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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
Integrated management of foliar fungal disease of culinary melon (Cucumis meloL. var. acidulus Naudin)
(Department of Plant Pathology, College of Agriculture, Vellayani, 2016) Narmadhavathy, S; KAU; Kamala Nayar
The project entitled “Integrated management of foliar fungal disease of culinary melon (Cucumis melo L. var. acidulus Naudin)” was undertaken with the objective of making a comparative evaluation of the efficacy of foliar application of fertilizers, micronutrients, bio-control agents and newer fungicide for the management of Colletotrichum leaf spot (Colletotrichum sp.) disease of culinary melon. Surveys conducted during September 2013 to December 2013, in ten culinary melon fields located at Instructional Farm (IF), College of Agriculture (CoA), Vellayani as well as in farmers’ fields near, CoA, Vellayani, in order to assess the prevalence of major diseases such as Colletotrichum leaf spot and downy mildew disease affecting the crop. Highest disease incidence (DI) and percentage disease index (PDI) of Colletotrichum leaf spot were observed, 75 days after sowing, at Chavadinada (70.00 per cent and 64.44 per cent respectively). Incidence and index of downy mildew disease were recorded in four out of the ten locations surveyed (Palapoor, Papanchani, Kalliyoor and Punjakari). Maximum disease incidence and percentage disease index of downy mildew disease (36 per cent and 33.33 per cent respectively) were observed at Papanchani. The most virulent isolate of anthracnose leaf spot pathogen (IF, Vellayani isolate), obtained during the survey was identified as Colletotrichum fructicola by molecular characterization. The treatment NPK 19:19:19 (0.5 per cent) combined with the fungicide mancozeb (0.4 per cent) and adjuvant was most effective in inhibiting the mycelia growth of the pathogen C. fructicola, in vitro, (100 per cent) over control as well as in suppressing artificially induced anthracnose disease and improving the growth parameters of the plants, in the two greenhouse experiments conducted at the CoA, Vellayani during March to June 2014 and August to October, 2014. Results of two field trials conducted at CoA, Vellayani, during January to March, 2015 and April to June, 2015 for testing four most effective treatments screened from the greenhouse experiments, indicated that NPK 19:19:19 (0.5 per cent) + azoxystrobin (0.15 ml/l) + adjuvant (DI 40.00 and PDI 13.05 respectively) and NPK 19:19:19 (0.5 per cent) + mancozeb (0.4 per cent) + adjuvant (DI 40.00 and PDI 13.47 respectively) were most effective in managing the disease and also increasing total yield of plants, when compared to the remaining treatments. Trials were conducted in farmers’ fields at three locations (Venganoor, Vavamoola and Venjaramoodu) for confirming the efficacy of the two most effective treatments screened from the field trials conducted at CoA, Vellayani and pooled analysis of the results indicated that the lowest PDI (12.22) and DI (28.50) were obtained in plants treated with NPK 19:19:19 (0.5 per cent) + azoxystrobin (0.15ml/l) + adjuvant, which was significantly superior to the other treatments. Results of the microbial studies indicated that there was decline in fungal flora of the plants treated with foliar fertilizer NPK 19:19:19 (0.5 per cent) + azoxystrobin (0.15 ml/l) + adjuvant, days after application of treatments whereas bacterial population was higher in plants applied with the same treatment when compared to the application of combination of foliar fertilizer NPK 19:19:19 (0.5 per cent) + mancozeb (0.4 per cent) + adjuvant. There was indication of higher induction of systemic resistance in plants treated with NPK 19:19:19 (0.5 per cent) + azoxystrobin (0.15 ml/l) + adjuvant due to the higher activity of defense related enzymes, such as phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO), β-1,3glucanase, super oxide dismutase (SOD) and the compound phenol, all of which, reached maximum level on the 15th day after treatment. Leaf samples obtained from plants treated with foliar fertilizer NPK 19:19:19 (0.5 per cent) + azoxystrobin (0.15 ml/l) + adjuvant indicated highest nutrient use efficiency in all three locations of the confirmation trials while highest pigment status due to this treatment was observed in the trial conducted at Venganoor. Relative water content was generally high in leaf samples collected from all plants irrespective of the treatments, although it was comparatively low, in leaf samples obtained from plants of absolute control plot. Epicuticular wax content was slightly lower in the plants treated with combination of the foliar fertilizer NPK 19:19:19 (0.5 per cent) and fungicides, either azoxystrobin (0.15 ml/l) or mancozeb (0.4 per cent) + adjuvant. Stomatal frequency on the upper and lower surfaces of leaves was not much affected by application of foliar fertilizer NPK 19:19:19 (0.5 per cent) combined with the fungicides. B:C estimated ratio revealed that the highest returns were obtained from the plants treated with foliar spray of NPK 19:19:19 (0.5 per cent) + azoxystrobin (0.15 ml/l) + adjuvant, in all three locations of the farmers’ field trials. This study presents the first report of the pathogen Colletotrichum fructicola causing anthracnose leaf spot disease of culinary melon in India. In field conditions, combination of the foliar fertilizer NPK 19:19:19 (0.5%) and azoxystrobin (0.15 ml/l) along with adjuvant applied twice at 15 days’ interval was most effective in controlling anthracnose leaf spot disease of culinary melon and also increasing the yield of the crop.
Open Access
Strain improvement of oyster mushrooms- pleurotus cystidiosus O.K. Mill and pleurotus opuntiae (Durieu and LEV.) SACC.
(Department of Plant Pathology, College of Agriculture, Vellayani, 2018) Krishnapriya, P J; KAU; Geetha, D
The present study entitled “Strain improvement of oyster mushrooms: Pleurotus cystidiosus O.K.Mill and Pleurotus opuntiae (Durieu and Lev.) Sacc.” was carried out in College of Agriculture, Vellayani during 2015-2018, with the objective to standardize the techniques for production of oyster mushrooms: P. cystidiosus and P. opuntiae; and to study their morphological, physiological and cultural characteristics as well as nutritional and organoleptic qualities; and to undertake genetic improvement by protoplast fusion. The mushrooms were collected from two locations of Thiruvananthapuram and three fast growing isolates of Pleurotus spp. viz., PC2 (Vellayani), PNC1 (Chirayinkeezhu) and PO1 (Vellayani) were selected for the study. These isolates were identified as P. cystidiosus subsp. abalonus, P. cystidiosus and P. opuntiae using internal transcribed spacer (ITS) primers and subsequent sequencing; and registered at Genbank database with accession numbers KY214254, KY887023 and KY214255 respectively. The fast growing isolates of P. cystidiosus (coremial), P. cystidiosus (non-coremial) and P. opuntiae recorded maximum growth on PDPA amended with one per cent yeast under dark condition. The optimum temperatures for the growth were 30 0C, 25 to 30 0C and 25 0C respectively whereas, the optimum pH were 8, 8 and 7 to 8 respectively. Studies with different substrates and amendments for spawn production revealed that sorghum with one per cent yeast was the best for P. cystidiosus (coremial) and P. opuntiae whereas, paddy grains with one per cent yeast for P. cystidiosus (non-coremial). Experiments with different substrates and amendments for mushroom production revealed that rubber wood sawdust sprayed with 2.5 per cent of 1 M potassium dihydrogen phosphate recorded the maximum BE for P. cystidiosus (non-coremial) (192.76 per cent). P. opuntiae recorded the maximum BE in rubber wood sawdust amended either with 4 per cent neem cake (91.38 per cent) or wheat bran (91.37 per cent). Major insect pests observed in the beds of Pleurotus spp. were phorid flies, spring tails, black ants and staphylinid beetles. The competitor moulds observed were different species of Coprinus, Aspergillus, Penicillium and Trichoderma. Sporocarps soaked in one per cent CA for 15 minutes followed by mechanical drying and powdering was the best post harvest treatment for both P. cystidiosus (non-coremial) and P. opuntiae. Mycelium of P. cystidiosus (coremial) showed black coremial structures, representing its asexual stage (Antromycopsis broussonetiae Pat. & Trab.). The coremia comprised of elliptical (16.31 µm x 7.48 µm) and round conidia (8.06 to 8.49 µm). The black colour of coremia was due to melanin which was extracted (255.56 mg l-1) and characterized. The performance of long duration P. cystidiosus (non-coremial) and short duration P. opuntiae was compared with two ruling mushrooms of Kerala viz., long duration P. florida (Mont.) Singer and short duration P. eous (Berk.) Sacc. The study revealed that P. cystidiosus (non-coremial) and P. opuntiae showed higher BE compared to P. florida and P. eous, respectively. P. cystidiosus (non-coremial) recorded maximum moisture (94.05 per cent), starch (200.55 mg g-1), protein (30.2 mg g-1), fat (4.25 per cent), antioxidants (485.45 μg equivalent gram of ascorbic acid-1), beta-carotene (25.69 µg 100 mg-1), polyphenols (7.55 mg g-1) and energy (359.45 Kcal) compared to other Pleurotus spp. Sensory evaluation of mushroom products made from the species of Pleurotus was done and masala curry prepared from P. cystidiosus (non-coremial) scored the maximum value for overall acceptability. Shelf life of P. cystidiosus (non-coremial) was higher (5 days) compared to P. opuntiae, P. florida and P. eous (3 days each) in perforated poly propylene covers stored under refrigeration. Vanillin (0.05 per cent) and carbendazim (1 mM) were selected as dual biochemical markers for the PEG mediated protoplast fusion. Three days old P. cystidiosus (non-coremial) and four days old P. opuntiae recorded the maximum protoplast yield at five and four hours after incubation respectively with 0.6 M KCl and 30 mg ml-1 of enzyme consortium. Eight fusant lines with varied mycelial characters were obtained. Among fusants, F6 and F8 did not segregate in the second generation whereas, F4 segregated. F6 and F8 recorded higher BE of 168.05 and 99.95 per cent respectively compared to the parental lines and other fusants. Sporocarp of F6 and F8 was morphologically similar to P. cystidiosus (non-coremial) and P. opuntiae respectively; and F8 also exhibited low temperature adaptability. The present investigation indicated the exploitability of two promising isolates viz. P. opuntiae for tropical areas and P. cystidiosus (non-coremial) for cooler regions of Kerala using locally available materials and the standardized cultivation practices. The present study also standardized the protoplast fusion technique between P. cystidiosus (non-coremial) and P. opuntiae; and two fusant lines (F6 and F8) recorded higher BE which can be used for future breeding programmes.
Open Access
Strain evaluation and production technology of shittake mushroom ( Lentinula edodes ( Berk. ) pegler)
(Department of Plant Pathology, College of Agriculture, Vellayani, 2016) Deepa Rani, C V; KAU; Lulu Das
The present investigation on "Strain evaluation and production technology of Shiitake mushroom (Lentinula edodes (Berk.) Pegler' was conducted at Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during the period 2012-2015. The aim of the experiment was to exploit various strains of Lentinula spp. for novel production technology and their phylogeny analysis through physiological and molecular studies. Surveys were collected during pre and post monsoon periods of May to December from different parts of Thiruvananthapuram, Kollam, Wayanad, Idukki, Pathanamthitta, Kannur and Kasargode districts. Six isolates of sp. (VLYN- 1 to VLYN-13) obtained during the survey were identified and compared with procured reference strains of Lentinula edodes (LE-1 to LE-5 from GB Pant University of Agricultural and Technology, Pantnagar, Uttarakhand) and LE-6 strain (Maharana Pratap University of Agriculture and Technology, Udaipur) . Morphologically the native isolates of Lentinus spp. had concave, funnel and convex pileus with varying colors and were leathery in nature.L. edodes strains in contrast had convex pileus with chocolate brown and golden yellow sporocarps which were fleshy and edible. Phylogenetic analysis of all six strains of L. edodes using RAPD markers confirmed the variability between the strains. Maximum similarity coefficient of 74.10 per cent was observed between LE-2 and LE-6 strains while LE-2 and LE-4 strains showed a minimum similarity coefficient of 35.70 per cent. Further studies by ITS sequencing showed that all the L. edodes strains tested in the study showed 99- 100 per cent similarity with the known sequences off L. edodes available in NCBI database while that of native isolates showed 99- 100 per cent similarity to Lentinus tuber-regium and Lentinus connatus thus confirming the variability between Lentinus and Lentinula sp. All the six strains of L. edodes, showed maximum mycelial growth in malt extract peptone dextrose agar in solid and oat meal broth in liquid medium. L. edodes strains preferred temperature of 20 °C with an acidic pH of 6. Dark and ambient light conditions favored maximum mycelial growth and biomass production for L. edodes culture. Although a minimum period of 16.33 days was required for full mycelial run in maize grains but due to comparatively less contamination rate in paddy grains which took 18.33 days for completion of mycelial run were selected as best substrate for further studies. Different substrates were evaluated for the development of a cultivation package for shiitake mushroom. Results showed that LE-1 strain took minimum of 71.00 days for initiation of sporocarp in sawdust supplemented with 20 per cent wheat bran. Hard wood sawdust especially of teakwood was used in the study. The substrate based on paddy straw and banana pseudo stem were not found effective for pinhead initiation and thus failed to produce sporocarps. LE-1 produced maximum sporocarp (11.33) in sawdust + 20 per cent wheat bran which was followed by LE-3 (10.63) in sawdust + 20 per cent rice bran. Maximum yield of 290.66 g/ 500 g substrate was obtained in sawdust + 20 per cent wheat bran by LE-6 strain. Maximum biological efficiency of 58.13 per cent was also recorded in LE-6 in sawdust supplemented with 20 per cent wheat bran substrate. Substrates like paddy straw and sawdust amended with 20 per cent wheat bran substrates were evaluated for the development of native isolates of Lentinus tuberregium and Lentinus connatus . Results showed that maximum biological efficiency of 58.00 per cent was obtained by Lentinus tuber-regium whereas 36.60per cent biological efficiency by Lentinus connatus in sawdust amended with 20 per cent wheat bran substrate. Nutrient analysis of all the six strains showed that carbohydrate content ranged between 35.29 per cent to 40.23 per cent, protein 18.33 per cent to 21.66 per cent, crude fibre 22.33 per cent to 27.33 per cent, Vitamin- C 2.53 per cent to 3.50 per cent, ash 2.70 per cent to 4.40 per cent and lipid 2.46 per cent to 3.60 per cent. Mineral content of L. edodes included Ca (11.00 mg to 19.00 mg/ 100 g), Mg (0.46 to 1.10 mg/ 100 g), Fe (1.36 mg to 1.80 mg/ 100 g), Mn (1.53 mg to 2.63 mg), P (1.65 mg to 2.87 mg), K (16.33 mg to 25.20 mg), Na (13.00 mg to 23.66 mg) and Zn (19.66 mg to 28.33 mg/ 100 g). Sensory evaluation of mushroom products made from L. edodes was carried out by a panel of judges for various characters of which mushroom masala scored maximum for texture, taste, flavor and overall acceptability when compared to other recipes like mushroom cutlet, scramble, soup, baji and biscuit. As part of the study, paddy grain was found to be the most suitable substrate for spawn production of L. edodes and teakwood sawdust amended with 20 per cent wheat bran was the most efficient bed substrate. LE-6 strain was superior in terms of yield and biological efficiency. Therefore findings of the above investigation recommends the adoption of a suitable cultivation package for shiitake mushroom by using low cost substrates (hardwood sawdust) available in Kerala in plains and hilly regions.
Open Access
Endophytic microorganism mediated systemic resistance in Cocoa against Phytophthora palmivora (Butler) Butler
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2011) Sainamole Kurian, P; KAU; Koshy Abraham
The study on 'Endophytic microorganism mediated systemic resistance in cocoa against Phytophthora palmivora (Butler) Butler was carried out during 2005-2010. The pathogen causing pod rot of cocoa was isolated from infected pods , and its pathogenicity established. Based on cultural and morphological characters, it was identified as Phytophthora palmivora (Butler) Butler. Endophytes were isolated from samples of feeder roots, tender shoots, leaves and pods of cocoa collected from various locations of major cocoa growing area of the state. The population of endophytic microflora varied among different locations and parts of the plant, and in general, the population was more in roots. Bacteriaand fluroscent pseudomonads were more abundant than filamentous fungi and yeasts. Out of the 325 endophytic isolates comprising of 116 bacteria, 153 fluorescent pseudomonads, 34 years and 22 fungi, 82 were found exerting antogonism towards the pathogen. These antagonistic endophytes were further evaluated in In vitro by dual culture and by inoculation on detached cocoa pods, and leaves. It was found that, 25 isolates were more efficient antagonists.
Open Access
Integrated management of viral diseases of bittergourd (momordica charantia L.)
(Department of Plant Pathology, College of Agriculture, Vellayani, 2018) Radhika, N S; KAU; Umamaheswaran, K
The present research work entitled ‘Integrated management of viral diseases of bitter gourd (Momordica charantia L.) was carried out in the College of Agriculture, Vellayani during 2014-2017, with the objectives to study the occurrence and distribution of viruses in bitter gourd in Thiruvananthapuram, Idukki and Palakkad, immunomolecular characterization of the viruses, and screening of antiviral chemicals, antiviral principles of animal, plant and microbial origin for the management of the disease. In the suvey conducted at five locations in Thiruvanaanthapuram district, Pappanchani area recorded highest incidence of viral disease (60%) while highest Vulnerability Index (V.I) was recorded from Vellayani (56.00). In Idukki district, six major bitter gourd cultivating areas were surveyed among which Rajakumary area recorded the highest disease incidence (100%) and V.I (82.00). In Palakkad district, five locations were surveyed, among which panackatri and Thekkepotta recorded highest disease incidence of 88% and highest V.I (69.00). The major insects associated with the crop were whitefly (Bemisia tabaci (Genadius) with an incidence of 10-25%, aphids (Aphis gossypii glover) with an incidence of 10-40%, Jassids (Empoasca (Empoasca) motti Pruthi) with an incidence of 10-30% and mites with an incidence of 10-50%. Phyllody and little leaf symtoms (20% incidence) were also recorded in bittetgourd form Rajakumary and Rajakkad areas in Idukki. Flat limb and multiple proliferation of shoot tip were observed at many fields in Idukki. Symptoms associated with the disease include yellow mottle, mosaic,blistering, leaf curl and reduction in leaf size. Yellow mosaic and blistering is seen in severe infection finally leading to stunting of the plant, reduced flowering an fruiting and hairyness on stem. Mechanical transmission of the virus on Datura stramonium produced yellow lacal lesions indicating the presenceof Bean Golden mosaic virus (Begomo) in the infected leaf extract. This leaf extract also produced local lesions on othe indicator hosts like Chenopodium amaranticolor and Gomphrena globosa indicating the presence of Cucumber mosaic virus (CMV) or Potato virus Y (PVY). The viruses were transmitted by whiteflies (20%) and aphids (30%) from infected bittetgourd plants to healthy seedlings. Whiteflies (Bemisia tabaci Gennadius)) and aphids (Aphis gossypii Glover) are the vectors of the respective viruses Wedge grafting diseases scion on to 3-5 leaf stage healthy seedling of bittergourd produced symptoms of infection within ten days. KAU varieties Preethi and Priyanka were found to be susceptible to infection with preethi expressing a V.I of 70.80 and Priyanka expressing a V.I of 62.50 respectively. Ensyme linked immunosorbent assay (ELISA) and Dot immunobinding assay (DIBA) revealed the presence of three viruses belonging to Begomo, CMV and PVY group causing an mixed infection in bittergourd. The presence of all the three viruses were also confirmed in electron micrograph, Begomovirus as twin particles of size 18-20 X 30nm,CMVas single particles of 18nm and PVY as lonog flexuous rod of size 750nm. PCR amplification of coat protein gene (cp gene) of virus isolates from all the three districts yielded an amplicon of size approximately equal to 570 bp. Idukki and Palakkad isolates showed 94% identity to Tomato leaf Curl Virus isolate TNUDU BGI Coat Protein (AVI) gene while Trivandrum isolate showed 95% identity to Tomato leaf Curl Virus isolate TNPDU BG4 Coat Protein (AV1) gene . Phylogenetic tree constructed using multiple sequence alignment programme showed close relation between Begomo viruses identified in bittergourd from different districts. Studies on defense related enzymes such as peroxidase (PO), polyphenol oxidase (PPO) and phenyl alanine ammonialyase PAL) showed significant activity of PO and PPO in diseased plants than in healthy plants and the activity was on par in healthy and diseased for PAL. Protien profile of healthy and diseased at different days after virus inoculation through grafting indicated the production of novel proteins in diseased. There was no difference in the native profile of peroxidase in healthy and diseased at 15 days after virus inoculation. An additional isozyme band with a Rm value of 0.5 was observed in diseased at 45 days after virus inoculation. Management of the disease with antiviral chemicals and antiviral principles of plant, animal and microbial origin was undertaken as pot culture studies with pre and post inoculation of treatments. Twelve treatments with three replications each were laid out in completely randomized design for the evaluation. The treatments included Aspirin at two levels of 100 and 150 ppm, Salicylic acid (SA) at two levels of 100 and 150 ppm and Acibenzolar S methyl (ASM) at 50 and 75 ppm concentration, and two commercial formulations viz., Perfect and virus –Ex at 0.5 and 1.0 ml concentrations. The treatments were applied three times at 10 days interval. Pre application of thrice sprapying of Acibenzolar S methyl (ASM), 75 ppm concentration (V.I-35.00) at ten days interval was statistically significant over other treatments followed by ASM-50 ppm (V.I-41.33). Post application of antiviral chemicals also showed a statistically significant effect of three times spraying ASM-50 ppm(V.I-25.00) at ten days interval followed by spraying of Virus Ex 1ml L-1 (VThe best eight treatments with control was laid out as Randomised Block Design at the Instructional Farm, College of Agriculture, Vellayani during February to May 2017 as a field trial to study the effect of treatments on natural incidence of the viruses in the susceptible variety Preethi. The treatment, three sprays of ASM-50 ppm (V.I-28.33) at ten days interval ws on par with buttermilk (Three times dilution of curd) (V.I-39.16). Yield was also significantly high in ASM-50 ppm (437g plant-1) followed by Pseudomonas fluorescens talc based formulation (2%) (233 g plant-1)among the treatments.

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