracnose (Colletotrichum dematium and C. lindemuthianum (Philip et al., 1969, Dwivedi and Saksena, 1974., and Grewal,1988) Among these diseases, leaf blight (Macrophomina phaseolina) has been identified as an economically important disease in this region which causes considerable losses (10.8 per cent) in grain yield to the crop (Kaushik et al., 1987). Very little attention has been paid on the epidemiology and management of leaf blight caused by Macrophomina phaseolina which has become a serious problem in hampering the production in all the mungbean growing areas particularly in the sandy & sandy loam soils. Therefore, the work has been under taken on the following aspects. 1. Survey, collection, isolation, pathogenecity and identification of pathogen. 2. To standardize techniques of disease production and multiplication of inoculum. 3. To study the survival and transmission of the pathogen. 4. To study the role of environmental factors on disease development. 5. To study the biochemical changes in infected plant or plant parts. 6. To evolve suitable strategy for disease management using (a) Chemicals (b) Bio agents (c) Botanicals and (d) Host resistance
2. REVIEW OF LITERATURE
History and occurrence
Rhizoctonia bataticola (Taub.) Butler as a plant pathogen was recognized by Halsted (1890). In India, Shaw (1912) described a sterile fungus with black sclerotia causing seedling disease in jute, cowpea, groundnut and cotton. He identified the fungus as Rhizoctonia solani. Taubhanus (1913) gave the name of genus Sclerotium because of absence of spores and the species name as bataticola because it was pathogenic to Ipomea bataticola (L.) Lam. Briton- Jones (1925) transferred the fungus to the genus Rhizoctonia based on the identification of cultures by Butler (1918). Ashby (1927) accepted Macrophomina and rejected the binomial Macrophomina phaseoli and proposed a new binomial M. phaseolina as the pycnidial stage of Rhizoctonia bataticola. He was not aware that Tassi had earlier described Macrophomina phaseolina. Haigh (1930) suggested that R. bataticola be used for sclerotial isolate and pycnidial strains should be called M. phaseolina. Goidanich (1947) examined the original material of Tassi and compared with Macrophomina phaseoli, M. corchori, M. cajani, M. sesami, M. philippinesis, Dothorela cajani and D. phaseoli and found all of them indentical. He correlated the mistake made by Ashby and according to the International code of Botanical Nomenclature the bionomial Macrophomina phaseolina is the valid name for pycnidial stage of R. bataticola. Macrophomina phaseolina (Tassi) Goid the incitent of root rot of mungbean, also causes leaf blight (Philip et al., 1969) is one of the most important disease. The pathogen causes root rot in a number of crops including groundnut, sesamum, soybean and kidney bean (Dhingra and Sinclair, 1977). The pathogen was also reported to cause leaf blight, seedling blight & charcoal rot of guar, moth bean, urdbean, mungbean, groundnut , soyabean and sunflower (Singh, 1953., Kumar et. al, 1969 a&b, Senecha and Srivastava 1982, Shukla and Bhargava, 1976, Philip et al., 1969, Shanmugam & Govindaswamy 1971, Gangopadhyay et. al. 1973, Udit Narain, 1983 and Grover & Sakhuja, 1981).
Symptomology
Philip et al (1969), Grover & Sakhuja (1981) and Singh & Srivastava (1988) reported leaf blight phase of mungbean with the information that disease usually makes its appearance when the crop is 4-6 weeks old and the older leaves are first affected. Initially small, circular to irregular brown to reddish brown lesions appears on or near the margins which enlarge and coalesce. Under hot and humid conditions the entire plant may be blighted. The dried lesions are yellowish brown and become papery in texture. Severely affected leaves fall-off prematurely. Singh and Srivastava (1988) reported that in affected young seedlings of cowpea, discolouration & rotting occurred from young root tip and proceed back wards. The cotyledonary leaves are completely blighted and necrotic. Stem decay occurs in advance stage.
Economic importance
Leaf blight of mungbean and other crops, caused by M. phaseolina, is a serous disease responsible for heavy loss in yield. Kaushik et al (1987) reported 2.2-15.7% infection by R. bataticola (Macrophomina phaseolina) causing 10.8% reduction in grain yield and 12.3% reduction in protein content of the seed.
Pathogenecity test
Deshkar et al. (1973) gave a new and better technology for screening varieties of Phaseolus aureus for resistance to R. bataticola (Macrophomina phaseolina), which involves bringing one surface of a sterilized paper towel in contact with a previously prepared suspension of R. bataticola. Surface sterilized seeds of P. aureus are then dipped in the inoculum suspension and placed on the paper towel which is then transferred to moist chamber. Higuera (1991) used three methods to established pathogenecity of Macrophomina phaseolina in cowpea i.e. toothpick inoculation method, inculation using dry sclerotia of M. phaseolina, and inculation using rice seeds colonized by M. phaseolina sclerotia. Grover and Sakhuja (1981) used two methods for establishment of disease in mungbean, a foliar spray of 4-8 days old inoculum on the foliage by hand atomizer till run-off and through adding mycelial suspension to the sterilized soil as per method applied & used by Kataria and Grover (1976)
Multiplication of the pathogen
For various studies, mass or bulk production of mycelium and sclerotia is necessary to create artificial epiphytotics as the large number of sclerotia are required to study their survival in nature. Mayee and Garud (1978) used sorghum grain for mass culturing of M. phaseolina. Dandnaik et al. (1986) used sorghum, bajra, mung, wheat, maize and rice grain for mass culturing of sorghum charcoal rot fungus M. phaseolina. A mixture of sand and maize was used for mass inoculum production of R. bataticola (Byadgi and Hegde, 1988, Muthukrishnan et at., 1995b). Grover and Sakhuja (1981) used Czapek’s nutrient solution for mass production of mycelial growth for the establishment of the disease.
Age of culture
Age of culture has been correlated with virulence. Ayanru and Green (1978) reported that sclerotia of R. bataticola mature within 3-6 days after their formation and are most infective. With advance in age, the pathogenecity of sclerotia decreases. Grover and Sakhuja (1981) reported that 4-8 days old culture of M. phaseolina caused a severe blight of Vigna radiata. Hooda and Grover (1982) reported that the seedling mortality with 5 days old inoculum and foliage blight with 3 days old inoculum were maximum in mungbean.
Survival of the pathogen
Survival and perpetuation of the pathogen in various sexual/asexual form helps in spread of the inoculum year after year in same locality or nearby area. Sheikh and Ghaffar (1978) reported that Macrophomina phaseolina persisted in the soil in the form of black sclerotia which are produced in large number in infected host tissues and are subsequently dispersed in soil during tillage operations. They have also reported increase in disease percentage with an increase in the inoculum density in guar, black gram, cotton and okra but disease severity varied with different levels of soil moisture. Singh and Chohan (1973) isolated Macrophomina phaseolina from guar seeds using blotter and agar plate method. Kaushik et al. (1987) reported 2.2-15.7 per cent seed infection by Macrophomina phaseolina in 20 cultivars of Vigna radiata. They also reported that seeds from diseased pods of cultivar varsa showed infections of 61.8 per cent in unsterilized seeds and 47 per cent in sterilized seeds. Bhatia et al. (1998) observed the incidence of R. bataticola which ranged from 0.5 to 19.5 percent in guar seeds collected from different parts of Rajasthan. They also reported that mycelium present in seed coat caused seedling and plant infection and in case of heavy seed infection the pathogen also causes pre and post emergence mortality. Sinha and Khare (1977) reported that mycelim of M phaseolina was present in cotyledons, plumule and radicle in the naturally infected seeds of cowpea. Sharma and Singh (2000) reported that twenty four per cent seed samples of mungbean colleded from 11 districts of Rajasthan showed 0.5-38 percent infection range of R. bataticola. Sandhu and Singh (1988) found that infected seeds of cowpea act as an important source of primary inculum to cause charcoal rot (M. phaseolina) in new areas. They also reported that the pathogen invaded the seed coat, cotyledons, plumule and radicle, thus causing pre-emergence rot. Grover and Sakhuja (1981) reported that the Rhizoctonia bataticola causing leaf blight of mungbean was externally seed borne. Lodha et al. (1991) observed highest population and viability of sclerotia of M. phaseolina at 0-5 cm depth. Muthukrishana et al. (1995) reported that sclerotia of M. phaseolina remained viable for 34 months in plant debries buried up to 10 cm soil depth. They further observed that highest germination of sclerotia was found in stem and root pieces buried at 0-5 cm soil depth. Songa and Hillocks (1998) reported M. phaseolina in bean seed and crop residue. They also reported that sclerotial of M. phaseolina remained viable up to depth of 20 cm and observed that sclerotia germination decreased up to 50 per cent after 6 month of storage of infected seeds.
Factors affecting pathogen and disease development
Effect of temperature
Uppal (1934) found that soil temperature from 30º to 34º C was the most favourable for M. Phaseoli to invade cotton plants while in case of sorghum seedling blight, 35º C was most favourable and no disease development was observed at 30ºC and below or at 40ºC. Cook (1955) reported that severity of M. phaseolina on castor bean was greater during the extended hot and dry weather followed by warm and moist spring. Agarwal et al. (1973) reported that charcoal rot of soyabean occurred only at 25 to 40ºC. Ayanru and Green (1978) studied the germination of M. phaseolina sclerotia obtained from 3 to 20 day old PDA cultures. It was 98 to 25 per cent and 75 to 30 per cent, when incubated at 32ºC and 24ºC, respectively. Grover and Sakhuja (1981) reported that the fungus M. phaseolina obtained from blighted leaves of mungbean, grew well at optimum temperature of 35 0C and disease development was best at 30-35 0C on detached mungbean leaves. Dwivedi and Dubey (1987) reported that survival of M. Phaseolina was greatly reduced at a soil depth of 1 cm when the soil was covered with polythene sheet under full sunlight that increases soil temperature upto 54 0C. Mehrotra (1988) reported that cotton plants became susceptible to R. bataticola at 35ºC or more with soil moisture ranging between 15-20% in alluvial soil and sandy soil. Byadgi and Hegde (1988) demonstrated the saprophytic activity of R. bataticola in soil and found that it was maximum at soil temperature of 30ºC. Ratnoo et al. (1997) reported that the infection and development of ashy grey stem blight in cowpea caused by M. phaseolina was most favoured by high temperature 25-40ºC. They also observed that disease development was low in flooded soil as compared to dried soil with 40-60% moisture. Singh (1998) reported that charcoal rot of sorghum appeared at temperature of 28-35ºC and its blighting symptoms appeared when soil temperature was 30ºC or more.
Effect of relative humidity
Grover and Sakhuja (1981) reported that under hot and humid conditions the entire plant of V.radiata became blighted. More than 50 per cent seedlings developed lesions on the foliage when inoculated plants were kept for 12 hrs or more under 100 per cent relative humidity. Humidity plays in important role in growth and sclerotial production. Kushi (1977) reported 76 per cent RH to be optimum for the growth of M. phaseolina in sesamum. Suhag and Rana (1984) reported that growth and sclerotia production of M. phaseolina was maximum in the range of 40-100 per cent RH and the same RH was favourable for causing the infection of ginger plants. Patel and Patel (1990) observed that maximum incidence of M. phaseolina on sesamum occurred at 35ºC and RH 76 per cent. Muthukrishnan et al. (1995) reported that the incidence of root rot of Vigna mungo had a positive correlation with maximum temperature (r=0.165) and maximum RH (r=0.226) and a significant negative correlation with rainfall 9r=0.328) and the number of rainy days (r=0.298). Cheema (1997) reported that inoculated seeds of cowpea with M. phaseolina incubated at different combinations of temperature and RH had maximum seedling mortality at temperture 30ºC and RH 70 per cent.
Biochemical changes in leaves on pathogenesis
In fungus infected plants the total nitrogen and protein content of the host pathogen complex has been reported to be generally increased during the early stage of disease development, either due to the presence of the pathogen or a newly synthesized proteins in the host (Uritani, 1971). An increase in the protein contents of rice after infection by Helminthosporium oryzae has been noticed by Vidhyasekaran et al. (1973). Yamamoto et al. (1976) and Tani and Yamamoto (1979) have also reported increase in protein synthesis during the first 14-20 hrs after inoculation in inocompatible interaction in oat Puccinia caronata f.sp. avenue system but not in a compatible interaction. Increased protein syathesis in incompatible interactions in certain host- parasite system has been considered to reflect accelerated biosynthesis of enzymes and other proteins involved in plant deference (Vance and Sherwood, 1976; Yoshikawa et al., 1978; Heath, 1979 and Goodrnan et al., 1986a) have proposed that infection by pathogens is accompanied by the biosynthesis of new pathogen proteins, which may be few in number in case of the simpler viruses and several hundreds in case of bacteria or fungi. There may be both qualitative and quantitative changes in the pattern of protein biosynthesis or degradation which may further change as infection progresses In lima bean Macrophomina phaseolina infected plants, contents of phenols and amino acids have been reported to be increased (Jadeja and Patel, 1989). Total sugar, reducing sugar and non reducing sugar contents were found to be higher in healthy heaves of susceptible munbean genotypes than of resistant during Cercospora leaf spot pathogensis (Sindhan et al., 1999). Similar type of observations have also been reported by Sindhan and Parashar (1996) while working on early and late leaf spots of groundnut. The host infectional increase in phenolic compounds and decrease in carbohydrates have been assumed due to enhancement of synthesis of phenolic compounds and hydrolysis of phenolic glycosides by fungal glycosidases to yield free phenols (Sharma et al., 1983) and rapid hydrolysis of sugars during pathogensis (Jaypal and Mahadevan, 1968). Considerable reduction in sugar contents of sunflower leaves was noticed after 40 and 70 days of Alternaria inoculation (Kumar and Singh, 1996). Obvious reducion in reducing and non reducing sugars was due to utilization of the nutrients (present in plants in greater propertion) by the pathogen (Padamnabhan, 1973). Singh et al. (1998) while working on rust infected safflower leaves have reported higher amount of chlorophyll, amino acids and orthodihydroxy phenols in tolerant cultivars and that of total soluble and reducing sugars in susceptible cultivars. The chlorophyll, amino acids and suagr content was found to be decreased while that of phenolics increased upon rust infection. Significantly less amount of total soluble sugars and free amino acids but higher amount of total phenols in the resistant genotypes than the susceptible genotypes were observed in chickpea in relation to gray mould infections (Mitter et al., 1997). Post infectional decrease in chlorophyll and reducing sugar content were observed in the leaves of both susceptible and resistant cultivars of pea upon powdery mildew infections.Whereas, the total sugar and non reducing sugar content increased upon infection (Guleria et al., 1997). The infection of cotton plants by Helminthosporium specifenum has been shown to disturb the metabolism of sugars, nitrogen, amino acids etc. In general, sugars decreased and the total amount of nitrogen increasesed in the diseased plants. The reduction in total sugar and non reducing suagr was found to be more pronounced in comparatively susceptible genotypes (Bedi et al., 1977). Tomiyama (1963) has thoroughly reviewed the physiology and biochemistry of disease resistance in plants emphasizing the higher activities of the oxidases in the infected resistant tissues than in the infected susceptible ones or the uninfected healthy ones. Higher levels of activity of peroxidase enzyme in the leaves and stems of mungbean upon leaf blight infection have been reported by Chandra and Tyagi (1992) both in resistant and susceptible cultivars but more pronounced in the resistant cultivars. In number of cases, increased peroxidase activity or activity of new isozymes may be reflection of symptom response or may be associated with degradative processes caused by infection (Farkas and Lovrekovich, 1965; Maxewell and Bateman, 1967). Changes in peroxidase isozymes or rate of their synthesis may be responsible for determining resistance or susceptibility to a given plant pathogen (Farkas and Stakmann, 1966; Stakmann et al. 1966 and Fehrmann and Dimond, 1967). Batra and Kuhn (1975) noted increase in the levels of protein, polyphenoloxidase and peroxidase which were concomitant with the development of acquired resistance in soybean leaves. Attempts to correlate peroxidase or polyphenol oxidase with resistance have been inconsistent leading some investigators to conclude that increases of these enzymes must be the result rather than the cause of resistance (Bell, 1981).
Effect on seedling vigour
The successful stand establishment in field is dependent upon the quality of planted seed. Kriton and Panner (1977) defined seed quality as the chemical composition of seed and its ability to germinate and produce vigourous healthy seedlings. In 1977, ISTA accepted seed vigour as “the sum of those properties of seed which determines the level of activity and performance of seed or seed lot during germination and seedlings emergence”. Seeds which perform well are termed high vigour seeds and seeds which perform poorly are called low vigour seeds (Perry, 1981). Seed vigour is highly complex. At the biochemical level it involves biosynthesis of energy and metabolic compounds. At the germination level it involves speed and totality of germination under all conditions. There are a number of factors, pathological, entomological and agronomical which may affect the seed quality such as seed germination, seedling emergence and seed vigour (Hydekar 1972; Mc Donard 1975 and Perry 1981). Among pathological factors, seed borne fungi have been associated and correlated with decresed seed germination of soybean and many other crops (Gupta et al., 1993). As the percentage of seed infection increse the percentage germination and field emergence decreases (Gupta and Cheema, 1990). Soil microflora including facultative parasite like Pythium, Phytophthora, Fusarium and Rhizoctonia also influence seed performance either by casuing seedrot and pre-emergence damping-off or by causing stunting of seedlings and delayed emergence (Gupta et al., 1993).
Management of Disease
Management of soil borne pathogen is very diffult but disease incidence can be reduced by various methods of disease management viz, chemical, biological, cultural and host resistance. Sinha and Khare (1977) found carbendazim, benomyl, and thiram effective against Macrophomina phaseolina in lab, pot and field tests. Taneja and Grover (1982) compared the efficacy of fungicides and found benomyl and carbendazim most inhibitory to M. phaseolina. Kotashthane and Gupta (1983) reported that seed treatment of mungbean with bavistin followed by 1 or 2 spray of bavistin was most effective in reducing leaf blight caused by M. phaseolina. Gangopadhyay and Grover (1985) reported that seed treatment and soil drenching with carbendazim and thiophanate methyl was effective against R. solani and R. bataticola causing root rot of cowpea. Bhate et. al. (1985) found benlate, captan and dithane M-45 effective in reducing the damping off of mungbean caused by M. phaseolina. Singh and Lodha (1986) reported carbendazim, benomyl and phenylmercury acetate as most promising fungicides when used as seed dressing for reducing the incidence of dry root rot of cowpea caused by M. phaseolina. Gaikwad and Sokhi (1987) reported that M. phaseolina, the seed borne pathogen of cowpea, was effectively checked by seed dressing fungicides namely benomyl and thiram. Ramadoss and Sivaprakasam (1993) reported that carbendazim, quintozene and carbendazim + carbosulfan seed treatment were highly effective in the control of root rot and stem fly on cowpea. Vir (1987) reported that benomyl and carbendazim were effective against M. phaseolina in the soil to a depth of 4 cm. Fungicides were inactive at 5 cm depth possibly due to their adsorption by soil particles. Hooda et al. (1988) found carbandazim, thiophanate methyl, captafol, thiram and phenyl mercury acetate effective against M. phaseolina of mungbean and cowpea. Singh et al. (1990) tested 10 different fungicides as seed dressers against M. phaseolina of cowpea and observed that bavistin @ 3g/kg was most effective for controlling the stem and root rot followed by thiram, vitavax and topsin-M. Ratnoo and Bhatnagar (1990) reported that ashy grey stem blight pathogen M. phaseolina of cowpea was effectively checked by treating the seed with bavistin, benomyl, brassicol, difolatan, thiram and thiabendazole. Shivanna et al. (1992) found thiram and captan most effective against root rot of guar when used as seed treatment. Ramadoss and Sivaprakasam (1993) reported that carbendazim, quintozene and thiram, as seed treatment, were most effective in controlling the root rot of cowpea caused by M.phaseolina. Lodha (1993) found carbandazim, benomyl, thiram, thiophanate methyl were most effective in controlling the dry root rot of mungbean, cowpea, clusterbean and mustard caused by M. phaseolina Ramadoss and Sivaprakasam (1994) reported that sclerotial production of M. phaseolina was completely inhibited by carbendazim and thiram. Arjunan and Raguchander (1996) found that seed treatment with carbendazim (2g /kg) and thiram (4g /kg) seed were most effective in reducing the root rot of cowpea caused by M. phaseolina. Chippa et al. (2000) reported that carbendizim, mancozeb and phenylpyrrole were most inhibitory to M. phaseolina at concentration of 500 ppm. Dubey (2003) reported that mancozeb and bavistin inhibited the radial growth of M. phaseolina by 86.3 and 86.4 per cent respectively after 96 hours of incubation at 30±1ºC. A variety of chemicals having antibacterial and antifungal properties found in different parts of plants, have been reported to be effective (Atal et al., 1978). Dubey and Dwivedi (1991) studied the fungitoxic properties of Acacia arabica, Allium cepa and A. sativum against vegetative growth and sclerotial viability of Macrophomina phaseolina and reported that bulb extract of A. sativum was more effective than its leaf extract in inhibiting mycelial growth even at 0.1 per cent concentrations. Upadhyaya and Gupta (1990) observed antifungal activities of A. sativum, Ocimum sanctum and Datura alba on mycelial growth and sclerotia germination of Macrophomina phaseolina. Raja and Kurucheve (1998) reported that 10 percent concentration of garlic clove extract completely inhibited the mycelial growth and sclerotial germination of M. phaseolina. Sindhan et al. (1999) reported that bulb extract of Allium cepa, A. sativum and leaf extract of Azadirachata indica were most toxic and inhibited the mycelial growth of Rhizoctonia solani and R. bataticola even at 5 percent concentration. They also observed that as the concentrations of extracts increased in the medium the effectiveness of the extracts also increased and maximum growth inhibition was recorded at 20 percent concentration. Chippa et al. (2000) reported that clove oil inhibited mycelial growth of M. phaseolina effectively. In the modern era, where hazards of pollution are increasing day by day, the biological control should be preferred over chemical control of plant disease. Several mycoparasites and hyperparasites have been tested for their effectiveness by different workers in vitro or in vivo studies. Ghaffar (1968) studied the interaction of some soil fungi with M. phaseoli and observed that Trichoderma viride inhibited growth of M. phaseoli and grew over its colony. He further observed that the hyphae of T. viride coiled around the hyphae of M. phaseoli. Papavizas and Levis (1981) reported that Trichoderma harzianum, T. viride and Bacillus subtilis inhibited growth of M. phaseolina considerbily. Byadgi and Hegde (1988) reported that Trichoderma sp., Bacillus sp. and Streptomyces sp. suppressed the growth of M. phaseolina in culture. Algarsamy and Sivaprakasam (1988) reported that pelleting cowpea seeds with Trichoderma viride either alone or in combination with carbendazim inhibited the growth of M. phaseolina in vitro. Hussain et al. (1990) found that Trichoderma harzianum and Gliocladium virens reduced infection of M. phaseolina of sunflower and mungbean. Kehri et. al. (1991) reported that Trichoderma viride when applied as seed coating reduced mortality of Vigna radiata Cv. T-44 and Pusa Baisakhi due to M. phaseolina from 19 to 8 percent and from 19 to 10 per cent, respectively in unsterlized soil under green house conditions. Deshmukh and Raut (1992) reported that Trichoderma harzianum and T. viride overgrew colonies of Macrophomina phaseolina in in vitro test. They further observed that T. harzianum and T. viride were effective against M. phaseolina in pot trials. Raguchander et al. (1993) reported that dry root rot of Vigna radiata caused by M. phaseolina was reduced by application of Trichoderma viride as row treatment 2 days before sowing. Lodha (1993) reported that coating chickpea seeds with Bacillus sp. and seed of many legumes and oil seeds with Trichderma harzianum and T. viride were effective in reducing dry root rot incidence caused by M. phaselina. Singh et al. (1995) reported that Trichoderma harzianum and T. viride inhibited the growth of M. phaseolina in vitro test but T. viride caused greater inhibition. Majumdar et al. (1996) reported that Trichoderma viride, T. harzianum and Bacillus subtilis exhibited antagonistic activity against M. phaseolina, the incitant of leaf blight of mothbean. They further observed that T. harzianum caused maximum growth inhibition of pathogen. Ushamalini et al. (1997) reported antagonistic activity of Trichoderma viride and T. harzianum against M. phaseolina causing charcoal rot of cowpea. They recorded a root rot incidence of 17.0 and 17.6 per cent, respectively compared with 38 per cent in control when used as seed treatment. Sheela and Packiaraj (1999) reported that Trichoderma viride + neem cake reduced root rot incidence of groundnut caused by M. phaseolina in field conditions. Rajeswari et al. (1999) reported that carbendazim tolerant isolate of Trichoderma harzianum increased seed germination, plant height and total biomass of the mungbean plant when applied to soil infested with M. phaseolina compared to application of T. viride and T. virens. They also reported that seed treatment with 108 conidia/ml of T. harzianum was effective against root rot of mungbean. Chippa et al. (2000) reported that Gliocladium virens exhibited least mean growth of M. phaseolina causing leaf spot disease of bottle guard in vitro.
Effect of date of sowing
Singh (1957) reported that mortality of guar due to Rhizoctonia bataticola is influenced by planting date. He found that incidence of dry root rot of guar was high from mid July to end of July. Paramjit and Gupta (1993) reported that the severity of stem rot of sesamum caused by M. phaseolina was reduced by sowing sesamum on 10-20 July, resulting in increased yield as compared with crop sown on 1st July. Ratnoo and Bhatnagar (1993) reported that cowpea plants were more susceptible to ashy grey stem blight caused by M. phaseolina at young age. The disease was most severe upto the age of 45 days. However, disease index gradually decreased with increase in the age of the plant.
Host Resistance
Varietal resistance is one of the best methods to manage the disease as the use of resistant variety saves expenses incurred on chemicals and causes no environmental hazard. Resistant sources against M. phaseolina have been identified in several crops by various workers. Inheritance of root rot resistance have been studied in mungbean. Gangadharan et. al. (1981)reported that only CO-3, CO-9385 and LM 220 were resistant in infested field of M. phaseolina. Zote et al., (1983) tested 14 cultivars of vigna radiata agaist M. phaseolina observed that no cultivar was immune whereas, five cultivars were found resistant. Bhate et al. (1985) reported that out of 24 cultivars of vigna radiata tested in infested plots of M. phaseolina, 12 cultivars were moderately resistant and 11 moderately susceptible and 1 highly susceptible. Shivshikar and Deshmukh (1991) reported that out of 30 cultivars of V. radiata tested only BCG-1 was found resistant against M. phaseolina under artificial conditions.
3. MATERIALS AND METHODS
3.1 Survey and collection of diseased material
To know the extent of disease prevelance of mungbean leaf blight in Rajasthan, a survey of mungbean fields was carried out during the year 2000 and 2001 in some of the major mungbean growing district of the Agroclimatic zone II A and III A. These zones include of Churu, Jhunjhunu, Sikar, Jaipur and Tonk districts having largest cultivated area under this crop. During the survey, both prevalence and intensity of the disease were recorded on crop plants. Five fields were surveyed in each selected tehsil. In each field diseased and healthy plants were counted on randomly selected five spots each of 1 sq. meter area and per cent disease incidence was calculated using the formula given below: Number of diseased plants Per cent disease incidence = ------X 100 Total number of plants observed
Infected plant material including stem and leaves were collected from surveyed fields and brought to the laboratory for further studies. The diseased materials were collected from following locations. District Tehsil Zone II A Churu Churu Ratangarh Jhunjhunu Jhunjhunu Nawalgarh Sikar Fatehpur Piprali Zone III A Jaipur Sanganer Dudu Tonk Malpura Tonk
3.1.1 Isolation and purification of pathogen:
Isolations were made from the infected stems and leaves showing typical symptoms. The stems and leaves were thoroughly washed with tap water to remove soil. Infected plant parts were cut in to small pieces (0.5-1.0 mm) and surface sterilized with 0.1 per cent mercuric chloride solution for 2 minutes followed by three serial washings with sterilized glass distilled water and blot dried. One bit was placed aseptically in each potato dextrose agar (PDA) slant. Culture tubes were then incubated at a temperature of 30±1ºC for four to five days for the growth of the pathogen..
3.1.2 Identification of the pathogen
Identification of the pathogen was done on the basis of cultural and morphological characteristics of the fungus mycelium and sclerotia.
3.1.3 Pathogenicity
For pathogenicity test, mungbean plants of a susceptible cultivar K-851 (21 days old) grown in 9 x12 inch earthen pots, were inoculated using mycelial suspension of 3 days old culture grown on potato dextrose broth which was filtered and homogenized to give 1X103 viable propagules per ml. This suspension was sprayed on the foliage of the mungbean plants by hand atomizer till run-off. The inoculated seedlings were kept at high humidity for 48 hrs, after which pots were transferred to cage house. The disease incidence was observed after a week of inoculation. The same fungus was re-isolated from these artificially inoculated plants. To test pre emergence and post emergence, mungbean seeds of K-851 were grown in 9X12 inch earthen pots containing sterilized soil inoculated with 5 days old culture of the fungus. Soil inoculation technique employed by Kataria and Grover (1976) was used with slight modification, that the inoculum was raised on sorghum grains. For multiplication of inoculum, 20 gm sorghum grains were moistened with 10 ml distilled water in 250 ml Erlenmeyer flask and autoclaved at 1.045 kg/cm2 for 2 hours and colled before inoculum was added aseptically. The autoclaved sandy loam soil (2.5 kg/pot) was filled in 9x12 inch earthen pots which were inoculated with fungus in a proportion of 1:10 (Semeniuk, 1944) by thoroughly mixing it in the upper 4-5 cm layer of soil and allowed to stabilize for one week. A check was maintained without inoculum. After a week of colonization of soil, seeds were sown in these pots. Initial seedling emergence was recorded. Seedling infection was recorded after 15 days of sowing. The fungus was re-isolated from the seedlings showing rotting symptoms in these artificially inoculated pots.
3.2 Multiplication of inoculum for inoculation:
Following approaches were made to finally select the most effective method(s) for raising inoculum (mycelium and sclerotia) for inoculation and other studies in present case.
3.2.1 For Soil inoculation
Five different grain media viz., sorghum, bajra, mungbean, guar and maize were tested for their suitability for mycelial growth and sclerotial production. For obtaining inoculum, 100 gm of grain mentioned above were filled seperately in 250 ml conical flasks. Sufficient quantity of water was added in each flask for soaking the grains. After 12 hrs of soaking seeds, these flasks were then autoclaved at 1.045 kg / cm2 pressure for 20 minutes and inoculated with 4 days old mycelial bit of M. phaseolina growing on PDA slants and then incubated at 30+1ºC for 4-5 days. Observations with regard to time of initation of mycelial growth, initiation of sclerotia formation and growth characters were recorded for each medium.
3.2.1.1 For foliar inoculation
Thirty ml of potato dextrose broth medium was poured in each 150 ml conical flask, auotoclaved at 1.045 kg/cm2 pressure for 20 minutes. After proper cooling, medium was inoculated with 4 days old mycelial disc (5mm) of pathogen and incubated at 30 ± 1ºC for 3 days.
3.2.2 Standardization of inoculation techniques for disease development
3.2.2.1 Seed inoculation
Two hundred apparently healthy surface sterilized seeds were rolled on 7 days old culture of M. phaseolina thriving on PDA in petridishes. Inoculated seeds were sown in 9x12 inches earthen pots containing sterilized soil @ 15 seeds per pot. The uninoculated surface sterilized and apparently healthy seeds served as check. These pots were kept in cage house and watered as and when required. Observation for seed germination was recorded after 1 week of planting and seedling mortality was recorded after 15 days of sowing.
3.2.2.2 Soil inoculation
Autoclaved soil was inoculated using 10 % inoculum, multiplied on sorghum grains as described earlier, using modified method of Kataria and Grover (1976) and allowed to multiply for a week before seeds were planted. 3.2.2.3 Foliar inoculation
Mycelial suspension of 3 days old culture grown on potats dextrose broth was filtered and homogenized to give 1x103 viable propagules per ml. This suspension was sprayed on 21 days old seedlings by hand atomizer till run-off. After inoculation plants were kept in moisture chamber for 48 hrs and then transferred to cage house. Observations for development of symptoms were recorded 7 days after inoculation.
3.2.3 Effect of age of culture
To assess the effect of age of culture on disease development, the fungus M. phaseolina was grown on potato dextrose broth for varying period (3,5,7,9 and 11 days). Twenty one days old seedling were inoculated through foliar spray by hand atomizer with culture suspension of fungus of different age having 1x103 cfu/ml and kept in moisture chamber for 48 hrs before transferring to cage house. Observations on development of symptoms were recorded 7 days after inoculation.
3.2.4 Effect of duration of humidity
Twenty one days old plants of mungbean (10 plants/pot) were inoculated by foliar application of inoculum suspension and kept under humid chamber for varying time period i.e. 12,24,36 and 48 hours and observations on disease development were recorded after 7 days of inoculation.
3.3 Survival and perpetuation
Viability of the pathogen propagules plays an important role in re- occurrence of the disease.
3.3.1 Detection of the pathogen in or on the seed Seed samples colleted from all the surveyed locations were tested for the presence of pathogens in the years 2000 and 2001, employing methods described by ISTA (1985). Both blotter method and agar plate methods were used. Component plating was also done on seeds showing dark brown to black lesions to know the place of presence of mycelium if any in seed components.
3.3.1.1 Standard Blotters Test
Four hundred seeds of a sample were analysed. Three layers of moistened blotter paper were placed in the bottom of a petriplate. Ten seeds were placed at equidistance in each petriplate. The plates were then incubated at 30 ºC under alternating cycles of 12 h light and 12 h darkness. The seeds were examined on 10th day of incubation, when the formation of sclerotia took place.
3.3.1.2 Ager plate test
Four hundred seeds were surface sterilized with 0.1 per cent mercuric chloride for 3 minutes followed by three serial washings with sterilized glass distilled water. Sterilized petriplates, each containing 20 ml PDA and cooled, were used. Ten seeds were placed at equidistance in each petriplate. The plates were then incubated at 30 ± 1 ºC for 10 days when the formation of sclerotia took place.
3.3.2 Component planting method
The various components of mung bean seed viz., seed coat, cotyledons and embryonal axis were examined by agar plate method. Suspected infected seeds were surface sterilized using method described earlier and rinsed with sterilized water and soaked for 12 hrs in sterilized water to facilitate removal of surface contamination and seed coat from cotyledons. The seed coat and cotyledons were separated aseptically with forceps. These were then surface rinsed with sterilized glass distilled water, blot dried and placed on PDA in petriplates. The plates were then incubated for 10 days at 30±1ºC. The seed parts sowing presence of mycelial growth were recorded.
3.3.3 Sclerotia as a source of primary inoculum
The mature slerotia, collected from 30 days old culture, grown on 2 per cent potato dextrose agar, were kept in nylon net and placed in earthen pots filled with oven dried autoclaved soil at different depts (0,5 and 10 cm). Burried sclerotia were harvested and observations were taken for their germination on PDA from one month to 12 months period. Five hundred grams surface sterilized mungbean seeds were coated with mature sclerotia, collected from 30 days old culture (on PDA). Such inoculated seeds were stored in laboratory conditions in glass bottles. Samples were drawn periodically at an interval of 2 months and role of sclerotia on seed in causing seedling infection was observed by sowing these inoculated mungbean seeds in earthen pots filled with oven dried autoclaved soil. Ten seeds were seeded per pot and replicated thrice. Observations on seedling mortality was recorded.
3.4 Role of environmental factors on disease development
Meteorological data viz., temperature, relative humidity and rainfall were recorded and correlated with the disease incidence. The disease incidence in the field was recorded in the sick plot conditions at regular interval and disease severity was correlated with weather parameters.
3.5 Biochemical changes in infected leaves
3.5.1 Sampling and preparation of leaf extracts for estimation of sugar and amino acid contents Healthy and infected leaves (2nd and 3rd) from top of the plant were collected from each varieties viz., K-851, RMG-268 and MUM-2 after 10, 20 and 30 days of inocululation. Leaf samples were first air dried at 55 0C and then powedered in a grinder. The powdered leaf sample (one g) were extracted in 80 per cent ethanol on a water bath and finally the aqueous extracts were prepared.
3.5.1.1 Estimation of suagr contents
Properly diluted aqueous extracts were used for estimation of sugar contents as per the method of Dubois et al. (1956) using 5 per cent phenol and conc H2SO4. In 2.0 ml aliquot of the leaf extract, 1.0 ml of 5% phenol solution was added and mixed well. This was followed by addition of 5 ml of concentrate sulphuric acid and shaked vigorously. It was kept standing for 15 min and the absorbance of the pinkish colour developed was read at 490 nm. The sugar contents were expressed as mg/g dry weight of the leaf tissue with reference to glucose standard curve (10-100 mg). Five replications were used for each estimation taking average value as the estimated sugar content.
3.5.1.2 Estiamtion of amino acid contents
Properly diluted aqueous extracts of leaves were used for estimation of amino acids contents as per the widely used ninhydrin method of Moore and Stein (1948). In 1.0 ml aliquot of the leaf extract, 1.0 ml of ninhydrin reagent (prepared by mixing aqual volume of ninhydrin solution and stannous chloride solution- [prepared by dissolving 400 mg of ninhydrin in 10 ml of methyl cellosolve
(ethylene glycol monomethyl ether) and 16 mg of SnCl2 (stannous chloride) in 10 ml of citrate buffer- pH 5.0 respectively] was added and the mixture was heated for 20 min on a water bath. Later, this was cooled and diluted by mixing 5 ml of the diluting solution of n-propand and water (1:1 v/v). Optical density of the purple colour developed was read at 570 nm. The amino acid content in terms of glycine as standard (5-20 g) was expressed as mg/g dry wt of the leaf tissue. Five replications were used for each estimation taking average amino acid value as the estimated content.
3.5.2 Sampling for estimation of oxidative enzyme (peroxidase activity)
Healthy and infected leaves (2nd and 3rd) from top of the plant were collected from each variety upon 10, 20 and 30 days of inoculation. Leaf samples were cut into smaller pieces in the laboratory at normal temperature. From among this mixture, 1 g of leaf tissue was homogenized in 10 ml of 0.05 m Tris HCl buffer (pH 7.6) in a chilled pestle mortar. The homogenate was centrifuged at 10, 000 x g for 15 min in a refrigerated centrifuge at 4 0C. The supernatant so obtained was used for enzyme assay and protein estimation.
3.5.2.1 Peroxidase activity assay
Peroxidase activity was assayed by the modified method of Shanon et al. (1966). The reaction mixture contained 0.2 ml of 0.2% dianisidine in methanol,
0.1 ml of 0.2% hydrogen peroxide (H2O2), 3.6 ml of 0.05 M phosphate buffer (pH 6.5) and 0.1 ml of properly diluted enzyme extract. The enzymatic activity was initiated by adding H2O2 and the subsequent change in absorbance was recorded upto 2 min at 430 nm. The enzyme activity was expressed as units of activity per mg protein, whereas one unit was taken as change in optical density @ 0.01 per min. The enzyme assay was carried out in five replications.
3.5.2.2 Protein estimation of enzyme extract
The protein content of the enzyme extract were estimated by following the method described by Lowry et al. (1951). To a total of 1.0 ml (0.9 ml of H2O + 0.1 ml of properly diluted extract) was added 5.0 ml of the freshly prepared reagent C [Prepared by mixing 50 ml of reagent A (2% sodium carbonate in 0.1
N NaOH) with 1.0 ml of reagent B (0.5% CuSO4 in 1.0% sodium potassium tarate] and then adding 0.5 ml of the folin-Ciocalteu reagent diluted with water (1:1 v/v). The contents were rapidly mixed and were allowed to keep for half an hour in dark. Later, the intensity of the blue colour developed was read at 620 nm and the protein content was estimated in mg per g fresh weight taking average of the five replications.
3.5.3 Seed infection on sedling vigour in In vitro
Seed lot used for testing survival of the pathogen on seed, described earlier, were also tested for changes in seed germination and seedling vigour due to fungal infection of the seed. Samples were drawn periodically at 2 months interval upto 12 months. The seeds without inoculation were kept as check. The germination of seeds was determined by paper towel method (ISTA, 1996) as described below. Seed samples were placed on two moist paper towels of 23 x 30 cm size. Seeds were kept at appropriate spacing and covered with another moist paper towel and rolled up. The rolled paper towels were kept in an upright position in an incubator at 30 + 1 0C. The germination counts were taken after 7 days. Seedling vigour was determined by measuring the shoot length, root length, total length and dry weight of seedlings. The seedlings were dried in butter paper bags at 60 0C in oven for 24 hrs before taking dry weight (Agarwal, 1980).
3.6 Management of the disease
3.6.1 Management through chemical Follwing four fungicides were used for their comparative effectiveness in management of disease. Trade Common Chemical Name Formulation Name Name per cent Bavistin Carbendazim Methyl-2-Bengemidazole carbamate 50 WP Benlate Benomyl Methyl-N (1-butyl carbamayl)-2- 50 WP benzimidazole carbamate Captan Captafol N-Trichloromethyl thio-4-cyclohexene–1,2- 75 WP dicarboximide Thiram Thiram Tetramethyl thiuram disulphide 75 WP
3.6.1.1 Efficacy of fungicides, In vitro
Above fungicides, each at 250, 500, 1000 and 2000 ppm concentrations, were evaluated for their comparative efficacy on the growth of Macophomina phaseolina using the poisoned food technique. The treatment without fungitoxicant served as control. Desired quantity of fungicide was mixed thoroughly in cooled PDA, just before pouring in sterilized pertri plates and were allowed to solidify for 12 hrs. Each plate was inoculated with 2 mm disc of mycelial bits taken from the periphery of 4 days old colonies of M. phaseolina growing on PDA. The inoculated perti plates were then incubated at 30±1ºC. After 5 days of incubation colony diameter was measured on both the diagonals. Four replication (petri plates ) were used for each concentration of a fungicide and check. Per cent growth inhibition was calculated by Vincent’s (1947) formulae as follows C-T Growth inhibition (%) = ------X 100 C Where, C= Diameter of the colony in check (average of both diagonals) T= Diameter of the colony in treatment (average of both diagonals)
3.6.1.2 In vivo efficacy of fungicides
The comparative efficacy of above fungicides were evaluated as seed treatment, foliar spray and both seed and foliar application against leaf blight of mungbean. The field experiments were laid out during Kharif 2000 and 2001 at Agriculture farm of SKN College, Jobner. Seeds of susceptible cultivar K-851 was used. The experiment was laid out using RBD with four replications. Each plot was of 2.5 m x 2 m = 5 m2 size. In one set only seed treatments were applied. In other set, seed were treated with fungicide and again foliar application applied on 21 days old plant. In third set no seed treatment were given but plants were sprayed with above fungicides when 21 days old. All plots, where fungicidal sprays were given on foliage, were inoculated with 3 days old culture of M. phaseolina having 1 x 103 colony forming unit/ml, one day in advance. Seed without fungicidal treatment served as check. Observations for seedling mortality, foliar blight severity and grain yield were recorded.
3.6.2 Efficacy of different bio plant extracts
In recent years, due to environmental hazards from pesticides usage, extracts of many plants have been used in place of fungicides for the control of various plant pathogens. In the present investigations extract of different plants were evaluated individually for their effect on the M. phaseolina of mungbean. The list of plant extract evaluated are as under. Common name of plant Botanical name Aak Calotropis procera Bougainvillea Baugainvillea spectabelis (L.) Datura Datura alba (L.) Garlic (Bulb) Allium sativum (L.) Neem Azadirachta indica (A.Juss.) Onion (Bulb) Allium cepa (L.) Tulsi Ocimum sanctum (L.) Extracts were prepared from fresh leaves/cloves washed with tap water followed by sterilized water. These were then finaly grinded with sterilized distilled water @ 1 ml g-1of tissue (1:1/v/w) with pestle and mortar and filtered through a muslin cloth. The extract was taken as standard (100 per cent) and used either as such or after dilutions.
3.6.2.1 Efficacy of plant extracts in pot and field condition
To test the efficacy of plant extract apparently healthy surface sterilized seeds of cultivar K-851 were inoculated with 5 days old culture of M. phaseolina by seed roll method. Inoculated seeds then were incubated at 30±1ºC for active growth of test fungus on seed for 48 hrs. After incubation, these seeds were treated by soaking for 5 hrs with plant extract (50 per cent) seperately for each treatment. Inoculated and treated seeds were planted in pots. They were also sown under field conditions and sprayed with relevant plant extract (10 per cent) one day after inoculation. Seedling mortality and leaf blight severity was recorded.
3.6.3 Biological control
3.6.3.1 Efficacy of bioagents In vitro
Several bioagents have been found effective in controlling many plant pathogens. In the present study four antagonistic bioagents viz., Trichoderma viride, T. harzianum, Gliocladium virens and Bacillus subtilis were tested against M. phaseolina under In vitro conditions. For testing antagonism of fungal bioagents one week old growth 2 mm mycelial disc of antagonistic fungi and 4 days old growth of M. phaseolina were placed 2 cm apart from periphery of PDA plates in opposite directions. Plates were then incubated at 30±1º C. The zone of inhibition developed due to lysis and antibiosis in each treatment were measured after five days of incubation. The number of sclerotia formed were also recorded in each treatment. For measurement of efficacy of bacterial biocontrol agent (Bacillus subtilis) sterilized petri plates containing 20 ml PDA were first inoculated with 4 days old culture of M. phaseolina and incubated at 30±1º C for 24 hrs. These plates were again inoculated with 2mm disc of sterilized filter paper dipped in suspension of B. subtilis. Five such disc were placed at equidistance/ plate and incubated at 30±1º C for five days. The zone of inhibition was measured after incubation and the number of sclerotia formed were also recorded. Each treatment was replicated 5 times and whole experiment was repeated twice.
3.6.3.2 In vivo
In order to study the effect of these biocontrol agents as seed treatment and foliar spray alone and in combination on seedling mortality and leaf blight incidence, field experiments were laid out during kharif 2000 and 2001 at Agriculture farm, SKN Agriculture College, Jobner in randomize block design. Seeds of highly susceptible cultvar K-851 (dressed with required quantity of bioagents) were sown in 5 m2 plot in two sets and four replications. In one set spray of bioagents applied one day after the plants were inoculated by spraying mycelial cum sclerotial suspension of 3 days old growth of M. phaseolina on 21 days old plants. In another set, seeds were sown without seed treatment and bioagents were sprayed on foliage one day after spraying mycelial suspension of 3 days old growth of M. phaseolina on 21 days old plants. In third set treated seeds were sown and foliar spray applied one day after inoculation. The seed sown without any bioagents treatment served as check. Observations on seedling mortality, leaf blight severity and grain yield kg/ha were recorded.
3.6.4 Effect of date of sowing
The field experiments were conducted during kharif 2000 and 2001 to find out the effect of sowing dates on seedling mortality and leaf blight severity. The experiments were laid out in randomized block design. Sowing of mung bean variety K-851 was done from 14th July to 13th August at an interval of 15 days in sick soil condition with the spacing of 30x10 cm in 5 m2 plots for each treatment. Observations on leaf blight severity was recorded after 35 days of sowing.
3.6.5 Host resistance
Fourty genotypes of mungbean received from Agricultural Research Station, Durgapura Jaipur were evaluated against leaf blight for two years (kharif 2000 and 2001) following standard agronomical practices. The inoculum of the pathogen was prepared by multiplying culture of the pathogen on sterilized boiled sorghum grain medium. The inoculum was mixed in soil at the rate of 200 gm per row of 3m, one week prior of sowing at 5-10 cm depth. The each genotype were sown in three replications with two rows of 3 m length at SKN College of Agriculture farm Jobner. Observations were recorded after 15,30 and 45 days of sowing. The genotypes were categorized according to their disease reaction based on scale suggested by Stonehouse (1994). The details of reaction and leaf area covered by leaf blight is as follows: Disease reaction Percentage leaf area infected Highly resistance No lesions on leaves Moderately resistant 1-25% area covered by lesions Moderately susceptible 25.1-50% area covered by lesions Susceptible 50.1-75 % area covered by lesions Highly susceptible 75.1-100 % area covered by lesions Twenty five leaves were randomly selected from each plot. Leaves were rated as per the above scale. The percent severity was calculated according to the following formula: Sum of all disease rating Per cent disease severity = ------X 100 Total number of rating X maximum disease index
4. RESULTS
4.1 Survey and collection of diseased material
Mungbean [Vigna radiata (L.) Wilczek] crop was surveyed for incidence of leaf blight in Churu, Jhunjhunu and Sikar districts in agroclimatic zone II A, and Jaipur and Tonk district in zone III A, of Rajasthan during kharif season of 2000 and 2001. Five crop fields in each of the mentioned tehsils of these district was surveyed between Aug-16 to 15 September each year. During the field survey it was observed that disease was present in all fields and the disease incidence was more in the fields where farmers were continuously taking pulse crops. Observations recorded on the disease incidence are presented in Table-1. Disease was found to be prevalent in all the crop fields surveyed but per cent disease incidence varied. Average disease incidence was 19.11% for all fields. The disease incidence was maximum (26.0%) in Dudu tehsil followed by Jhunjhunu (23.7%), Sanganer (21.5%) and Nawalgarh (21.3%). At other places it was less than 20%. In both the years disease incidence was almost at the same level in all fields.
4.1.1 Isolation and purification of pathogen
Isolations were made aseptically from diseased leaves and stems from collected samples during survey utilizing all samples. The fungus obtained was purified by adopting single hyphal tip culture method. Observations on growth characteristics, morphological characteristics and sclerotial formation were recorded. Generally infected tissues yielded appressed, gray to pale gray and dark gray mycelium on potato dextrose agar medium. Sclerotia with black colour were formed after 4-8 days (Plate-1) in all cultures. 4.1.1.1 Cultural and morphological variability among M. phaseolina isolates
All the 10 isolates made from different places in zone II A and III A showed cultural and morphological differences. Based on cultural characters on PDA and morphological differences in the colour of hypha, type of growth, colour of the colony and sclerotia formation, they were grouped into five groups and named as MB-1, MB-2, MB-3, MB-4 and MB-5. All the five isolates were grown on PDA and incubated at 30 + 10C and growth characters observed. From observations presented in Table-2 it is concluded that mycelial growth varied from aerial to appressed with fine texture to course texture, gray dull white to pale gray, smoke gray to dark gray in colour. All the five isolates formed sclerotia in culture. Maximum number of sclerotia were observed on 8th day. The size of the sclerotia varied from 40-218 m and the shape also varied from irregular to oval and spherical.
4.1.1.2 Identification of the pathogen
Identification of the isolated fungus was done on the basis of cultural and morphological characteristics. The young hyphae of the fungus was observed to be hyaline, thin walled, septate, turning to pale grey and later dark gray in colour and having more septa as mycelium grew older. Branches from the main hyphae was generally found at right angle to parent hyphae with contraction at the point of origin. The colour of the sclerotia was light brown and turning finally brown to black. Sclerotia varied in shape from irregular to oval or spherical, measuring 40-113 x 72-218 m in size. Pycnidia was not observed on PDA in any culture but was found on rotted seed and at the collar region of rotted seedling. These pycnidia were lighter in colour than sclerotia, dark brown to black, rough, globose and irregular in shape. Pycnidiospores were one celled, hyaline, thin walled oval to elliptical measuring 19.6 x 7.2 m (Plate-1). On the basis of morphological characters observed and the measurement of various fungal structures under study, the pathogen was identified as Macrophomina phaseolina (Tassi) Goid.
4.1.1.3 Pathogenicity
All five isolates group MB-1, MB-2, -----MB-5 were found to be pathogenic on mungbean variety K-851. The inoculated plants showed typical symptoms within 36-48 hours causing 42-65 per cent disease severity (Table-3). Affected leaves showed initially smaller circular to irregular brown to reddish brown lesions which later enlarge and coalesce (Plate-2) Table 3 Pathogenic reactions of different isolate groups of Macrophomina phaseolina on mungbean in pot condition . Isolate Time taken for initiation of symptoms Disease severity group (hrs) after 7 days of inoculation (%) MB-1 36 56 MB-2 48 42 MB-3 48 40 MB-4 36 65 MB-5 48 50 * Average of five replications The pathogen was re-isolated from the infected plant parts and purified on PDA medium. The diseased spots yielded the fungus culture, identical to original fungus. The isolates MB-1 and MB-4 produced symptoms early (36 hrs) with high disease severity of 56 and 65 per cent, respectively, whereas isolate MB-2, MB-3and MB-5 produced symptoms within 48 hrs but disease severity was medium. Isolate MB-4 produced symptoms within 36 hrs and disease severity was maximum and represented isolates of Piprali, Fatehpur, Dudu and Sanager, was used for all further studies.
4.1.1.4 Symptomatology and nature of damage
In mungbean plants infection due to M. phaseolina was observed on all growth stages from pre-mergence to fully grown stage causing seed rot, seedling blight and leaf blight. The entire seedling was blighted. Lesions were also observed on the cotyledonary leaves of the seedlings (Plate-3).
Maximum disease appeared when the crop was 4-6 week old. Usually the older leaves were first affected. Initially small, circular to irregular brown to reddish brown lesions appeared which later enlarged and coalesced to cover entire leaf. Under hot and humid condition the entire plant was blighted. The infected leaves become yellowish brown and papery in texture. Severely affected leaves fall off prematurely(Plate-4).
4.2 Multiplication of inoculum
In order to find out the suitable medium for production of mass inoculum, five different grains namely sorghum, bajra, mungbean, guar and maize were tested for their efficiency in favouring growth of the fungus(Table 4). These grain media were inoculated with isolate MB-4. Out of these, fungus developed well on sorghum grains followed by mungbean and guar grains. The fungus showed excellent sclerotial production on sorghum whereas bajra and maize responded poorly for sclerotial production. Table 4 Cultural characters of Macrophomina phaseolina on different grain media used for mass multiplication Medium Mycelial growth Sclerotia formation on 8th day * Sorghum Aerial, white, profuse ++++ Bajra Submerged, scanty and dirty white ++ Mung Aerial, superficial, white +++ Guar Aerial, white, scanty +++ Maize Aerial, white, scanty ++ ++++ Excellent +++ Good ++ Fair
Sorghum grain medium supported excellent vegetative growth and sclerotial formation and was used for soil inoculation.
4.2.1 Efficacy of inoculation techniques on disease development
To find out an easy technique for creating maximum disease development, three different inoculation techniques were tested. Germination of healthy seeds was significantly superior than seed inoculation and soil inoculation. Poor seedling emergence (81.95 %) accompanied by increased seedling mortality (34.85 %) was recorded in seed planted in inoculated soil than check, which showed 96.8% emergence and no seedling mortality. Leaf blight disease severity was recorded highest (63.70 %), when plants were inoculated 21 days after the emergence with 3 days old culture. Foliar inoculation technique was found significantly superior in causing leaf blight than seed and soil inoculation techniques (Table-5). 4.2.2 Influence of age of culture on disease development
Age of culture is one of the important determinants in influencing the development of disease. Inoculum of different ages were mixed in soil @ 100 gm /kg soil and also sprayed on foliage. It was observed that soil inoculation with 5 days old culture was significantly superior in causing more disease and seedling mortality (49.6%) as compared to other methods employed. Leaf blight severity was observed maximum when plants were inoculated with three days old culture (56.7%) and was significantly superior to other growth periods tested. Tabulated data in Table-6 revealed that there was a significant and steady decline in virulence of pathogen with increase in the age of the culture. Table 6 Effect of age of culture of Macrophomina phaseolina on disease development in pot conditions Age of culture (days) Soil inoculation * Foliar inoculation* Seedling morality (%) Disease severity (%) 3 38.2 (38.17) 56.7 (48.85) 5 49.6 (44.77) 48.3 (44.03) 7 40.3 (39.41) 44.2 (41.67) 9 23.6 (29.06) 31.9 (34.39) 11 15.7 (23.34) 26.4 (30.92) 13 14.2 (22.14) 21.7 (27.76) 15 12.4 (20.62) 18.5 (25.48) Check 0.00 (0.00) 0.00 (0.00) SEm+ 0.91 1.15 CD 5% 2.64 3.32 * Average of five replications (Figure in parentheses are angular transformed values) 4.2.3 Influence of duration of exposure to higher relative humidity on disease development
To study the effect of exposure to higher humidity for different durations on disease development, inoculated plants were kept in humid chamber for varying period of time i.e. 12, 24, 36 and 48 hrs before transfer to cage house.
Observations on disease severity were recorded after 7 days of inoculation and presented in Table-7. The data revealed that the percent disease severity increased significantly with the increase in the duration of exposure to humidity after foliar inoculation. Maximum disease severity (72.40%) was observed when the inoculated plants were exposed under humid chamber for 48 hrs followed by
36 hrs, (63.00%) and 24 hrs (51.20 %). Minimum disease severity (46.7%) was observed when the inoculated plants were exposed under humid chamber for 12 hrs.
Table 7 Influence of duration of relative humidity on leaf blight of mungbean Relative humidity (hrs) Disease severity (%)* 12 46.70 (43.11) 24 51.20 (45.69) 36 63.00 (52.53) 48 72.40 (58.31) SEm+ 1.52 CD 5% 4.50 * Average of six replications (Figure in parentheses are angular transformed values)
4.3 Survival, perpetuation and transmission of the pathogen
The study of survival of pathogen in soil, for perpetuation and transmission of pathogen in/on seed is important for understanding disease development in crop through soil and seed acting as primary source of inoculum.
4.3.1 Detection of pathogen in/or on the seed
Pathogen survival in/or on the seed was carried out in petriplates. To know the efficient method of detection of pathogen in seed, two ISTA methods viz., Seedling Blotter Test and Agar Plate Method were employed. Maximum seed infection of M. phaseolina was observed in the seed samples collected from Sanganer tehsil (24%) in SBT, and (11%) in Agar plate methods followed by samples collected from Jhunjhunu (22%) and (11%), Churu (21%) and (9%), respectively. The lowest seed infection percentage was found in seed samples collected from Malpura tehsil (10%) and (5%), respectively in SBT and Agar plate methods (Table-8). From all samples tested 17.1 % seed showed presence of pathogen in blotter test and 7.5% in agar plate method. Table 8 Detection of M. phaseolina in seed samples collected from the surveyed fields of zone II A and III A. Agroclimatic District Tehsil M. phaseolina (%) zone Blotter test Agar plate method II a Churu Churu 21 9 Ratangarh 16 7 Jhunjhunu Jhunjhunu 22 11 Nawalgarh 14 6 Sikar Fatehpur 19 7 Piprali 13 6 III a Jaipur Sanganer 24 11 Dudu 18 8 Tonk Malpura 10 5 Tonk 14 5 Average 17.1 7.5 4.3.2 Component plating method
To detect the pathogen harbouring in seed coat, cotyledons and embryonal axis, seed component plating method was used. Following ISTA, a samples of 400 seeds was used in these studies. It was observed (Table-9) that seed coat has maximum sites of infection as component of seed (12.8%) followed by cotyledons (6.7%) and embryonal axis (2.0%). The data obtained in above studies, based on all samples, indicate that seed coat infection varied from 8 to 15.5%, colylendon 4-8.5 % and embryonic axil from 1.5 to 2.5% in mungbean seeds collected from infected crop. Table 9 Detection of M. phaseolina in seed components in samples collected from zone II A and III A Agroclimatic District Tehsil Presence of M. phaseolina (%) zone Seed coat Cotyledon Embryonal
axis
II A Churu Churu 17 9 2
Ratangarh 14 8 2
15.5 8.5 2
Jhunjhunu Jhunjhunu 17 7 2
Nawalgarh 10 6 1
13.5 6.5 1.5
Sikar Fatehpur 13 7 3
Piprali 10 5 2
11.5 6 2.5 III A Jaipur Sanganer 18 10 3
Dudu 13 7 2
15.5 8.5 2.5
Tonk Malpura 7 3 1
Tonk 9 5 2
8 4 1.5
Average 12.8 6.7 2.0
4.3.3 Survival of the pathogen
Surface sterilized seeds of mungbean cultivar K 851 were coated with mycelium and sclerotia of M. phaseolina by method described earlier. Such seeds were stored in laboratory at ambient temperature. The seed samples were tested periodically at an interval of 2 months for survival of the pathogen and its ability to cause seedling infection. The observations revealed that there is significant decline in the survival of pathogen after 4 months of storage of inoculated seed at ambient temperature (Table-10). The seed infections decline from initial 20.4% to 7.1 % after a storage for 12 months. The decline was slow in the beginning but with passage of time their was sharp decline. Seed store for 12 months still showed that 7.1% seeds harbour pathogen. Table :10 Effect of Macrophomina phaseolina on seedling mortality in pot Storage period (months) Seedling mortality (%) * 2 20.4 (26.85) 4 18.5 (25.48) 6 15.6 (23.26) 8 12.4 (20.62) 10 10.4 (18.81) 12 7.1 (15.45) SEm+ 0.54 CD 5% 1.57 * Mean of six replications (Figure in parentheses are angular transformed values)
4.3.4 Viability of sclerotia in soil at different depth
The study was carried out to ascertain the role of sclerotia in perpetuation of the disease from one crop season to next crop season in soil under pot condition. For this, the sclerotia buried in soil at different depth in pots filled with sterilized soil were dug out from pots and placed on PDA to record germination of sclerotia of the fungus. The results presented in Table -11 regarding the viability of sclerotia in soil at different depth indicated that there is progressive decline in the viability of sclerotia of M. phaseolina with the passage of time. The sclerotial germination decline from 75.66% to 57.6% in 12 months. The difference are significant at 3 months interval. Soil depth is important in survival of the sclerotia. They survive better at 0 cm depth i.e. at soil surface (73.62%) and the survival decreased significantly at 5 cm depth (64.9%). Minimum germination was observed when sclerotia was burried at 10 cm depth (61.43%). There was no significant difference in survival at 5 cm to 10 cm depth upto 9 month of storage but later the differences are significant.
4.4 Role of environmental factors on disease development
Relationship of various environmental factors viz. temperature, relative humidity and rainfall on the disease development were studied during the year 2000-2001 (Annexure 1). The comparative weekly temperatures observed during both the years reflect that the mean temperature was almost equal in both years 2000 and 2001 and has no significant effect on severity of leaf blight. Relative humidity has a positive correlation to disease severity in both the years 2000 and 2001. Maximum leaf blight severity (46.8 per cent) was observed at RH 77.5 per cent in the year 2000 and 40.2 per cent at relative humidity 74.5 per cent in the year 2001 (Table -12). However, the minimum leaf blight severity 5.2 per cent was observed when the prevailing RH was 57 per cent in the year 2000 and 8.7 per cent when the RH was 55.5 per cent in the year 2001. As far rainfall is concerned, it has significant and positive correlation to leaf blight severity. In the year 2000, a maximum 46.8 per cent disease severity was observed when there was 24.4 mm rainfall. In the year 2001, maximum disease severity 40 .2 per cent was observed when the rainfall was 15.0 mm. Disease severity was minimum i.e. 5.2 per cent and 8.7 per cent in the year 2000 and 2001 respectively, when there was no rainfall in the year 2000 and 3.0 mm rainfall in 2001. Similar results were observed in pooled analysis also. The temperature has no positive correlation to disease severity whereas, relative humidity (0.792) rainfall (0.549) have shown highly significant and positive correlation with disease severity.
4.5 Biochemical changes in leaves during pathogenesis
4.5.1 Total sugar estimation
Total sugar contents (Table-13) were found to be increased through 10 days stage to 30 days of growth in healthy leaves of all three genotypes; however, the moderately susceptible genotype RMG-268 indicated the highest increase of 62.2% as compared to highly susceptible genotype K-851 (31.7%) and the moderately resistant genotype MUM-2 (40.8%). Upon inoculation, the total sugar content was found to decrease in all the genotypes at all stages except at 30 days after inoculation. In case of the susceptible genotype, K-851 sugar content was higher as compared to its healthy state. The overall sugar contents got increased in the diseased leaves in all three cases with progressive blight pathogenesis when studied through 10 to 30 days stage. However, the increases were more pronounced from 10 days to 20 days stage just like in case of healthy leaves. The susceptible genotype, K-851, the moderately susceptible genotype RMG-268 and the moderately resistant genotype MUM-2 reflected an increase of 96.7%, 83.8% and 68.8%, respectively from 10 days to 30 days of inoculation during the course of disease development. Table 13 Total sugar contents (mg/g dry wt) of mungbean leaves during leaf blight pathogenesis Inoculation Genotypes period K-851 (H. susceptible) RMG-268 (M. MUM-2 (M. (day) susceptible) Resistant) Healthy Inoculated Healthy Inoculated Healthy Inoculated 10 41.0 + 2.81 30.6 + 2.12 36.3 + 3.10 28.4 + 1.32 48.0 + 2.05 37.2 + 2.81
20 52.0 + 1.25 45.8 + 1.23 50.0 + 2.05 46.2 + 3.06 62.0 + 1.85 58.4 + 1.35
30 54.0 + 2.75 60.2 + 2.56 58.9 + 1.25 52.2 + 2.06 67.6 + 3.25 62.8 + 2.05
Values are the average of five replications readings (+ S.D)
4.5.1.1 Amino acid estimation
In general total amino acid content (Table-14) were observed to be increased in both healthy and infected leaves, in all the genotypes through differential stage of growth of the plants from 10 days to 30 days of the study. However, the trend of increase from 10 days to 20 days stage was much effective as compared to 20 days to 30 days stage. More pronounced profile of increase in the moderately resistant genotype MUM-2, both in healthy and inoculated leaves during the aforesaid period. In healthy leaves from 10 days to 30 days of stage of study, the highest increase of 58.3% was observed in case of moderately susceptible genotype RMG-268 as compared to 18.5% in case of highly susceptible genotype K-851 and 16.0% in case of the moderately resistant genotype MUM-2. The inoculated leaves presented a differential scenario of 22.8% increase in case of the moderately resistant genotype as compared to 7.9% and 18.3% of the highly susceptible and moderately susceptible genotype respectively. A significant increase of 34.4% in amino acid contents in the diseased leaves of the moderately resistant genotype MUM-2 from 10 days to 20 days stage spoke about significant metabolic consequences during course of leaf blight pathogensis of mungbean leaves. Table 14 Total amino acid contents (mg/g dry wt) of mungbean leaves during leaf blight pathogenesis Inoculation Genotypes period K-851 (H. susceptible) RMG-268 (M. MUM-2 (M. (day) susceptible) Resistant) Healthy Inoculated Healthy Inoculated Healthy Inoculated 10 27.5 + 1.32 31.5 + 0.99 21.6 + 0.89 30.6 + 0.79 32.3 + 1.21 34.6 + 1.27
20 30.6 + 0.91 36.5 + 1.26 30.8 + 1.81 38.5 + 1.26 41.6 + 0.67 47.4 + 2.04
30 32.6 + 1.21 34.0 + 1.05 34.2 + 2.05 36.2 + 0.66 37.5 + 1.05 42.5 + 1.08
Values are the average of five replicate readings (+ S.D.)
4.5.1.2 Protein estimation While going through the picture of protein profile, it got increased through 10 days to 30 days stage in case of both healthy and inoculated leaves in all the three genotypes but the intensity of increase was of higher value during 10 days to 20 days phase at one instance in case of the moderately resistant genotype MUM-2, where the increase was much higher during the later phase (Table-15). As a result of infection the highly susceptible genotype K-851 did not reflect much increase of 30% in protein content as compared to the 76.1% and 96.7% in case of moderately susceptible and the moderately resistant genotype respectively. An overall higher status of protein content was observed in case of moderately resistant genotype MUM-2 in healthy and inoculated leaves as compared to the highly susceptible matured genotypes. Table 15 Protein content of mungbean leaves during leaf blight pathogensis (mg /g fresh wt) Inoculation Genotypes period K-851 (H. susceptible) RMG-268 (M. MUM-2 (M. (day) susceptible) Resistant) Healthy Inoculated Healthy Inoculated Healthy Inoculated 10 19.0 + 1.25 26.0 + 1.99 21.8 + 1.01 23.0 + 0.77 22.7 + 0.79 24.0 + 0.85
20 25.0 + 1.07 31.0 + 2.01 28.7 + 0.99 36.5 + 1.08 29.7 + 1.92 38.5 + 1.02
30 30.6 + 2.01 33.8 + 1.98 35.0 + 1.23 40.5 + 2.06 37.0 + 0.73 47.2 + 2.35
Values are the average of five replicate readings (+ S.D.)
4.5.2 Estimation of oxidative enzymes
The enzymatic peroxidase activity pattern in mungbean genotypes is shown in Table-16. Peroxidase activity got increased through 10 days to 30 days stage of study, in general, in both healthy and diseased leaves in all the genotypes. Once again the comparative increases were more during first phase of 10 days to 20 days. Upon inoculation, the peroxidase activity was found to be increased in all cases of genotypes and pathogen interactions. However, at 20 days stage of determination the blight affected leaves delivered significantly higher values of activity and that too of greater extent in case of moderately resistant genotype MUM-2 (where it was from 14.0 to 23.5, 67.8% more). The differential nature of host pathogen interaction was quite clear in terms of elevated levels of peroxidase activity in terms of elevated levels of peroxidase activity in case of moderately resistant genotype over the moderately susceptible matured counterparts. Table 16 Enzymatic peroxidase activity (Units*/ mg protein) in mungbean leaves during leaf blight pathogensis Inoculation Genotypes period K-851 (H. susceptible) RMG-268 (M. MUM-2 (M. (day) susceptible) Resistant) Healthy Inoculated Healthy Inoculated Healthy Inoculated 10 8.2 + 0.28 10.5 + 0.42 9.8 + 0.13 12.7 + 0.22 12.1 + 0.09 16.2 + 0.19
20 11.8 + 0.35 14.6 + 0.41 12.8 + 0.24 14.8 + 0.38 14.0 + 0.23 23.5 + 0.65
30 12.8 + 0.45 14.2 + 0.25 13.6 + 0.32 15.8 + 0.60 13.2 + 0.17 20.2 + 0.32
Values are the average of five replicate readings (+ S.D.) * One unit of enzyme activity is a change of 0.01 per min in optical density
4.5.3 Effect of seed infection on seedling vigour When seeds were inoculated with M. phaseolina culture and tested for
seed germination immediately after inoculation, there was a sharp drop in seed
germination from 96% to 82%. The differences in root shoot length and the dry
weight of the seedling are also reduced significantly in inoculated seeds (Table-
23). With the passage of time during storage of inoculated seed, the seed
germination improved progressively to 94% after a storage of 12 months
indicating inactivation of fungus. Average root length, shoot length and dry
weight also increased progressively with the passage of time in the storage. Data
revealed that seed vigour is greatly reduced in seeds inoculated with M.
phaseolina (Plate-5).
Table 17 Effect of seed inoculation with M. phaseolina on seed germination of seed vigour of mungbean Storage Seed Root Shoot Seed vigour period germination length length Total Dry months (%) (cm) (cm) length of weight/ 10 seedling seedling (cm) (g) Uninoculated seed (check) 0 96 10.5 19.5 30.0 0.290 Inoculated seed (check) 0 82 6.1 11.2 17.3 0.218 2 83 6.6 11.4 18.0 0.227 4 85 7.5 13.1 20.6 0.241 6 85 7.9 14.6 22.5 0.256 8 89 8.4 15.9 24.3 0.274 10 91 9.1 16.8 25.9 0.277 12 94 9.8 18.2 28.0 0.289 Data based on four replications of 100 seed each
4.6 Management of the disease 4.6.1 Management through chemicals
4.6.1.1 Efficacy of different fungicides against M. phaseolina in vitro
Four fungicides at four concentration (250, 500, 1000 and 2000 ppm) were tested in vitro by poisoned food technique to see their efficacy against mycelial growth of M. phaseolina. All fungicides were found significantly superior over check in reducing the mycelial growth (Table-18) The data revealed that bavistin and benomyl inhibit 100 per cent growth of Macrophomina phaseolina at 500 ppm concentration following by thiram and captan. At 1000 ppm or above all fungicides completely inhibited the growth of fungus. At 250 ppm bavistin (97.9%) and benomyl (96.4%) inhibited growth and were better than captan and thiram.
4.6.1.2 Effect of fungicidial seed treatment, foliar spray and in combination with foliar spray
Though all the fungicides as seed treatments alone and in combination with foliar sprays of same fungicides reduced the incidence of seedling mortality and leaf blight severity (Table-19) but the best control of seedling mortality was obtained by treating the seed with bavistin (5.35%) followed by benlate (7.40%), thiram (10.65%) and captan (14.65%) against check (29.35%). The severity of leaf blight was best managed by seed treatment and its combination with foliar spray. The minimum disease severity was recorded in bavistin ST + FS (7.5%) followed by benlate ST + FS (11.75%) and thiram ST + FS (14.3%) against check (57.8%). Captain as seed treatment alone and its combination with foliar spray was the least effective. Thus, the fungicides found effective against seedling mortality were also effective against leaf blight when used as foliar spray to reduce foliage blight bavistin and benomyl was best in reducing foliage blight to 19.8 and 21.05% respectively but at par followed by thiram. Treatments significantly improved grain yield. Maximum yield was recorded in bavistin (ST + FS) followed by benlate (ST + FS), bavistin (FS) and bavistin (ST) as 562 kg/ha, 512 kg/ha, 486 kg/ha and 472 kg/ha, respectively.
4.6.2 Effect of plant extract as seed treatment on seedling mortality under pot condition
Data collected on seven plant extract used @ 50 per cent of stock solution and were tested as seed treatment for their effect in reducing the seedling mortality in pot condition are tabulated in (Table-20). The data revealed that all plant extract used as seed treatment effectively reduced the seedling mortality significantly over untreated check. The lowest seedling mortality (15.2%) and maximum control 68.53% was observed with neem leaf extract seed treatment followed by garlic bulb, datura, aak, tulsi, onion and bougainvillea controlling seed blight by 61.98, 60.12, 58.26, 55.7, 50.8, 40.9% respectively.
Table 20 Effect of plant extract as seed treatment on seedling mortality of mungbean under pot condition
Plant extract Seedling mortality Per cent control over (%)* check Aak (leaf extract) 20.2 (26.71) 58.26 (49.75) Bougainvillea (leaf extract) 28.6 (32.33) 40.90 (39.75) Datura (leaf extract) 19.3 (26.06) 60.12 (50.83) Garlic (bulb extract) 18.4 (25.40) 61.98 (51.93) Neem (leaf extract) 15.2 (22.95) 68.53 (55.87) Onion (bulb extract) 23.8 (29.20) 50.80 (45.46) Tulsi (leaf extract) 21.4 (27.69) 55.70 (48.27) Check 48.4 (44.08) - SEm+ 0.76 CD 5% 2.34 * Average of six replications (Figures in parentheses are angular transformed values)
4.6.2.1 Effect of plant extracts on seedling mortality and leaf blight severity
Under field conditions all the plant extracts used as seed treatment followed by foliar spray for their relative efficacy on seedling mortality and leaf blight severity were found significantly superior over check in reducing the disease in both the years (2000 and 2001) of testing under field condition (Table -21). The neem leaf extract were found superior in reducing seedling mortality (9.95%) followed by aak, garlic, tulsi, onion, datura and bougainvillea over check (43.25%). Neem extract were found most effective in controlling the leaf blight severity (20.5%) an increase in yield 5.53 q /ha. Similar trends were observed in seedling mortality, leaf blight severity and yield for other plant extracts.
4.6.3 Efficacy of bioagents
4.6.3.1 In vitro:
Relative efficacy of four antagonist bioagents in reducing growth of M. phaseolina were tested by dual culture method in PDA plates. The observations recorded regarding comparative zone of inhibition and sclerotial formation are given in Table –22. Trichoderma viride was found most effective in inhibiting the radial growth and sclerotial production of test fungus. The zone of inhibition for T. viride was 6.2 mm followed by T. harzianum, Gliocladium virens and B. substilis. The sclerotial production was minimum in T. viride followed by T. harzianum G. virens. Bacillus subtilis was least effective in checking the growth of fungus and sclerotial production. Table 22 Effect of biocontrol agents on inhibition of radial growth of M. phascolina (In vitro)
Antagonist * Zone of inhibition Number of sclerotia (mm)
Bacillus subtilis 2.7 19
Gliocladium virens 3.1 14
Trichoderma harzianum 5.4 9
T. viride 6.2 5
Check 0 Abundant
* Average of five replications
4.6.3.2 Efficacy of bio agents under field condition
Though, all the bioagents used as seed treatments alone and in combination with foliar sprays reduced the incidence of seedling mortality and disease severity significantly over check (Table -23), by a similar and greater extent yet best control of seedling mortality was obtained by seed treatment + foliar spray with T. viride (16.10%) followed by T. harzianum, by seed treatment (22.45%), G. virens (ST + FS), B. subtilis (ST + FS). The severity of leaf blight was best managed by seed treatment followed by foliar spray (ST + FS) of T. viride (13.90%), followed by T. harzianum (18.10%), G. virens (18.10%) and B. subtilis (23.75%) over check (56.20%). The disease severity was best controlled by ST+ FS followed by FS and ST. The grain yield was also influenced and the similar trends were observed as for disease management. Maximum yield was recorded in ST + FS of T. virens (495 kg/ha) and the minimum yield observed when only foliar spray applied with B. subtilis (301 kg/ha) as compared to check (259 kg/ha).
4.6.4 Effect of dates of sowing
Results of different sowing dates on the incidence of seedling mortality and leaf blight severity during 2000 and 2001 are presented in Table -24. The crop sown in sick plot on 14th July recorded minimum seedling mortality (23.2%) and leaf blight severity (35.25%) followed by sowing in 13th August and 29th July. The yield was highest in 14th July sown crop (375 kg/ha) as compared to other dates. First sowing date of 14th July is significantly superior in getting highest crop yield inspite of seedling mortality. Minimum yield were observed (274 kg/ha) in the crop shown on 13th August whereas the maximum seedling mortality (34.35%) and diseases severity (42.39%) was observed when crop was shown on 29th July.
4.6.5 Host resistance
On the basis of leaf blight severity, the mungbean varieties/genotype were categorized under artificial soil and foliar inoculation conditions as highly resistant, moderately resistant, moderately susceptible, susceptible and highly susceptible. Evaluation of 40 genotypes of mungbean in field revealed that none of the line was highly resistant in both years under study (Table -25). However, 6 genotypes were categorized as resistant, while 9 genotypes were categorized as moderately resistant, 13 as moderately susceptible, 8 as susceptible and 4 genotypes categorized highly susceptible.
Table 11 Survival of sclerotia of Macrophomina phaseolina in soil buried at different depth Period of Soil depth survival 0 cm 5 cm 10 cm Mean (in months) % Germination 1 80.2 74.6 72.2 75.66 (63.58) (59.74) (58.18) (60.40) 2 79.3 72.0 69.8 73.70 (62.94) (58.05) (56.66) (59.15) 3 76.9 71.2 68.6 72.23 (61.27) (57.54) (55.92) (58.18) 4 74.6 69.4 67.3 70.43 (59.74) (56.42) (55.12) (57.04) 5 74.0 68.3 66.2 69.50 (59.34) (55.73) (54.45) (56.48) 6 73.5 65.3 63.4 67.40 (58.02) (53.91) (52.77) (55.18) 7 72.6 64.1 62.6 66.43 (58.44) (53.19) (52.30) (54.57) 8 72.0 61.7 58.9 64.20 (58.05) (51.77) (50.13) (53.25) 9 71.3 60.2 57.4 62.96 (57.61) (50.89) (49.26) (52.48) 10 70.5 58.4 52.6 60.50 (57.10) (49.84) (46.49) (51.06) 11 70.0 57.6 50.3 59.30 (56.79) (49.37) (45.17) (50.36) 12 68.6 56.3 47.9 57.60 (55.92) (48.62) (43.80) (49.37) Mean 73.62 64.92 61.43 66.65 (59.08) (53.67) (51.53) (54.70) SEm+ CD 5% Period 0.66 1.85 Depth 0.33 0.92 P x D 1.14 NS * Mean of four replications each of 100 sclerotia (Figure in parentheses are angular transformed values) Table 1 Per cent disease incidence of leaf blight of mungbean caused by M. phaseolina observed during survey in zone II A and III A of Rajasthan Agroclimatic District Tehsil Per cent disease incidence zone Kharif Kharif Mean 2000 2001 II A Churu Churu 16.90 18.10 17.50
Ratangarh 18.30 19.70 19.00
Average 17.60 18.90 18.25
Jhunjhunu Jhunjhunu 22.90 24.50 23.70
Nawalgarh 20.70 21.90 21.30
Average 21.80 23.20 22.50
Sikar Paprali 17.30 18.70 18.00
Fatehpur 14.50 17.10 15.80
Average 15.90 17.90 16.90
III A Jaipur Dudu 26.80 25.20 26.00 Sanganer 20.40 22.60 21.50
Average 23.60 23.90 23.75
Tonk Malpura 14.60 16.00 15.30 Tonk 11.40 14.60 13.00
Average 13.00 15.30 14.15
Mean 18.38 19.84 19.11
Table 2 Morphological and cultural characters of five isolate groups of Macrophomina phaseolina collected from zone II A and III A
Isolate Size of Shape of Colony characters on PDA Sclerotial group sclerotia sclerotia formation (m) on 8th day MB-1 80-160 x 60- Oval Mycelium coarse and appressed, gray to ++++ 120 dark gray MB-2 72-218 x 50 Irregular Mycelium fine and profuse, dull white +++ - 122 turn pale gray MB-3 78-192 x 72- Irregular Mycelium coarse and completely +++ 169 appressed, gray turning pale gray MB-4 87-142 x 56- Spherical Mycelium coarse and completely ++++ 137 appressed, pale gray MB-5 73-157 x 40- Oval Mycelium profuse aerial with fine texture, +++ 113 smoke gray
* ++++ Excellent Size of sclerotia – mean of 25 Sclerotia measured +++ Good ++ Fair
Table 5 Effectiveness of different inoculation techniques of Macrophomina phaseolina on seedling emergence, seedling mortality and leaf blight severity of mungbean Techniqu Germination (%) Seedling mortality (%) Disease severity (%) e * 2000 2001 Mean 2000 2001 Mean 2000 200 Mean 1 Seed 85.9 84.3 85.1 27.50 29.00 28.25 40.20 38. 39.3 inoculatio (67.94) (66.66) (67.29 (31.63) (32.58 (32.08) (39.3 40 (38.82) n ) ) 5) (38. 29) Soil 83.20 81.70 81.95 34.60 35.10 34.85 49.50 52. 50.85 inoculatio (65.80) (64.67) (64.82 (36.03) (36.33 (36.15) (44.7 20 (45.46) n ) ) 1) (46. 26) Foliar 96.40 96.00 96.2 0.00 0.00 0.00 65.00 62. 63.70 spray (79.00) (78.46) (78.76 (0.00) (0.00) (0.00) (53.7 40 (52.95) ) 3) (52. 18) Check 97.00 96.60 96.8 0.00 0.00 0.00 0.00 0.0 0.00 (healthy (80.02) (79.37) (79.69 (0.00) (0.00) (0.00) (0.00 0 (0.00) and ) ) (0.0 sterilized 0) seed) SEm+ 1.80 1.72 1.24 0.88 0.83 0.60 1.50 1.3 1.01 7 CD 5% 5.31 5.08 3.52 2.59 2.45 1.71 4.41 4.0 2.87 4 * Average of 6 replications (Figures in parentheses are angular transformed values)
Table 12 Simple correlation coefficient, regression equation and coefficient of determination between different epidemiological factors and disease incidence during 2000 and 2001
Y Correlation Regression equation Coefficient of coefficient determination (%) 2 2001 Pool 2000 2001 2000 2001 Pooled 0 ed 0 0
(Y) - -0.166 -0.419 Y = 10.208 + 313.608 X1 Y = -8.062 + 17.38 2.75 17.55
Disease 0 256.288 X1 incidence . x 4 Temperat 1 ure (X1) 7
Disease 0 0.906** 0.792* Y = 2.259 - 134.88 X2 Y = 1.185 – 71.91 82.08 62.72 incidence . * 53.946 X2 x relative 8 humidity 4
(X2) 8 * *
Disease 0 0.633 0.459* Y = 0.860 + 12.495 X3 Y = 0.292 + 26.01 40.06 30.14 incidence . * 20.972 X3 x 5 Rainfall 1
(X3) 0 ** Significant at the 0.01 level (2 tail) * Significant at the 0.05 level (2 tailed)
5. DISCUSSION
In spite of adopting best of technology full productivity of pulses have not been exploited. This is mainly due to higher insect and disease incidence in properly fertilized crop. Mungbean occupies most prominent place among pulse crops of north west India. Leaf blight caused by Macrophomina phaseolina is widely prevalent disease causing seedling mortality and leaf blight of mungbean in tropical parts of the India. It is a polyphagous fungi having a wide host range covering around 290 plant species (Dhingra and Sinclair, 1977). Its vigorous ability to thrive in soil, besides having lethal pathogenic potential on unlimited number of hosts causing different types of disease symptoms is mainly responsible in soils having sandy to sandy loam texture and accompanied by moderate temperature of 30 0C and less soil moisture as prevalent in zone II A zone III A of Rajasthan. The pathogen causes seedling mortality as well as leaf blight. The leaf blight phase makes its appearance when the plants one 4-6 weeks old. Initially starting from a circular to irregular brown lesions on or near the margin, gradually covering almost entire foliage, giving blighted appearance. Disease starts appearing from end of July in this part of Rajasthan and is almost endemic causing serve economic losses. Kaushik et al. (1987) have reported 2.2 to 15.7 per cent infection of leaf blight causing 10.8 per cent reduction in grain yield. Lodha et al. (1986) reported 31.1 per cent disease severity in guar, 64.5 per cent in cowpea and 43.89 per cent in sesame. To know the prevelance of this disease a survey was under taken in mungbean growing areas covering 5 districts of Rajasthan namely Churu, Jhunjhunu, Sikar, Jaipur and Tonk belonging to agroclimatic zone II A and III A of Rajasthan state. Disease was prevalent in all the districts, severity ranging from 11.4-26.8 per cent and 14.6-25.2 per cent in the year 2000-2001 and 2001- 2002, respectively. Highest disease incidence was observed in Dudu tehsil of Jaipur district, corroborating the findings of Philip et al. (1969) and Grover and Sakhuja (1981) reporting variable incidence of leaf blight in different areas. Isolations from diseased plants collected from ten tehsils of five above mentioned districts revealed presence of dull white to dark gray, septate mycelium having profuse aerial to appressed growth forming irregular, oval to spherical sclerotia measuring from 72-218 m x 40–169 m forming pycnidia and pycnidiospores on rooted seed and on infected stem at collar region. All these morphological and cultural characters resesmbles with the characters of Macrophomina phaseolina originally described by Shaw (1912) from India and Butler (1918), Briton-Jones (1925) and Ashby (1927). Morphological and cultural variability of ten isolates of M. phaseolina exhibited marked differences in colony colour, colony shape, scleriotial size and sclerotial production. Isolates were grouped in to five groups (MB-1, MB-2, MB- 3, MB-4 and MB-5). Group MB-4 was most prevalent isolated from Piprali, Fatehpur, Dudu and Sanganer and was used in all the studies. Cultural and morphological variations in different crop isolates, isolates from same crop, different plant parts were also observed by Hooda and Grover, 1988), Devi and Singh (1998) and Bansal et al. (1990).. Three different inoculation techniques were tested for creating maximum disease. Plants inoculated 15 days after emergence with three days old culture @ 1 x 103 propagules/ml as foliar spray till run-off was found most suitable technique to create foliar blight disease and similar results were made by Grover and Sakhuja (1981) and Hooda (1988). Seed inoculations was better than soil inoculation in creating seedling blight. Sorghum seeds found superior for mass multiplication of inoculum both in the form of mycelial growth and formation of sclerotia. Mayee and Garud (1978) also reported the use of sorghum grains as a medium for mass culturing of M. phaseolona. Dandnaik et al. (1986) and Shekhawat (2002) used sorghum, bajra, mung, urd, wheat, maize and rice for mass culturing of M. phaseolina and reported good mycelial growth and sclerotial formation of the pathogen on sterilized grains of mungbean and sorghum. Age of culture influenced seedling mortality as well as leaf blight incidence. Present findings suggest that 5 days old inoculum led sorghum grain proved capable of causing high seedling mortality which decline with the increasing age of culture. The 3 day old vegetatively active fungal mycelium was capable of creating maximum foliage blight, which reduced with increased age of culture. Ayanru and Green (1978), Grover and Sakhuja (1981) and Hooda and Grover (1982) reported more disease similarly found in the present findings. Seedlings after inoculation exposed to moist chamber with high humidity for 48 hrs produced maximum leaf blight. Our results are in confirmity to Grover and Sakhuja (1981), Hooda and Grover (1990) who reported 48 hrs duration of 70 per cent RH suitable for collar infection in mungbean. Sandhu and Singh (1998) found a period of 96 hrs duration of humidity caused maximum seedling mortality at 70 per cent RH in cowpea. Studies made for the survival of the pathogen in/on seed and soil have revealed that M. phaseolina survived on seed, infection ranging from 10-24%, 5- 11% in blotter and agar plate method, respectively, indicating that pathogen is carried by seed externally as well as internally. The results are in confirmity with Nath et al. (1970), Singh and Chohan (1973) Kaushik et al. (1987), Bhatia et al. (1998), Sharma and Singh (2000) and Shekhawat (2002). Studies conducted to reveal the presence of M. phaseolina in/or seed parts i.e. seed coat, cotyledon and embryonal axis, revealed that 12.8 per cent seed coat, 6.7 per cent cotyledons and 2 per cent embryonal axis harboured fungal mycelium in the seed. Seed may harbour fungus on one site or more than one site in the same seed. Results are in support of findings of Kaushik et al. (1987), Sharma and Singh (2000) on mungbean. Ushamalini et al. (1998) working with cowpea reported M. phaseolina infection upto 13.3%, 8.33% and 1.66% in seed coat, cotyledon and embroyo, respectively in discoloured seeds. Sinha and Khare (1977), Sandhu and Singh (1998) reporting results of component planting showed the presence of M. phaseolina in seed coat, cotyledons and radicle plumule. Infected seeds along with healthy seed when planted in field may be a source of inoculum to soil. Sclerotia of M. phaseolina survive on seed for more than 12 months under laboratory storage conditions but there is significant progressive decline in their capacity to cause seedling infections. These findings are in aggrement with Sandhu and Singh (1998) reporting 12 months survival of M. phaseolina on cowpea seeds. Dhar and Sarbhoy (1989) observed that the viability of sclerotia depends on range of temperature during storage. The optimum range of 20-30 0C seems most favourable. There was gradual reduction in viability at temperatures below 20 0C and above 30 0C. Studies were carried out in vitro for survival of scleroria in soil. Fungus sclerotia were buried at different depth of soil in pots. The results revealed that sclerotia could remain viable for 12 months in a soil upto depth of 10 cm. Sclerotia survive better at surface level and this may be due to less availability of moisture at soil surface than at 10 cm depth. The microbes present in the soil may also either parasites sclerotium or create antibiosis hampering sclerotial germination indicating less survival at greater depths. The findings are in aggrement with Muthukrishnan et al. (1995) who reported 50 per cent reduction in the viability of selerotia after 24 months of burial upto 10 to 15 cm depth. Mishra and Bais (1985) reported that sclerotial may remain viable for 30 months but higher viability was found at the depth of 0 and 5 cm for six months. Singh (2002) also reported that fungus survive for 12 moths in soil upto 10 cm depth in Macrophomina guar interaction. They observed maximum viability at 0 and 5 cm for 6 months. The relationship of various environmental factors viz. temperature, relative humidity and rainfall to disease severity revealed that the mean temperature during both the years had no significant effect on severity of leaf blight as there was not much variations in temperature for most period mean temperature 28.2 to 30 0C . This was also evident from infections observed during both years viz., 2000-01 under study. Grover and Sakhuja (1981) who observed 30 – 35 0C as the optimum temperature for mungbean leaf blight. Gupta and Chohan (1970) reported that the damage to peanut plant occurred by M. phaseolina between 21 0 – 30 0C. Ratnoo et al. (1997) also reported that Ashy grey stem blight in cowpea caused by M. phaseolina was favoured by higher temp of 30-35 0C. Singh (1998) reported that charcoal rot of sorghum appeared at 28-35 0C temperature. Rainfall has a significant positive correlation to leaf blight severity with correlation coefficient being 0.459 and coefficient of determination being 34 per cent. Relative humidity for both the years has a significant and positive correlation to leaf blight severity with correlation coefficient being 0.792 and coefficient of determination being 62.72 per cent. Heavy rainfall during early period of susceptible crop growth stage favours the germination of pathogen and subsequent infection. The rain splashes also disseminated the propagules from soil, infected part or plant to adjacent healthy plant and parts. The results are in confirmation to Grover and Sakhuja (1981) for mungbean, Kushi (1977) for sesamum, Suhag and Rana (1984) for ginger and Muthukrishnan et al. (1995) for urdbean. Sharma and Tripathi (2001) also reported similar observations on web blight of urdbean. The increased sugar content of the healthy leaves of all three genotypes through the period of observation from 10th to 30th day indicated an accelerated carbohydrate anabolism corresponding to growth and development of the plant. The reduced total sugar content upon inoculation, in general, indicated higher catabolic rate of the carbohydrates as a result of process of blight pathogenesis of the mungbean leaves. Parallel observation of reduction of sugar contents in mungbean leaves during cercospora leaf spot pathogenesis by Sindhan et al. (1999) and earlier in case of leaf spot pathogenesis of groundnut (Sindhan and Parashar, 1996) deserve significant mention in context to the present observations such a post, infectional decrease in carbohydrates has been attributed to rapid hydrolysis of sugars under act of pathogenesis (Jaypal and Mahadevan, 1968). The significant decrease during the early course of blight pathogenesis of mungbean leaves is a characteristic observations of the present study. Differential variation of the leaf sugar content, decrease at various stages of observation in relation to susceptibility index, indicated the highest rate of decrease in case of the highly susceptible cultivar K-851, followed by the moderately susceptible and moderately resistant. The higher decrease in sugars in the susceptible plant pathogen interactions have been well document in various studies. On cotton (Bedi et al., 1977) on chickpea (Mitter et al., 1997) and mungbean (Sindhan et al., 1999). The increased amino acid content observed in both healthy and inoculated leaves in all three cultivars and pathogen interactions through 10 day to 30 day stage (with more effective trend from 10 to 20 day) might be considered as a characteristics feature of plant morphogenesis. The inoculated leaves presented a differential scenario of amino acid increase (the highest in the highly resistant genotype MUM-2). Such an increase of amino acids and phenolics has been reported in Macrophomina phaseolina infected limabean leaves (Jadeja and Patel, 1989). A general decrease of sugar and an increase of nitrogen in diseased cotton plants upon Helminthosporium infection as reported by Bedi et al., 1977; once again may be considered as a supportive of the present observations while focusing on amino acid-nitrogen link in context to plant metabolism. An increase of 34.4 % in amino acid content in the diseased leaves of the moderately resistant genotype MUM-2 as compared to the other two comparatively susceptible mungbean genotype during the present study speaks well about significant consequences during course of leaf blight pathogenesis. Singh et al. (1998) while working on rust infected safflower leaves have reported higher amounts of amino acids chlorophyll and orthodihydroxy phenols in tolerant plants. The increased protein profile through 10 days to 30 days stage in case of both healthy and inoculated leaves in all three genotypes recorded in this study, is well in accordance to the observation of Uritani (1971) who has indicated increased level of both nitrogen and protein content of the host-pathogen complex. Similar observation in increased protein synthesis in incompatible interactions in certain host parasite system has been considered to reflect accelerated biosynthesis of enzymes and other proteins involved in plant defense (Heath 1979, Vance and Sherwood, 1976 and Saini, 1989). As a result of macrophomina infection of mungbean leaves the highest increase of 96.7% through 10 to 30 day stage by the highly resistant genotype MUM-2 may rightly be taken as a genetic differential by relation to the present host pathogen interaction. Such an increase in protein synthesis in incompatible interactions have been reported by Yamamoto et al. (1976), and Tani and Yamamoto (1979). Further, such type of increases considered due to accelerated biosynthesis of enzymes and other proteins involved in plant defense systems can be highlighted (Vance and Sherwood, 1976). The elevated levels of peroxidase activity through 10 to 30 days stage of study, in general, in both healthy and diseased leaves reflected dynamic metabolism during first phase of not only growth and pathogenesis. The differential mature of variable host-pathogen interactions reflected during the present study are notable in relation to host genetics and metoblism as a consequence of pathogensis impact. Increased level of proteins, polyphenol- oxidases and peroxidase concomitant with the development of acquired resistance of soybean leaves have been reported by Batra and Kuhn (1975). Significantly higher values of peroxidase activity by case of the highly resistant cultivar MUM-2 at 20 day stage might be taken as an indicative of the result rather than the cause of resistance as reported by Bell (1981) while making an attempt to correlate peroxidase or polyphenoloxidase with resistance. Higher levels of activity of peroxidase upon leaf blight inoculation have been reported by Chandra and Tyagi (1992) both in resistant and susceptible cultivars but more pronounced. The resistant genotype Raised levels of peroxidase activity by the host wheat seedlings against different pathotypes of the brown rust pathogen have also been noted (Saini et al., 1988) furnishing the same genetics of the host to differential virulence of the rust pathotypes. Seed inoculations with M. phaseolinai reduced seed germination. The seed vigour is also reduced significantly. Storage of such inoculated seed for 12 months showed regain germination and seed vigour probably due to inactivation/ death of mycelial propagules on seed coat. Initial poor performance of inoculated seed may be either due to seed rot or pre emergence damping off and delayed germination or emergence. The observation of the present studies on reduction in germination and seed vigour of diseased seed are in confirmation to the studies made earlier by Gupta (1984), Gupta and Cheema (1990), Gupta et al. (1993). Management of a pathogen which is seed borne, soil borne and capable of surviving for long periods in adverse condition is very difficult. With the development of newer organochemicals, there is a hope that effective chemical can be identified which when used in small concentrations can reduce the pathogen. Four fungicides namely bavistin, benomyl, captan and thiram which have been found most useful in reducing pathogen infestion in seed, plant and soil by various worker in literature and recommendations of research scientists was tested here. These were tested in vitro by food poison technique at four concentrations. All fungicides were capable of inhibiting complete growth of Macrophomina phaseolina when used at 1000 and 2000 ppm. At lower concentration of 250 ppm bavistin and benomyl were at par and superior to captan and thiram inhibiting growth of fungus more than 95 %. At 500 ppm there are minor differences but trend remain the same. Other workers Menten et al. (1976), Taneja and Grover (1982), Jadeja and Patel (1988) and Giri and Peshney (1993) also observed similar trends in their studies against this pathogen from different crop hosts.. Pot and field studies have revealed that all fungicides used as seed treatment, foliar spray and in combination reduced seedling blight and leaf blight severity and increased grain yield. Bavistin used as seed treatment followed by foliar spray reduced maximum seedling mortality and leaf blight and increased maximum yield, followed by benomyl. Results obtained with bavistin are inconfirmity with findings of Thakur et al. (1991) who reported that best results were obtained when the seed treatment was followed by the foliar spray of the same. Kotasthane and Agarwal (1978), Kotasthane and Gupta (1983), Tiwari and Kotasthane (1986), Singh and Lodha (1986), Ramadoss and Sivaprakasam (1987), Hooda et al. (1988) and Singh et al. (1990) also reported effectiveness of bavistin, benomyl and captan in reducing seedling mortality, foliage blight caused by Macrophomina phaseolina on mungbean, cowpea, urdbean and sunflower. Exclusive reliance on fungicides for the control of diseases of various crops has resulted in residue problems and environmental hazards. Therefore, in recent years, efforts are being diverted to employ higher plants/botanicals and their derivatives as a tool for integrated disease management, because they do not cause bio-accumulation, bio-magnifications and environmental pollution. Plant extracts of five commonly found plants and bulbs of garlic and onion were tested for their role in reducing seedling mortality and leaf blight in pot and field conditions. Neem extract was most effective in reducing seedling mortality and leaf blight and increased yield followed by garlic extract. The results obtained in these investigations are in aggrement with Singh et al. (1980) who reported that plant extract of garlic and neem reduced the growth of some soil borne pathogens including Macrophomina phaseolina. Lakshmanan and Mohan (1989) reported that plant extracts of Allium sativum and A. indica significantly reduced the seedling mortality caused by Rhizoctonia solani in green house condition, which again supported the present investigations. As clear from the literature both beneficial (bioagents) and harmful (pathogenic) fungi co-exist in the rhizosphere of agricultural crops with a competitive tendency between each other. The effect on the host depend on the marking of the one over the other. In the present study four bioagents were evaluated for their comparative efficacy in reducing M. phaseolina growth and disease causing ability in in vitro and in vitro. Trichoderma viride was most effective in retarding the growth of Macrophomina pheseolina as anumerated by maximum zone of inhibition followed by T. harzianum. Gliocladium virens and Bacillus subtilis were least effective. All bioagents as seed treatment, foliar spray alone and in combination with foliar spray reduced the incidence of seedling mortality and leaf blight severity to a varying degree. Yet best control of seedling mortality and foliage blight was obtained by T. harzianum (ST + FS). The present finding lend support to observations of Harder et al. (1979), Papavizas and Levis (1981), Kehri et al. (1991), Majumdar et al. (1996), Ushamalini et al. (1997) and Mathew and Gupta (1998). However the information about the management of foliar blight by foliar application of T. harzianum is lacking in literature and con not be compared with. Disease is the manifestation of the interaction between pathogen, susceptible host and environment. Susceptibility of most host very from seedling stage to adult plant stage. Seedlings of mungbean are most susceptible at 4-8 weeks. Severe disease will appear if pathogen is present and environment is congenial. Changes in the date of sowing influenced the interaction. In the present study three date of sowing was evaluated and it was observed that seedling mortality was reduced by sowing mungbean crop on 14th July and resulting increased yield as compared with crop sown on 20th July and 13 August. The maximum leaf blight severity was observed when crop was sown on 29th July. Similar findings was also reported by Singh and Gupta (1994), thus supporting the present investigations. The genotype screening trails undertaken under field conditions revealed that non of the 40 genotypes tested under artificial inoculated conditions showed immune reactions to leaf blight pathogen. Our result revealed that the genotypes namely RMG-754, RMG-843, RMG-852, RMG-926, RMG-938, RMG-951 showed resistant reaction. Rest others were categorized as moderately resistant, moderately susceptible, susceptible and highly susceptible. Zote et al. (1983) tested 14 cultivar of mungbean and observed that no cultivar was immune. Deshmukh (1991) observed resistant in BCG-1 out of 30 cultivars tested.
6. SUMMARY
Mungbean is an important source of protein in vegetarian diet of Indian populace. The crop is grown in kharif and 3 jaid seasons in semi arid and arid regions of India. The crop suffers from many disease, among them, leaf blight caused by Macrophomina phaseolina has become a major problem in mungbean growing areas of zone II A and III A. High relative humidity and rainfall during early cropping season favours the development of disease. Surrey for disease incidence was under taken in five districts of II A and III A agroclimatic zones of Rajasthan and the disease was present in mild to sever from infecting plants, from 14.6 to 25.2%, affecting leaves, stems, pods and root portion. The symptoms appeared as circular to irregular brown lesions on or near the margin of leaf, gradually covering almost entire foliage, giving blighted appearance.
Isolations from infected leaves and stem yielded fungal growth with abundant sclerotial formation and resembled with Macrophomina phaseolina. Identification of pathogen was confirmed on the basis of morphological, cultural and sclerotial characters as Macrophomina phaseolina. Besides mungbean, the fungus Macrophomina phaseolina has been observed to be pathogenic on 290 plant species. Most kharif crops like guar, kidneybean, sesamum, sorghum, groundnut, soybean, urdbean, mothbean, cowpea are favoured host of the pathogen in this state. Isolations made from all collections from surveyed field was grouped into 5 groups, based on variations in the cultural characters. They were identification as isolate groups MB-1, MB-2, MB-3, MB-4 and MB-5, isolate group MB-4 was observed to be most commonly occurring and was highly virulent incausing maximum leaf blight severity. Different inoculation techniques were tested, for creating maximum disease. Foliar inoculation technique was found most effective in causing leaf blight. Out of five grain medium tested for rapid multiplication of fungus, sterilized sorghum grain medium proved superior for mycelial growth and abundant sclerotial formation, and was used for mass multiplication of the pathogen, used for creation of soil sick condition for various tests of this study. Five days old inoculum was found most effective in causing seedling mortality and 3 days old inoculum on 21 day old seedling for leaf blight. Exposure to 100 per cent relative humidity for 96 hrs favoured disease development. Blotter test proved most efficient in detecting the pathogen in/on seed. The pathogen was found both externally and internally seed borne, mostly in seed coat. The pathogen survived in/on seeds, stored at room temperature for 12 months. In potted soil sclerotia remained viable for 12 months upto the soil depth of 10 cm. Maximum viability was observed when sclerotia was present on soil surface. Total sugar, amino acid and protein content alongwith peroxidase activity got increased both in healthy and M. phaseolina inoculated leaves of three mungbean genotypes having differential susceptibility index (K-851, highly susceptible, RMG-268, M. susceptible, and MUM-2 moderately resistant), through 10 days to 30 days of study. The variations of susceptibility index of different genotypes was quite evident in terms of altered metabolism of these biochemical constituents as a reflection of the host genetics. The seed germination and seed vigour is greatly reduced due to seed infection of M. phaseolina. However, with passage of time in store leaf blight severity revealed significantly and positive correlation with rainfall and relative humidity. The fungicide bavistin, benomyl were found most effective in inhibiting mycelial growth and sclerotial formation. Field studies also exhibited that seed treatment followed by foliar spray with bavistin and benormyl controlled seedling mortality, leaf blight severity and increased yield. Out of seven plant extract used as seed treatment followed by foliar spray with neem leaf garlic leave extract were found effective in checking seedling mortality and leaf blight severity. Out of four bioagents tested in vitro Trichoderma viride produced maximum zone of inhibition followed by T. harzianum. In vitro testing of different antagonists as seed treatment followed by foliar spray also revealed that T. viride was most effective in controlling seedling mortality and leaf blight severity. Late showing in the month of July in 29th July increased seedling mortality and leaf blight severity. Forty varieties/genotypes were screened under artificial inoculations, none of them showed higher resistant against the pathogen. The genotypes RMG-754, RMG-852, RMG-926, RMG-938 and RMG-951 showed resistance. The varieties/ genotypes K-851, RMG-965, RMG-968 and RMG-970 were found highly susceptible.
Epidemiology and management of leaf blight of mungbean [Vigna radiata (L.) Wilczek.] caused by Macrophomina phaseolina (Tassi) Goid.
Suraj Mal Mehta* Dr. J.P. Goyal** (Research scholar) (Major Advisor)
ABSTRACT Leaf blight of mungbean caused by Macrophomina phaseolina (Tassi) Goid. has emerged as a serious problem to the growers of mungbean in zone II A and III A of Rajasthan for the last few years. Under natural condition disease incidence ranged from 14.15 to 25.2 % during survey in the year 2000 and 2001. The disease appeared initially as brown to reddish brown resious which enlarge and coalesce. Under hot and humid condition the entire plant blighted with sclerotial bodies on stem and leaves. Pycnidia were also observed on rotted seed and on infected stem at collar region. Sorghum grains were found most suitable for raising inoculum. For successful inoculation the foliar spray with three days old culture was found most effective for leaf blight development. The optimum mean temperature 28.2-30 0C favoured the disease development. The optimum relative humidity range of 45.7-77.5 per cent, and rainfall had positive role in foliage blight severity. The pathogen was found both externally and internally seed borne. The fungus survive in seed upto 12 months. The sclerotia were found to be the primary source of inoculum as they could survive for 12 months upto the soil depth of 10 cm in potted soil. Fungus infestation of seed reduced seed germination and seed vigour. Total sugars, amino acids, protein along with peroxidase increased both in healthy and inoculated leaves having differential susceptibility index. The similar trend was observed for highly susceptible, moderately susceptible and moderately resistant varieties. The fungicides, bavistin and benlate were found most effective in in vitro test. In field bavistin as seed treatment and foliar spray was found superior followed by benlate as ST+FS in controlling seedling mortality and leaf blight. There was significant increase in yield over check in treated plots. Neem leaves and garlic cloves extract were found most promising in checking the seedling mortality and foliage blight. Trichoderma viride produced maximum zone of inhibition against M. phaseolina in in vitro, T. viride as seed treatment and foliar spray found most effective in checking seedling mortality and leaf blight. Disease severity was found maximum when sowing of the crop was done on 29th July. Out of 40 varieties/ genotypes screened under artificial inoculation conditions, none of them was found highly resistant. * Ph. D. student, Department of Plant Pathology, (Rajasthan Agricultural University, Bikaner), S.K.N. College of Agriculture, Jobner. ** Thesis submitted in partial fulfilment of the requirement for the degree, Doctor of Philosophy in faculty of Agriculture in the subject of Plant Pathology under the supervision of Dr. J.P. Goyal, Ex. Professor and Head, Department of Plant Pathology, (Rajasthan Agricultural University, Bikaner), S.K.N. College of Agriculture, Jobner.
APPENDIX-I
Analysis of variance for table-5
Source of d.f. Mean sum of square
variation Germination (%) Seedling mortality (%) Disease severity (%)
Techniques 3 325.16** 355.91 2308.29** 2388.36** 3375.6** 3310.2**
Error 20 19.41 17.82 4.61 4.15 13.4 11.2
** Significant at 1 per cent level of significance
APPENDIX-II
Pooled analysis of variance for table-5
Source of d.f. Mean sum of square variation Germination Seedling Disease seveity (%) mortality (%) (%) Years 1 9.72 1.17 1.84
Techniques 3 641.06** 4688.46** 6677.15
Yrs x Tech. 3 40.01* 8.17 8.62
Error 40 18.62 4.38 12.33
* Significant at 5 per cent level of significance ** Significant at 1 per cent level of significance
APPENDIX-III
Analysis of variance for table-6
Mean sum of square
Source of d.f. variation Soil application seedling Foliar spray disease severity mortality (%) (%) Age 7 1003.73** 1151.76**
Error 32 4.18 6.62
** Significant at 1 per cent level of significance
APPENDIX-IV
Analysis of variance for table-7
Source of D.f. Mean sum of square variation Period 3 282.95**
Error 20 13.94
** Significant at 1 per cent level of significance
APPENDIX-V
Analysis of variance for table-10
Source of D.f. Mean sum of square variation Period 3 110.18** Error 20 1.78
** Significant at 1 per cent level of significance
APPENDIX-VI
Analysis of variance for table-11
Source of D.f. Mean sum of square variation Period 11 153.51**
Soil depth 2 726.80**
Period x Depth 22 15.24**
Error 108 5.25
** Significant at 1 per cent level of significance
APPENDIX-VII
Analysis of variance for table-17
Source of variation D.f. Mean sum of square
Fungicides 4 21304.96**
Concentration 3 92.71**
Fungicides x concentration 12 790.55**
Error 60 16.61
** Significant at 1 per cent level of significance
APPENDIX-VIII
Analysis of variance for table-18
Source of d.f. Mean sum of square variation Mortality (%) Disease severity (%) Yield (kg/ha) Replication 3 1.07 1.03 5.07 4.30 1297 1378
Fungicides 12 264.22** 242.10** 316.93** 272.12** 27157** 32081**
Error 36 2.23 1.88 4.34 3.92 982 1057
** Significant at 1 per cent level of significance
APPENDIX-IX
Pooled analysis of variance for table-18
Mean sum of square d.f. Mortality (%) Disease severity (%) Yield (kg/ha) Source of variation
Year 1 1.258 0.040 4929
Rep/year 6 1.047 4.701 1337
Fungicides 12 505.014** 584.053** 58303**
Yrs x Fungi. 12 1.305 5.003 936
Error 72 2.054 4.131 1020
** Significant at 1 per cent level of significance
APPENDIX-X
Analysis of variance for table-19
Source of variation D.f. Mean sum of square
Plant extract 7 259.69**
Error 40 2.31
** Significant at 1 per cent level of significance
APPENDIX-XI
Analysis of variance for table-20
Mean sum of square Seedling mortality Disease severity (%) Yield (kg/ha) Source of d.f. (%) variation 2000 2001 2000 2001 2000 2001
Replication 3 2.618 3.197 4.872 3.944 2291 2514
Extract 7 183.183** 228.016** 119.401 113.789** 29065** 30795**
Error 21 1.921 2.468 3.385 2.909 1230 1292
** Significant at 1 per cent level of significance
APPENDIX-XII
Pooled analysis of variance for table-20
Source of d.f. Mean sum of square variation Seedling Disease severity Yield (kg/ha) mortality (%) (%) Years 1 31.081** 52.056** 20 Rep/Yrs 6 2.908 4.408 2402
Extracts 7 410.483** 229.320** 59174**
Yrs x Extract 7 0.715 3.871 685
Error 42 2.195 3.147 1261
* Significant at 5 per cent level of significance ** Significant at 1 per cent level of significance
APPENDIX-XIII
Analysis of variance for table-22
Mean sum of square Seedling mortality Disease severity (%) Yield (kg/ha) Source of d.f. (%) variation 2000 2001 2000 2001 2000 2001
Replication 3 11.681 13.047 6.171 8.338 2980 2419
Bio-agents 12 194.676** 194.811** 188.303** 190.049** 20754** 17617**
Error 36 5.161 5.615 2.731 3.174 1440 1120
** Significant at 1 per cent level of significance
APPENDIX-XIV
Pooled analysis of variance for table-22
Source of d.f. Mean sum of square variation Seedling Disease severity Yield (kg/ha) mortality (%) (%) Years 1 6.906 4.081 670
Rep/Yrs 6 10.364 7.254 2699
Bio-agents 12 389.337** 375.785** 37310**
Yrs x Bio- 12 0.983 2.967 1061 agents Error 72 5.388 2.952 1290
** Significant at 1 per cent level of significance
APPENDIX-XV
Analysis of variance for table-24
Mean sum of square Seedling mortality Disease severity (%) Yield (kg/ha) Source of d.f. (%) variation 2000 2001 2000 2001 2000 2001
Replication 5 12.716 10.918 9.136 10.427 1013 1281
Dates 2 83.661** 86.834** 258.175** 253.822** 20522** 11894**
Error 10 7.819 7.192 4.502 6.256 850 1107
** Significant at 1 per cent level of significance
APPENDIX-XVI
Analysis of variance for table-24
Source of d.f. Mean sum of square variation Seedling Disease severity Yield (kg/ha) mortality (%) (%) Years 1 0.085 0.292 225
Rep/Yrs 10 11.817 9.982 1147
Dates 2 150.827** 510.024** 30892**
Yrs x Dates 2 19.668 1.972 1524
Error 20 7.506 5.379 978
** Significant at 1 per cent level of significance
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