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ThesisItem Open Access Characterization and integrated management of Fusarium oxysporum f.sp. cubense (E.F. Smith) synder and hansen causing fusarium wilt disease of banana(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2020) Lishma, N P; KAU; Anita Cherian, KFusarium wilt of banana caused by the soil borne fungus Fusarium oxysporum f. sp. cubense (Foc) is a serious constraint to banana cultivation in Kerala. The fungal species constitute four pathogenic races, of which Race 1 is the prevalent one in our country and Race 4 is one of the emerging threats, though not reported from Kerala yet. The present study was undertaken to characterize the associated pathogenic races and to develop an integrated package for the disease management. The project initiated with purposive sampling surveys in various districts viz., Thiruvananthapuram, Ernakulam, Thrissur, Palakkad, Kozhikode and Wayanad representing different agroclimatic zones of Kerala. The per cent disease incidence (PDI) and the per cent disease severity (PDS) ranged from 1.52 to 43.65 per cent and 20.34 to 49.57 per cent. The correlation analysis of PDI with weather parameters showed a positive correlation with rainfall. However, it was negatively correlated with temperature. The study on symptoms under natural as well as artificial conditions showed characteristic external and internal symptoms. The number of days taken for complete wilting under artificial inoculation was 29.67 in Rasthali (AAB), 47.99 in Njalipoovan (AB), 31 in Kadali (AA) and 37.67 in Chenkadali (AAA). Among the thirty isolates of the Foc collected, twenty three isolates were from Rasthali variety, four isolates from Kadali, two isolates from Njalipoovan and one from Chenkadali. Studies on identification of Foc races with the differential host assay revealed that the varieties such as Cavendish (assay host to Race 4), Nendran (assay host to Race 4), Heliconia sp. (assay to Race 3) and Monthan (assay to Race 2) did not produce any type of symptoms whereas, all the isolates produced symptoms on Rasthali (assay host to Race 1) variety. A non polymerase chain reaction (PCR) based quick molecular diagnostic technique with loop mediated isothermal amplification (LAMP) assay was developed for the detection of Races of the pathogen. All isolates showed positive reaction to the LAMP assay for Race 1 and negative for Race 4. A PCR was also standardised for the confirmation of the races. It is concluded that all the isolates collected from different agroclimatic zones belonged to the Race 1 category of the pathogen only. Cultural and morphological characterization of the isolates revealed white coloured aerial mycelium with pink pigmentation and cottony and fluffy mycelial mat. The mycelial growth rate in half strength potato dextrose agar (PDA) medium ranged from 0.83 to 2.40 cm/day and the length and breadth of macroconidia and microconidia measured about 15.01 - 20.20 μm x 2.14 - 5.07 μm and 4.49 - 7.42 μm x 1.35 - 3.13 μm respectively. The inter-septal length and breadth of hyphae ranged from 16.14 to 22.94 μm and 4.22 to 6.57 μm respectively and the size of chlamydospores varied from 5.68 to 9.58 μm in diameter. The PCR based molecular characterization of isolates using ITS (internal transcribed spacer) primers produced single bands of size approximately 580 bp. In silico analysis of the sequences showed 96 to 100 per cent homology to Foc. Based on cultural, morphological and molecular characters, the pathogen was identified as Fusarium oxysporum f. sp. cubense. The screening of accessions maintained in the germplasm of Banana Research Station (BRS), Kannara was done to assess their disease resistance to Foc Race 1 and were grouped into six categories. Fifteen immune varieties viz., Attunendran, Zanzibar, Big Ebanga, Nedunendran, Nendran, BRS II, Thiruvananthapuram, Pachanadan I, Cultivar Rose, Pisang Lilin, Pisang Jari Buaya, Yangambi Km5, Grand Naine, Chinese Cavendish and Nendran Hybrid and four highly susceptible varieties viz., Cheriya Poovan, Valiya Poovan, Kadali and Rasakadali were identified. The estimation of biochemical parameters for the assessment of host plant disease resistance against Foc Race 1 revealed that the activity of total phenols and defense related enzymes was more in resistant varieties compared to susceptible varieties and the activity of reducing and non reducing sugars was more in susceptible varieties. An in vitro experiment was conducted for the evaluation of chemical fungicides, biocontrol agents and botanicals for control of the pathogen. The effective treatments from in vitro evaluation were carried over to pot culture and field experiments for the disease management. Among the various treatments, an integrated package comprising of Pseudomonas fluorescens + arbuscular mycorrhizal fungi and Trichoderma enriched cow dung + tebuconazole (T6) was proved to be the best for yield and disease management. It is concluded that the present study has enlightened our knowledge on characterization, race identification and management of Fusarium wilt pathogen infecting banana.ThesisItem Open Access Management of early blight disease of tomato (Solanum lycopersicum L.) under protected cultivation(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2020) Sumbula, V; KAU; Sainamole Kurian, PTomato (Solanum lycopersicum L.) is one of the most remunerative and widely grown vegetables all over the world. With the coordinated efforts of central and state governments, protected cultivation of tomato is now gaining popularity in Kerala. Despite being a versatile crop adapted to various agroclimatic regions and seasons, cultivation of tomato is constrained by various fungal, bacterial and viral diseases. Among the fungal diseases, early blight caused by Alternaria solani is the most common, destructive and widespread in all the tomato growing tracts. Fungicides and bioagents are commonly used to manage plant pathogens. But little is known about their effects on the non-target microbial communities that inhabit inside and outside the plant. Hence, it has become necessary to consider the effect of different fungicidal and bioagent treatments on target and non-target microbial communities while formulating disease management strategies. So, the present investigation was carried out with the objectives to formulate suitable management strategies against early blight disease of tomato under protected cultivation and to assess their impact on culturable and non-culturable microflora associated with the plant. Isolation of the pathogen from infected tomato leaf samples revealed the association of the fungus, Alternaria sp. and its pathogenicity was established by inoculating on threemonth- old tomato seedlings. Symptoms observed on leaves, shoot and fruits were almost same under both natural and artificial conditions. Cultural and morphological characters of pathogen was studied on potato dextrose agar (PDA). Initially, pathogen produced greenish brown mycelium and later turned to grey colour. Hyphae are septate and the colony has aerial topography and irregular rough growth patterns with concentric zonation. Sporulation was observed after six days of incubation and conidiophores were straight or flexuous brown to olivaceous brown in colour. The conidia are solitary straight or muriform or oblong, pale or olivaceous brown, length 40-110 μm and 7-15 μm thick with 2-8 transverse and 0-3 longitudinal septa. The cultural and morphological characters of the pathogen completely fit into the description of Alternaria solani by Alexopoulos et al. (1996). Hence, it is confirmed that the symptom observed on tomato leaves are those of early blight disease caused by A. solani. In vitro evaluation of fungicides and bioagents showed complete inhibition of the pathogen with propineb (0.1%, 0.2% & 0.3%), hexaconazole (0.05%, 0.1% & 0.15%), iprodione + carbendazim (0.1%, 0.2% & 0.3%), difenoconazole (0.075%), Trichoderma viride (KAU), T. viride (PGPM mix), T. harzianum (PGPM mix) and plant growth promoting microbial consortium (PGPM mix of KAU). Among the bacterial antagonists, Bacillus subtilis (endophyte from cocoa) showed maximum growth inhibition of the pathogen. All the three bioagents recorded earliness in seed germination and enhanced seedling vigour compared to the fungicidal treatments and control. The results of field experiment under polyhouse and rain shelter conditions showed that all the treatments are superior to control in early blight disease management, of which, spraying of iprodione + carbendazim (0.2%) and propineb (0.2%) were the best among fungicides and PGPM mix application was the most efficient among bioagents. Moreover, the highest yield was recorded from iprodione + carbendazim treated plants. Biocontrol treated plants showed better performance in overall plant vigour of which PGPM mix application was the most effective. Residue analysis showed that degradation rate of fungicides was more under polyhouse condition. Analysis of population of phylloplane and endophytic microflora proved that there was drastic reduction in microbial population after spraying with chemical fungicides whereas population increased after bioagent application. The study on survival of bioagents on tomato phylloplane revealed that both Pseudomonas fluorescens and T. viride, survived on leaf surface up to 15 days after foliar application. Analysis of fungicidal residue on tomato fruits revealed that, the degradation of fungicides was faster in polyhouse compared to rain shelter. Metagenomic analysis of microbial diversity on tomato leaves revealed that spraying of chemical fungicides reduces microbial population and diversity while bioagent application enhances the same. However, microbial community structure was changed in both cases. This study also enlightened the new mode of action for fungicides and bioagents besides their direct effect that is shifting the microbial community structure so that it provides greater resistance against the pathogen. Interestingly, metagenomic results also showed association of Cladosporium, Corynespora, Pseudocercospora along with early blight pathogen Alternaria on tomato leaves that otherwise remain undetected. Another important observation was Clostridium in tomato leaf samples except in PGPM mix treatment, suggesting the possibility of plants as alternate host for major human and animal bacterial pathogens. Hence, considering the effects of treatments on per cent disease severity both under polyhouse and rain shelter condition, residue analysis, phylloplane and endophytic microbial enumeration study and metagenomics analysis of microbial diversity, the present study recommends spraying of propineb (0.2%) as the best treatment among the tested fungicides and spraying of PGPM mix among biocontrol agents for the management of early blight disease of tomato under protected cultivation. Further system-level analysis of the complex interaction that governs outcomes among community members in the context of the plant host is required, in order to identify microbial interaction and selection processes for beneficial communities at different concentrations of fungicides and pathogen pressures.
