Molecular Detection Of Colletotrichum Gossypii Causing Cotton Anthracnose And Biological Control Mechanism Of The Strain OE-04

Cotton plant(Gosypium spp.)linked with Malvaceae family.Previously reported that forty eight species from fungi caused the twenty different type of symptoms on cotton plants in the world.One of most destructive disease in cotton plant is anthracnose.Anthracnose disease in the cotton plant caused by fungal pathogen Colletotrichum gossypii Southw.C.gossypii is a member of C.gloeosporioides species complex.However,the member of C.gloeosporioides species complex has a high level of genetic similarity makes the development of a diagnostic assay for C.gossypii challenging.In this study,we examined the morphological variance,growth pattern,and pathogenicity of different strain of C.gossypii isolated from different cropping region of china,we found that different strains of C.gossypii have different morphological and cultural characteristics like some of C.gossypii isolated strains showed different growth rate and variation in their spores shape and spore size.Most of C.gossypii strains formed colonies with uniform or irregular edges.Mycelium was white to light dark gray on both sides of culture.Spores seemed normally at the center of colony surface.Spores masses color was reddish yellow to salmon pink,but,the color could be changed by changing the isolate.The conidia were oblong-elliptical shaped.Certain other shapes,like elliptical-fusiform and elliptical,were also detected.Conidium length(9.43 to 10.67 ?m)and width(2.7 to 3.2 ?m)varied significantly among isolates.After pathogenicity test,we also observed that pathogenic ability was also different among the different C.gossypii strains.Most of the C.gossypii isolates caused disease in leaves of both the resistant and susceptible cultivars of cotton.Some of strain showed diseased symptoms rapidly after 5th day of inoculation and some strains needs more time to cause disease in host plant.We also observed the host range of C.gossypii.After host range experiment we analyzed that some strains of C.gossypii have an ability to cause infection in different host but they are isolated from cotton host.In our experiment C.gossypii was successfully cause infection in mango plant leaves(Mangifera indica),rubber plants leaves(Ficus elastica)and both varieties of tomato plants leaves(Solanum lycopersicum).But these guava plants leaves(Psidium guajava),potato tuber(Solanum tuberosum),bell pepper(Capsicum annuum),banana fruit(Musa acuminata),pepper(Capsicum baccatum),carrot(Daucus carota)plants showed resistant against C.gossypii.C.gossypii produced irregular shaped anthracnose spots on host plants.So this ambiguity in C.gossypii characteristics motivate us to developed a molecular diagnostic assay to detect C.gossypii in cotton plants from different regions of China.To overcome any ambiguity in morphological and pathogenic characteristics,a set of primers targeting the ?-tubulin(TUB)gene of C.gossypii was designed.For species specific primer,we used TUB gene,A comparison of the TUB gene sequences of 77 C.spp.isolates deposited in GenBank and our 27 C.gossypii isolates revealed that the TUB gene of all these isolates showed more than 97%similarity and that there were only single nucleotide differences between them.For this reason,we focused only on the single nucleotide phenomenon.The single nucleotide polymorphism(SNP)difference was placed at the 5th prime ends of the forward primer SPSCG/F.The SNP used to design the primer pair is part of the start codon,but is missing in members of other species belonging to the Colletotrichum genus.The SPSCG/F and SPSCG/R primer pair only amplified C.gossypii,and was able to amplify C.gossypii in mixtures of other Colletotrichum species,even when DNA concentrations were up to 10 times lower than that of the other species.After successful molecular identification,we also examined the biological control of C.gossypii.The series of experiments were designed to assess the antagonistic activity of different strains of rhizobacteria.For this purpose,About 48 different rhizobacteria were isolated and performed qualitative and quantitative analysis for biocontrol of C.gossypii casual organism of cotton anthracnose.During qualitative and quantitative screening,we observed that 31 isolates of rhizobacteria have antagonistic activity and other seventeen isolates did not show any antagonistic activity against C.gossypii.Among the thirty one isolates of rhizobacterial isolates CE1 and OE-4 were selected as highly antagonistic.So for further study we used highly antagonistic rhizobacteria CE1 and OE-04.The rhizobacterial strains CE1 and OE-4 were identified on the basis of their cell morphology,colony structure and growth characteristics.In biochemical and growth characteristics test,both rhizobacterial strains CE4 and OE-4 showed positive result while in growth test at 55 ?,strain CE1 was negative so we were used OE-4 strain for further study.After molecular study,rhizobacterial strain OE-04 was identified as Bacillus licheniformis.We used biosurfactant released by strain B.licheniformis OE-04 against the anthracnose.We observed that OE-4 strain secretions produced inhibition zones for fungal mycelia and these zones were developed at periphery edges of mycelia.We noticed that secretions produced some morphological anomalies in C.gossypii cultures.We also estimated the heat stability and pH range and toxicity of biosurfactant produced by strain OE-04.The results showed that biosurfactant has maximum antifungal activity against C.gossypii.Moreover,the biosurfactant also has wide pH and temperature range.We analyzed that at less than 5 and more than 10 pH,the antifungal activity of biosurfactant of B.licheniformis OE-04 was significantly reduced and same antifungal activity reduction against C.gossypii was analyzed after 100 ?.The H2O2 production in host plant with different treatments showed that biosurfactant produced by strain 0E-04 have an ability to control the disease but it is also helpful to develop the immunity in host plant.We also observed the toxicity of biosurfactant produced by B.licheniformis against zebra fish(Danio rerio).We were noticed that biosurfactant have least harmful effect with maximum concentration.To check the fungicide sensitivities of C.gosssypii strain,prochloraz,phenamacril,mancozib,propiconazole,benomyl,Cyprodinil,Azoxystrobin,carbendazim and thiabendazole were assessed using an in vitro mycelia growth assay.We observed that Mycelial mass and spore production was severely affected with different treatment concentrations.When we performed experiment with replication then we also noticed that if we used inoculum from already fungicide exposed fungal culture then the significant variation was observed between first time exposed culture to fungicide and second time exposed culture fungal inoculum.This is a complete study of cotton anthracnose from their pathogen identification to control so this study can play a significant role in control of cotton anthracnose and biological control of cotton anthracnose through biosurfactant of B.licheniformis OE-04 can be a good replacement of chemical pesticides for cotton anthracnose biocontrol.