CHARACTERIZATION AND MANAGEMENT OF MACROPHOMINA PHASEOLINA (TASSI) GOID. ASSOCIATED WITH CHARCOAL ROT OF SESAME (SESAMUM INDICUM L.) IN PUNJAB, PAKISTAN

Abstract

Charcoal rot has been proved a serious threat to a wide range of field and horticultural crops in Pakistan but very little information on disease statistics on sesame are available. To investigate the updated status and associated factors for disease development comprehensive surveys were conducted during 2012-2013. Keeping in view importance of the crop and disease a project based on interlinked series of experiments on geographical distribution, pathogen virulence, host susceptibility, host pathogen interaction, genetics of host and pathogen, pathogen aggressiveness and varietal resistance trails was designed. Further plant residue incorporation, mulching and aqueous extracts of Cassia fistula, Azadirechta indica, Acacia nilotica, Parthenium hysterophorus, Datura metel, Calotropis procera, Chenopodium album, Alstonia scholaris, Ageratum conyzoides, Eucalyptus citriodora, Cronopus didymus Solanum nigrum, Convolvulus arvensis, Amaranthus viridis, Senna. occidentalis, Agremone mexicana were applied in pots trails under natural environment, in pots and field trails in 2× 2m plots. Data was recorded on post emergence mortality, sclerotia count and growth parameters. Field observations on disease index and associated issues were recorded on a structured questionnaire. Questionnaire was prepared in consultancy with all public and private sector stake holders of crop production and protection. Besides collecting disease observations disease specimens and soil samples were also collected as raw material for onward investigations on characterization and management. The surveys were conducted during the months of August–September in sesame growing areas of Punjab comprising on cotton zone, mix cropping, low rain fall, rice zone and irrigated plains. The cumulative description of disease statistics described that highest prevalence with 80-100 % prevalence, 3-9 severity and 62% incidence was observed in mix cropping zone. Whereas least 40% prevalence, 1-3 severity and 20-30% incidence was recorded in cotton zone. Dominant cultivation and fixed cropping pattern from decades resulted in highest index of 37% in mixed cropping zone and least of 17% in cotton zone. Major reason for lower disease levels in the cotton zone is lesser growing area and irregular cultivation of the crop. Pathogenicity of the isolates was confirmed by observing Koch’s Postulates. M. phaseolina isolate MP28 (1156FCBP) was deposited in the first Fungal culture bank of Pakistan. Wide range of aggressiveness patterns were observed and test population of the fungus was classified in to highly aggressive, aggressive, moderately aggressive and non-aggressive. None of the isolate falls in non-aggressive class whereas 9 isolates exhibited aggressive, 12 moderate and 22 highly aggressive behaviors in pots whereas 12 aggressive, 17 moderate and 14 highly aggressive isolates were recorded in field. RAPD analysis revealed 2 main groups with two subgroups each. Group 1 is more heterogeneous. The high level of heterogeneity may be related to aggressiveness behavior as highly aggressive isolates (MP8, MP28, MP19). Cluster analysis explains that isolates like MP 21 MP22, MP41 MP42, MP26 MP27, MP6 MP10, MP15 MP16, MP36 MP37 and MP1 and MP3 are genetically similar. It provided evidence that irrespective of their origin isolates are related. Collection of isolates MP17, MP41, MP42, MP24, MP36 and MP37 isolated from Mix cropping, Barani and Cotton zone at same similarity level could be linked with long distance distribution of pathogen through infected seed and predominant cultivation of similar sesame genotype in the region. A set of nine isolates MP8, MP28, MP19, MP14, MP 37, MP18, MP4, MP10, and MP33 that exhibit stable virulence pattern under field and pot experiments were selected for morphological and physiological studies. The investigation was conducted with the objectives of findings the relationship of virulence factor with pathogen growth attributes .Morphology is believed to have link with enzyme production, antioxidant activity and some other factors that determines the survival and growth of pathogen in the host. Morphological variation of moderately aggressive (MP8, MP28, MP19) aggressive (MP14, MP37, MP18) and highly aggressive (MP4, MP10, MP33) isolates was examined on the basis of colony color radial growth, type of mycelium presence, size and population of sclerotia. The test isolates on PDA medium exhibited variations in colony growing pattern and growth rate. Colony color of test isolates varied from light gray to black while colony pattern was classified as dense or sparse colony with aerial / appressed mycelium. Sclerotia count showed prominent variation that ranged from lowest 10 sclerotia/ per microscopic field in MP4 to highest range 38 sclerotia/per microscopic xxiii field for MP19 respectively. Dark colony color with highest sclerotia count was the key feature studied in highly aggressive isolate MP8, MP28 and MP19. This can be used as base for aggressive behavior of the pathogen and it can be assumed that microsclerotial population might play a role in determining virulence behavior as MP19 that exhibited highly aggressive behavior has high sclerotia count. Physiological studies were conducted to find best suited conditions for fungal growth and investigating relationship of physiological responses with virulence factor. In lab studies Physiological parameters involving selection of growth media, temperature response, pH level, carbon nitrogen and light source were investigated. Among the test growth media PDA medium was found best to support the mycelia growth whereas Czapek dox agar medium exhibited low radial growth and least number of sclerotia. The most favorable temperature and pH regimes for development of M. phaseolina isolates were 30ºC and pH 6. However Highly aggressive isolates MP19 and MP28 collected from mixed cropping zone survived and produced higher radial colony growth even at 45oC and pH 9 which is usual soil temperature and pH level in Punjab province. Dextrose as carbon source supported mycelial growth and sclerotia count of test isolates. Significant variation in radial growth and sclerotia count was also recorded for different nitrogen sources. Photoperiod 16hrlight/8hrdark supported optimum colony growth in test isolates. However isolates represented slow colony growth under 24h dark photoperiod. Similar response towards test photoperiods was observed in sclerotial population among test isolates. External symptoms of infection process showed light brown lesion above soil line which gradually turned dark brown and finally black. Disease progression leads to formation of sclerotia with wet shiny black charcoal mat on the outer surface of stem. The process of lesion blackening and malformation describes resistance scaling of germplasm. In resistant germplasm lesion blackening process is extended to 12 days instead of 5-7days for susceptible germplasm. Stem tissue examination of moderately resistant germplasm gave better understanding for invasion and movement of hyphal structure in cortex region of root and stem tissue. Large sclerotia bodies observed inside host tissue which results in mechanical disintegration of cells and created large spaces in xxiv the cortex region. Large no of swelled and lightly melanized sclerotial bodies were also observed. Resistance reaction studies were conducted with a set of 26 sesame lines comprising on commercial varieties, candidate lines and new breeding lines against M. phaseolina in pot under natural and field trials conditions. None of the test line was found completely disease free or immune and general resistance reaction of test varieties ranged from 5-9. Residue incorporation of dried powder of leaves and herbaceous branches of D. metel proved effective in residue incorporation in pot and field experiment and in aqueous extract application it showed significant antifungal inhibition in vitro and in vivo trials. Plant mortality of 12%, 18% and 9, 10 sclerotia/g soil was recorded in pot and field experiment respectively where D. metel was added as residue at the rate of 3 %. However in mulching A. scholaris showed significant reduction in post emergence mortality and sclerotia count and improving growth parameters in pot and field experiment. Whereas in field trials, mulching of C. procera appeared most effective in reducing post emergence mortality. In collaboration with Institute of chemistry, initially 14 new synthesized heterocyclic compounds viz., Chalcons, Pyrimidine quinolone and Pyrazoles quinolone derivatives were applied to examine mycelial and fungal suppression. On the basis of initial results seven compounds were investigated against M. phaseolina in vitro experiment at 25, 50 and 100 PPM. The objective of evaluation of fungi toxicity of new compounds was to examine their potential development as commercial fungicide against M. Phaseolina. Heterocyclic compound 3(PZQ2) exhibited highest reduction (73%) and 100% in colony diameter and sclerotia count at 100ppm respectively. whereas compound 11 (CHCN5) showed lowest reduction (14%) and (20%) in colony diameter and sclerotia count respectively.