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Open Access
Development of recombinant coat protein for immunodiagnosis of banana bunchy top and bract mosaic diseases
(Department of Plant Pathology, College of Agriculture, Vellanikkara, 2021) Darsana Dilip, K C; KAU; Vimi, Louis
The present investigation was undertaken to develop recombinant coat protein (rCP) of Banana bunchy top virus (BBTV) and Banana bract mosaic virus (BBrMV) for immunodetection of the viruses. The experiments were conducted at the Virology Lab, Banana Research Station, Kannara; Department of Plant Pathology, College of Agriculture, Vellanikkara, Kerala Agricultural University and Indian Institute of Science, Bengaluru during the period of 2016-2020. A roving survey in 10 districts of Kerala, divided into population subsets viz., North, Central and Southern zones were conducted for sample collection. After a preliminary DAC-ELISA, 17 and 12 representative samples respectively were selected and carried forward for further evaluations. The CP gene of BBTV was amplified from the total DNA isolated using reported primers by Polymerase Chain Reaction (PCR) and that of BBrMV by Reverse Transcriptase-PCR (RT-PCR). The CP gene sequences of these isolates were determined and submitted in the NCBI-GenBank Database. The 17 BBTV isolates were designated as MT174314-MT174330 and the 12 BBrMV isolates as MT818176- MT818187. It was inevitable to evaluate the molecular diversity of the viruses prior to devising nucleic- acid based and serological detection methods. The phylogeographic analysis depicted a clear demarcation of BBTV Kerala isolates based on geography whereas no such clustering was observed in the case of BBrMV isolates. Being an RNA virus, the molecular diversity of BBrMV (ranging between 1-12 %) was higher than BBTV. However, the 5’ and 3’ terminal of BBrMV CP gene was hypervariable and found unsuitable to be targeted for nucleic-acid based detection. Hence, forward primer was designed from the NIb region of ssRNA genome of BBrMV and reverse primer from 3’ UTR region upstream and downstream to the CP gene respectively. For nucleic-acid based detection of BBTV, highly conserved non-coding region of DNA-S upstream and downstream to the CP ORF was targeted. The primers were validated by detecting virus from the field samples collected from various parts of the state. The rCPs were chosen as a potential antigen for raising antibodies in order to develop serodiagnostic assays for the early detection of the viruses. The BBTV CP gene was clonedin to three expression vectors viz., pRSET-C, pGEX-4T-2 and pET32a(+) and transformed to expression hosts like BL21 (DE3) pLysS, Rosetta (DE3) pLysS and C41 strains of E. coli after amplification in DH5α. The 20 kDa recombinant BBTV CP (rBBTV CP) cloned in to pRSET-C, and overexpressed in various E. coli hosts had a hexa histidine (6X His) tag at the N terminal. Similarly, a 37 kDa fusion protein (pET/rBBTV CP) was overexpressed from pET/BBTVCP clone had a thioredoxin (Trx) tag (17 kDa) along with the 6X His tag. Whereas, a 45 kDa fusion protein (pGEX/rBBTV CP) with GST tag was overexpressed from pGEX/BBTVCP clone. These affinity tags in the fusion rCP enabled purification from other E. coli proteins. Although pRSET/rBBTV CP was soluble, the 20 kDa protein was highly unstable and partially degraded during purification at 4 °C. Curiously, pGEX/rBBTV CP dissociated from its GST affinity tag and the rCP without the tag degraded. On evaluating the protease cleavage sites in the fusion protein, trypsin cleavage sites were present between the C terminal of GST and N terminal of BBTV CP which might be the reason for cleavage of the ~20 kDa protein from its affinity tag. Thus, it was impossible to purify the protein from the pool of E. coli proteins. Restriction free (RF) cloning of BBTV CP to pGEX-4T-2 was attempted not only to replace these trypsin cleavage sites but also the thrombin cleavage site present in the vector with Tobacco etch virus (TEV) NIa protease site. Thrombin is a specific enzyme used to cleave off the tag from the fusion protein after purification. However, its specificity is not universal. Furthermore, the commercially available enzyme is costly. TEV protease on other hand was produced in the laboratory and was highly specific. However, the cleavage using TEV protease was unsuccessful apparently because of a steric hindrance contributed by the two extremely ordered regions flanking the TEV cleavage site present in the disordered region of the fusion protein. pET/rBBTV CP was highly soluble like ΔpGEX/rBBTVCP. Likewise, BBrMV CP gene was cloned into pRSET-C and pGEX-4T-2 to obtain pRSET/rBBrMV CP and pGEX/rBBrMV CP of size 34 kDa and 60 kDa respectively. The 34 kDa pRSET/rBBrMV CP was insoluble. Overexpression and purification of the protein was standardized in various conditions to increase solubility. On the contrary, pGEX/rBBrMV CP was highly soluble and was purified by GSH Sepharose affinity column chromatography. 360 μg/ml of untagged protein was obtained from 1 l culture. However, like any other potyviral CP, the exposed N and C terminal of BBrMV CP was also prone to proteolytic cleavage. It partially degraded when incubated with thrombin atroom temperature for GST tag cleavage. All these bands were detected by potyviral CP specific antibody in Western blot. Further on storage complete degradation of the protein was observed. Further standardisation of the protocol is necessary to either stabilise monomeric CP or develop BBrMV VLPs in vitro for immunising animal in order to raise the antiserum. The immunogenicity of the antigens (rBBTV CP and rBBrMV CP) was confirmed by Western blot using BBTV CP specific and potyvirus CP specific antibody procured from NRC, Banana and IISc, Bangalore respectively. The rCPs were also characterized by fluorescence spectroscopy, sucrose gradient ultra centrifugation and electron microscopy. The fluorescent spectra of tagged and tag less rBBrMV CP deviated from 330 nm which is typical for a partially disordered protein. However, the spectra of pET/rBBTV CP and ΔpGEX/rBBTV CP were different. The former depicted the spectra of a mostly globular protein. There were two λmax for the fluorescence spectra of ΔpGEX/rBBTV CP. The epitope prediction of BBTV CP with Trx tag gave interesting insights. A single linear epitope of 80 residues were detected in pET/rBBTV CP comprising of C terminal of the affinity tag and the N terminal of BBTV CP. This was expected to increase the immunogenicity of the antigen and administered for production of antiserum. The titre value of polyclonal antiserum produced against the 37 kDa pET/rBBTV CP was evaluated by DAC-ELISA and was found to be 1:128000. Titre value for serological assays of field samples was standardized as 1:10000 to be more inclusive for detecting virus even at early stages of infection. A total of 247 tissue culture samples and 10 field samples were screened for the presence of the virus using the antiserum and was compared with the procured antiserum. Seemingly, the latter non-specifically reacted with plant proteins which gave a higher absorbance value in negative control and correspondingly high absorbance in the infected samples. The polyclonal antiserum raised against rBBTV CP was used to standardize serological detection assays like IC-PCR, DIBA and TAS-ELISA apart from DAC-ELISA. DIBA and TAS-ELISA were the most sensitive assays which could detect up to 1:80 dilution of the antigen. In conclusion, due to the higher nucleotide variability of the CP gene, serological detection is preferred over nucleic acid based assays. However, the quality of antigen used for raising the antibody plays a major role in serodiagnostics. Hence, high quality rCPs of both BBTV and BBrMV were developed in the laboratory in various vector/host systems. ThepET/rBBTV CP overexpressed in C41 strain of E.coli (1.1 mg/ ml obtained from 1 L culture) was used for immunisation of the animal. A highly sensitive antiserum specific to BBTV with a titre ten fold higher than that of the commercially available antiserum was obtained. Using this antiserum raised against rBBTV CP, various serodiagnostic assays were standardised in the laboratory. Among these, TAS-ELISA was the most sensitive, detecting antigen even at higher dilution.
Open Access
Characterisation and management of sugarcane bacilliform virus (SCBV) causing leaf fleck disease in sugarcane
(Department of Plant Pathology, College of Agriculture, Vellanikkara, 2021) Sanju Balan; KAU; Anita Cherian
Sugarcane (Saccharum officinarum) is a monocotyledonous perennial cash crop cultivated worldwide both under tropical and sub tropical conditions. It is being cultivated in more than 120 countries in the world. Like any other crops, it is also susceptible to biotic stress. Of which, diseases caused by viruses not only pose serious threat to sugarcane cultivation but also result in deterioration and exclusion of elite varieties of the germplasm. One of the major viral disease which affects global exchange of sugarcane germplasm is leaf fleck disease caused by Sugarcane bacilliform virus (SCBV). The research project entitled ‘Characterization and management of Sugarcane bacilliform virus causing leaf fleck in sugarcane’ was initiated with purposive sampling surveys in selected sugarcane fields in districts of Kerala and Tamil Nadu in order to document the symptoms under natural conditions, to assess the disease incidence, severity and to collect infected samples for further studies. The per cent disease incidence of the leaf fleck disease in Kerala ranged from 12 to 51 per cent whereas severity ranged from 10 to 36.5%. In Tamil Nadu the per cent disease incidence ranged from 28 to 56 per cent while severity ranged from 28 to 50.41%. Major symptoms observed on leaves were mottling, chlorotic flecks, chlorotic patches streaks and stripes with general yellowing of the canopy. In the case of severely affected clones, there was reduction in tillering, internodal length, number of internodes and appearance of deep longitudinal cracks. In highly susceptible clones, stunted growth with bunchy top appearance was noticed. On the basis of phenotypic variability of symptom expression, genotypes were classified into five groups. The development of the symptoms was also studied under artificial condition through insect transmission of the virus using pink mealy bug, Saccharicoccussacchari. Morphological characterisation of the virus done using electron microscopy revealed the presence of bacilliform virus particles of size 30 X 130–150 nm which indicated that the virus belongs to genus BADNA and family Caulimoviridae and the etiology of the disease was confirmed as Sugarcane bacilliform virus. The molecular detection of SCBV was also standardized through polymerase chain reaction (PCR). PCR amplification of RNaseH/RT gene was done using BADNA specific and SCBV129 specific primers. The amplicons were sequenced and in silco analysis of sequences showed sequence homology of 99 to 100 percent identity to SCBV. Widespread occurrence of the disease was observed even in the early generation of varietal development and in newly developed varieties. The transmission of the virus was suspected through true seed (fluff) developed by biparental crossing during sugarcane varietal development programme. Hence, the study was conducted to establish possible transmission of the virus from sugarcane parents to their progenies and the role of maternal and paternal parents in disease transmission through true seeds to the progenies. Samples from eight months old seedlings, three months old seedlings and parental clones were tested positive to the virus in PCR assays. Real time PCR was also standardized to assay these clones. Immunodiagnostic technique was validated using DAC ELISA. The technique of immunocapture PCR was also standardized. Minimal dilution of antisera with which SCBV could be detected was 2:1000 (V/V). Plant extract (antigen) at a dilution of 1:5 was found to be optimal for the detection of SCBV. Molecular detection of SCBV from mealy bug vector was also standardized. Both phenotypic and molecular methods were utilized to identify potential sources of natural resistance against SCBV. Based on the severity of symptom expression and PCR assays these were further classified as highly susceptible (HS), moderately susceptible (MS) moderately resistant (MR) and resistant (R). For generation of RNAi hair pin construct, initially forward (SF) and reverse primer (SR) were used to amplify 700 bp fragment of RT/RNase H gene to be cloned in sense orientation of the vector, pHANNIBAL. The linearized vector and the insert were ligated, and the ligation mixture was used to transform competent cells of Escherichia coli and the transformants were selected. Later antisense forward (AF) and reverse (AR) primer pairswere used to amplify 700 bp fragment of RT/RNase H gene to be cloned in antisense orientation. PCR product ligated into antisense direction of the vector and transformed into competent cells of E. coli. The recombinant pHANNIBAL vector was digested with restriction enzymes. The recombinant pHANNIBAL vector harbouringRNase H /RT gene was released from the vector through Not I site and subcloned into plant expression binary vector. Thus, cassette for RNA silencing was prepared.130 Meristem tip culture was also standardized with antiviral chemical tenofovir. Recovery percentage of meristem varied from 70 to 75 per cent and the viral load was quantified using real time PCR. The outcome of the study would facilitate early detection and elimination of the source of infection and prevent the spread of the disease in the field. Information generated in the study could be utilized while planning biparental crossing and reduce the spread of the virus in varietal development programmes. The hair pin construct developed in this study could be further utilized to generate transgenic disease resistant plants.
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, K
Fusarium 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.
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, P
Tomato (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.
Open Access
Deterioration of oil cake by fungi
(Department of Plant Pathology, College of Agriculture, Vellayani, 1989) Naseema, A; KAU; Wilson, K I
Fungi causing deterioration of coconut, groundnut and sesamum oil cakes were studied. ficremonium implicatum, Asperdllus aculeatus, A. flavus, A. fumigatus, A. nlaer, A. terreus, A. versicolor, Bipolaris hawaiiensis, Curvularia clavata, Monascus ruber, Penicillium aurantioqriseum, P. Pinophilum, Pestalotiopsis palmarum, Rhizomucor £usillus and Ehizopus stolonifer were obtained from coconut oil cake. Aspergillus flavus, A. niaer, A. terreus, A. versicolor, Gliocladium sp. Penicillium pinophilum, RhizoEUS or^zae and Rhizopus stolonifer were noticed in groundnut and Aspergillus candidus, A. flavus, A. fumigatus, A. nlaer, A. tamarii, A. terreus, Curvularia clavata, Eurotium. chevalieri, F"sarium pallidoroseum, Monascus ruber, Fenicilliuiu pinophilum, Pestalotiopsls palmarum and Rhizopus or^zae in sesamum oil cake. Of these, Acremonium implicatum, Aspergillus aculeatus, A. caeslellus, A. .f"-igatus, Bipolaris hawaiiensis, Curvularia clavata, Monascus ru^, Penicillium anrantlogriseum, P. pinophilum, Pestalotiopsls palmarum and Rhizomucor pusillus from coconut oil cake, Aspergillus versicolor, Gliocladium sp., Penicillium pinophilum, Rhizopus oryzae and R. stolonifer from groundnut and Aspergillus candidus, A. fumigatus, A. tamarli, A. terreus, Curvularia clavata, Eurotium chevalieri, Fusarium pallidoroseum,Monascus ruber, Penicillium pinophilum, Pestalotiopsis palmarum and Rhizopus oryzae from sesamum oil cake have not been reported earlier. * Aspergillus flavus and A. niger were isolated from all the samples of groundnut and sesamum oil cakes. In coconut oil cake, these two fungi were present in 88.89 and 77.78 per cent of the samples. A. terreus was isolated from 66.67 per cent of groundnut and 55. 56 per cent of coconut and sesamum oil cake samples. Penicillium pinophilum was obtained from 66.67 per cent of groundnut, 44.44 per cent of sesamum and 27.78 per cent of coconut oil cake samples. Wide variation was noticed in the population of fungi present in the oil cakes collected from different regions during different periods of the year. Oil cakes collected during June-July had the highest population, of fungi. The central and the northern regions recorded higher population of fungi than the southern region. Positive and significant correlation could be obtained between weather parameters and population of fungi in different oil cakes. Maximum correlation was noticed in relation to total rainfall. Qood mycelial growth of fungi was obtained in all the oil cakes incubated at 27, 29 and 32°C. Maximum mycelial growth was noticed at 100 per cent relative humidity. This was followed by 96.1 per cent and 92.9 per cent in the descending order. The oil content of the oil cakes was considerably reduced due to the growth of all the fungi tested individually and in combination. Maximum reduction v/as noticed due to the growth of Pestalotiopsis palmarum in coconut oil cake, Rhizopus stolonifer in groundnut and Fusarium pallidorosem in sesamum oil cake. In the case of combinations, Aspergillus flavus, A. niger and Penicillium pinophilum together caused maximum reduction in oil content of coconut oil cake. In groundnut, combined growth of A. flavus, A. niger and A. terreus caused maximum reduction in oil whereas, A. niger and P. pinophilum together effected maximum reduction of oil in sesamum oil cake. Oil cakes inoculated with different fungi showed considerable reduction in total carbohydrates, crude protein, free amino nitrogen, crude fibre and ash to the extent of 6.11 to 76.95 , 4 . 28 to 68.03, 14.91 to 92.52, 1.25 to 92.55 and 0.17 to 65.16 per cent respectively. In the case of mineral nutrients like phosphorus, potassium, magnesium. calcium, copper and iron reduction ranging from 15.07 to 75.54, 23.13 to 94.41, 10.89 to 63.37, 28.78 to 90.20, 52.52 to 97.12 and 0.32 to 60.77 per cent respectively was noticed. Fourteen out of 2 0 isolates of Aspergillus flavus produced aflatoxins B^, and G2 in culture medium with maximum quantities being 1210, 1040 and 151 ppb respectively by the isolates from coconut oil cake. Eight out of 19 isolates of A. niger elaborated upto 222 ppb by the isolate from sesamum oil cake. When grown on the respective host material, A. flavus isolates from coconut oil cake produced maximum quantity of B^^, B^ and being 1517, 1092 and 272 ppb respectively. A. niger isolate from coconut oil cake produced B^^ upto 419 ppb. oil cakes treated with calcium propionate (0.6 per cent, w/w) were free from fungus growth throughout the period (180 days) of observation and showed minimum number of fungal propagules whereas, those kept as control had higher population of fungi than the treated ones, at all period.of observation. Oil cakes stored in polythene lined gunny bags had the least population of fungi, whereas those stored in ordinary gunny bag had very high population of fungi. These results revealed that fungal deterioration and spoilage of oil cakes could be prevented or reduced to the minimum by treatment with 0.6 per cent calcium propionate- and by using polythene lined gunny bags for storage arid transport.
Open Access
Fungal diseases of selected medicinal plants of Kerala
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 1991) Sukumara, Varma A; KAU; ; Abi, Cheeran
Open Access
Study of bacterial leaf spot of betel vine- biochemical changes and control
(Department of Plant Pathology, College of Agriculture, Vellayani, 1986) Koshi, Abraham; KAU; James, Mathew
The bacterial leaf spot is one of the most serious diseases of betel vine in Kerala. The bacterium is one of the most serious disease of betal vine. Confidering the seriouness of the disease , studies were undertaken on the different aspects of the disease and to find out a suitable control /management practice.
Open Access
Survival of Xanthomonas campestris pv. Oryzae and its Control in Kuttanad
(Department of Plant Pathology, College of Agriculture, Vellayani, 1996) Mary, C A; KAU; Sasikumar Nair
The present investigation was taken up to understand the factors responsible for the recurrence of bacterial blight disease in a severe from only during the additional crop season in Kuttanad. The mode of survival of the pathogen during and in between the two major cropping seasons of Kuttanad region were also studied in detail. An extensive survey was also conducted among 115 farmers in 12 Krishibhavans of Kuttanad taluk for this purpose to collect specific informations on existing cultural practices, crop variety, nature and distribution of weed flora and self sown rice plants in and around rice fields and on wether data from June 1992 to March 1994. The efficacy of two different methods of spraying, prophylactic and curative using streptocycline, mixture of streptomycin and oxytetracycline in the proportion 1:9, Bactrinol – 100 cowdung extract on the control of bacterial blight disease was tested under field condition at Nedumudi in Kuttanad. The survey showed that there was considerable variation in the incidence of bacterial blight in Kuttanad taluk. Among the 12 Krishibhavan areas the disease incidence was maximum in Ramankari and Nedumudi and minimum in Kavalam, Kainakary and Muttar. In Neelamperoor and Thalavadi areas there was no incidence of this disease during the period of survey. Between the two major cropping seasons the disease incidence was more during the additional crop season than during Punja season. Red Triveni and Jyothy were the most popular varieties cultivated in the area and more than 50% of the farmers cultivate Red Triveni. It was observed that the variety Red Triveni as highly susceptible to bacterial blight disease. The isolate of the pathogen Xanthomonas oryzae pv. Oryzae from the rice variety Red Triveni was capable of both gelatin liquefaction and starch hydrolysis. The pathogen X. oryzae pv. oryzae was found to survive for a maximum period of 42 days in infected seed, 105 days in infected straw, 56 days in infected stubbles at room temperature, 24 days in infected stubbles under dry land condition and 14 days under wet land condition. The pathogen did not survive in soil and water. Weeds like Oryza sativa var. fatua and Paspalum conjugatum served as alternate host for the pathogen. Bacterial blight infected self sown rice plants could be seen in Kuttanad during the cropping and non cropping seasons. Due to certain specific reasons, the cultivation practices were often found to extend beyond the normal cropping seasons in the region resulting in the chances of survival of bacterial blight pathogen in the host plant itself. The specific weather conditions during the additional crop season played an important role for the severity of bacterial blight desease in this season in Kuttanad. The pathogen X. oryzae pv. oryzae was tested for sensitivity to antibiotics, Bactrinol – 100 and cowding extract under in vitro conditions. The maximum growth inhibition was obtained with oxytetracycline followed by chloramphen icol which was statistically on par with oxytetracycline. The effect of increasing concentrations of oxytetracycline in combination with streptomycin on growth of X. oryzae pv. oryzae was studied with 100, 250 and 500 ppm concentrations. The growth inhibition increased not only with the concentrations of antibiotic from 100–500 ppm but also with increasing concentration of oxytetracycline. The maximum zone of growth inhibition was obtained with 1:9 proportion of streptomycin and oxytetracycline. The five treatments selected for field evaluation trial included streptocycline at 500 ppm, streptomycin + oxytetracycline (1:9) at 250 ppm and 500 ppm, Bactrinol -100 at 500 ppm and fresh cowdung extract at 20g/1. Two different spraying methods, prophylactic and curative were evaluated in two rice varieties, T(N) 1 and jyothy. The reduction in disease index by prophylactic and curative sprayings was maximum after spraying with cowdung extract 20g/1. As regards to two methods of spraying, significant reduction in per cent disease index was obtained with curative spraying. The maximum per cent increase in grain yield over control was obtained after curative spraying with 500 ppm streptomycin and oxytetracycline mixture in jyothy followed by cowdung extract 20 g/l. In T(N) 1 and jyothy both by prophylactic and curative spraying, the thousand grain weight was maximum with cowdung extract 20 g/1. As regards to two method of spraying, significant increase in grain yield and thousand grain weight was obtained after curative spraying. In T(N) 1, both by prophylactic and curative spraying the per cent increase in straw yield was maximum with a mixture of streptomycin and oxytetracycline at 500 ppm and jyothy with cowdung extract 20g/1. In T(N)1 significant reduction in chaff per cent was recorded by prophylactic spraying while in Jyothy no significant difference was obtained by the two methods of spraying. In both these varieties the reduction in chaff per cent was maximum by spraying with cowdung extract (20g/1). It was observed that two prophylactic spraying with selected bactericidal agents, neither resulted in any significant reduction in disease index nor increase in yield as compared to curative spraying. This could be due to the fact that in Kuttanad bacterial blight disease usually occurred only around the panicle initiation stage or even later. Therefore a need based curative spraying schedule would be most effective for the control of bacterial blight disease in Kuttanad. On working out the economic benefits of controlling bacterial blight it was observed that there will be economic return only from spraying infected plants of both (T(N) 1 and jyothy with cowdung extract 20g/1. The return from plants sprayed with all other treatments in the investigation was low when compared to unsprayed control plants. Thus it will be economically advantageous to use cowdung extract to control bacterial blight of rice.
Open Access
Management of foot rot of black pepper (piper nigrum L.) with va mycorrhiza and antagonists
(Department of Plant Pathology, College of Agriculture, Vellayani, 1998) Christin Robert, P; KAU; Sivaprasad, P
Extensive investigation was carried out to develop a native microbial inoculant based technology involving Arbuscular Mycorrhizal Fungi (AMF) and fungal antagonists for the foot rot disease management and growth improvement of black pepper in the nursery and field. Phytophthora capsici Leonian emend A. Alizadeh and P.H. Tsao, the foot rot pathogen isolated from Peringammala, Thiruvananthapuram district was found most virulent isolate. Seven native AMF cultures and fifty fungal antagonists were isolated from Kerala soils. AMF isolates were screened in the green house for plant growth improvement and disease tolerance in comparison with identified species-Glomus fasciculatum, G.clarum and Gigaspora margarita. Of the ten AMF tested isolates Is - 6, Pi - 11, Pi - 9, G. fasciculatum and Gigaspora margarita were very effective in stimulating growth and nutrient (P, K, Ca, Mg, Cu, Fe, Mn and Zn) uptake of black pepper. Regarding the ability of AMF in reducing the foot rot incidence, Glomus fasciculatum recorded the lowest plant mortality and root rot index (53.35% and 62.50%) followed by Is - 6 (60.00% and 64.77%) and Pi - 11 (60.64% and 68.18%) as against 100 per cent mortality and 98.60 per cent root rot index noticed in control. The above five cultures were subjected for further studies. Characterisation of AMF associated with different genotypes of black pepper grown in various soil types indicated the definite influence of soil type on AMF colonization. Sandy soil (oxyaquic quartpsamment) harboured maximum root colonization while forest soil (haplic argiustoll) had the lowest. Species of Glomus particularly G. fasciculatum was the predominant AMF associated with black pepper irrespective of soil type. As an exception Acaulospora and Gigaspora species were frequently noticed in sandy soils. Based on the ability of the fungal antagonists to suppress P. capsici in vitro either through mycoparasitism, antibiosis or soil fungistasis, 24 isolates were selected for green house studies. In the further testing isolates A1, A13, A21, A22and A35 significantly reduced the foot rot infection and increased the plant growth. They showed better population build up in the soil and suppressed the P. capsici population considerably. These native antagonists were further tested in combination with selected AMF in the green house and field. Under green house condition, combination of G. fasciculatum x A1 or A21 showed significant influence on growth stimulation, while Is - 6 x A22 recorded lowest mortality of 32.90 per cent due to foot rot incidence as against 97 per cent in control. The dual inoculation of Is - 6 x A21 and Pi - 11 x A1 was highly effective in plant growth stimulation and disease suppression. Both the combination recorded less than 60 per cent infection and mortality due to the disease, while control showed 95.66 per cent infection and plant mortality. Bordeaux mixture and copper oxychloride recorded 66.67 and 59.68 per cent mortality respectively. AMF colonization and multiplication of antagonists were also favoured by dual inoculation. The potential AMF isolates Is - 6 and Pi - 11 were identified as species of Glomus while, the antagonistic isolates A1, A13, A21, A22, and A35 were confirmed as aspergillus fumigatus Fres., Fusarium oxysporum Schlecht. Ex Fr. Aspergillus sydowii (Bain. & Sart.) Thom. & Church, Trichoderma viride Pers. Ex Gray. And Gliomastix murorum (Corda) Hughes respectively. A technique for AMF inoculation to established pepper vines was developed using ‘carrier plants’. Raising sorghum with AMF inoculation around the pepper vines was found effective to achieve intense colonization in pepper roots by the introduced AMF in the field. This technique developed for the pepper vines may be tried for extending to other perennial crops for AMF inoculation. Promising AMF cultures Pi - 11, Is - 6, G. fasciculatum and antagonists Aspergillus fumigatus, A. sydowii, Trichoderma viride were further tested on eight year old established pepper vines following ‘carrier plant’ based AMF inoculation and cowdung - neem cake based antagonist inoculation. The treatment Pi - 11 x A. Sydowii was most effective with no symptom development, followed by Is- 6 x T. Viride or A. sydowii with disease score of 2.0 as against 7.0 recorded for control. The disease score for bordeaux mixture and copper oxychloride application was 3.5 and 3 respectively. Neem cake-cowdung food base was highly favourable for multiplication and activity of fungal antagonists. The amino acids, total sugar and reducing sugar and total phenols and orthodihydroxy phenol content and activity of cellulose and chitinase were influenced by AMF colonization particularly by Is - 6 and Pi - 11. The positive change could be related with the relative disease tolerance recorded for various AMF isolates. The development of native AMF and antagonists through extensive testing in the green house and field and also the technology of AMF inoculation for established pepper vines are the first record of work.

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