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Open Access
Evaluation of Piriformospora indica against Piper yellow mottle virus in Black pepper
(Department of Plant Pathology, College of Agriculture, Vellayani, 2021) Lekshmi, R Krishnan; KAU; Joy, M
The study entitled “Evaluation of Piriformospora indica against Piper yellow mottle virus (PYMoV) in black pepper” was conducted in Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during 2019-2021, to evaluate the efficiency of the root colonizing endophytic fungus P. indica in managing PYMoV in black pepper, and to elucidate the role of Reactive Oxygen Species and antioxidant enzymes in the tripartite interaction. A survey conducted in the instructional farm of College of Agriculture, Vellayani, to identify and collect PYMoV-infected black pepper plants showed that the surveyed varieties viz., Panniyur 1 to 7 and Karimunda were moderately to highly susceptible to PYMoV infection, with the disease incidence ranging from 10 to 91 per cent and disease severity as vulnerability index from 30 to 58. PYMoV-infected black pepper plants showed symptoms like chlorotic flecking, mottling, light necrosis, crinkling and distortion of leaves, reduction in leaf size and stunted growth. Presence of the virus in the samples was confirmed by polymerase chain reaction (PCR) with oligonucleotide primers specific to open reading frame III (ORF III) of PYMoV. P. indica culture obtained from Department of Plant Pathology was maintained in potato dextrose agar (PDA) and potato dextrose broth (PDB) media. P. indica was mass multiplied in coirpith-dried farm yard manure (FYM) mixture (1:1) amended with two per cent gram flour and was allowed to colonize on rooted cuttings of black pepper. P. indica colonization enhanced different biometric characters at different intervals, compared to the non-colonized control plants. P. indica colonization resulted in 25 per cent more shoot length, 23 per cent more root length, 35 per cent more number of leaves, 23 per cent more leaf area and 27 per cent more number of primary roots than the control plants at 150 days after co-cultivation (DAC). A pot culture experiment was laid out to evaluate the efficiency of prophylactic as well as curative application of P. indica, in managing PYMoV of black pepper. P. indica colonization significantly reduced the incidence and severity of PYMoD, upon pre- as well as post- inoculation of the virus, compared to the control plants. Plants colonized with P. indica and later inoculated with PYMoV as well as plants infected by PYMoV and later colonized by P. indica, both recovered the loss in biomass caused by the virus infection, compared to the non-colonized plants challenged by the virus. However, prophylactic treatment with P. indica resulted in better control of the disease compared to the curative treatment. Amplification of the virus by PCR also revealed that the virus titer was the least in P. indica-colonized plants that were post-inoculated with the virus after 30 DAC, followed by pre-inoculated plants that were later colonized with P. indica. The virus titer was the highest in the non-colonized plants inoculated with the virus. In order to evaluate the performance of P. indica in modulating the biochemical responses of black pepper plants against PYMoV infection, the activity of various enzymes which are known to play a role in the antioxidant defense systems of plants viz. catalase, peroxidase, glutathione reducatse, glutamate synthase, phosphatase and superoxide dismutase were assayed. A significant increase in the activity of these antioxidant enzymes was observed in the P. indica-colonized plants compared to the control plants, upon pre- as well as post- inoculation of PYMoV. Thus, the better performance of P. indica-colonized plants upon PYMoV infection can be correlated to the increased activities of antioxidant enzymes. PYMoV-infected plants showed increased accumulation of reactive oxygen species like superoxide anions and hydrogen peroxide, which cause oxidative stress in the plants. P. indica-colonization was able to mitigate the increase in ROS concentration caused due to PYMoV infection, thus reducing the symptoms. Thus the study reveals that, co-cultivation of rooted cuttings of black pepper (obtained from disease-free mother plants) with P. indica and keeping them under insectproof conditions for around 30 days (for successful establishment of the fungus inside the root cortex) and then planting them in the main field is a reliable and chemical-free method to control PYMoD in black pepper. Bio-protective action of P. indica might be attributed to the increased activity of antioxidant enzymes, which in turn leads to suppression in ROS accumulation in the colonized plants, upon being challenged by the virus.
Open Access
Varietal screening and management of anthracnose of black pepper using new generation fungicides
(Department of Plant Pathology, College of Agriculture, Vellayani, 2021) Athira, K; KAU; Heera, G
The study entitled “Varietal screening and management of anthracnose of black pepper using new generation fungicides” was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during 2018 - 2020 with the objective to screen KAU varieties and most popular local cultivar Karimunda for resistance against anthracnose of black pepper caused by Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. and evolve management strategy using new generation fungicides. As a part of the study, diseased samples were collected from three black pepper growing tracts of Kerala viz., Thiruvananthapuram, Wayanad and Idukki. Sample collections were made from two locations from Thiruvananthapuram (Kowdiar and Vellayani), Wayanad (Meenangadi and Ambalavayal) and Idukki (Myladumpara, Pampadumpara, Kattapana and Kambilikandam). Disease incidence and severity were assessed from the surveyed locations. The highest percentage disease index was observed in Myladumpara (50.28%) followed by Kattapana (48.62%). Weather parameter viz., temperature, relative humidity and rainfall were recorded during the survey period. The weather parameters viz., low temperature, high relative humidity and heavy rainfall favoured the incidence of anthracnose. The symptoms of the anthracnose appeared as small necrotic spots with a yellow halo on the leaf lamina. Several lesions coalesce together resulted in leaf blight and defoliation. In Pampadumpara, spike infection was also observed along with leaf spot. Colletotrichum cultures were isolated from the diseased sample by tissue isolation technique and eight pure cultures of Colletotrichum sp. (C1 to C8) were obtained. The pathogenicity of the eight isolates of Colletotrichum sp. from different locations were proved by Koch postulates. The morphological characters of the eight different isolates were studied in potato dextrose agar (PDA) medium. The isolated cultures of Colletotrichum sp. produced whitish with yellowish orange centre to light pink, off white to greyish coloured colony having fluffy, cottony to sparse mycelial growth with regular margins. Days taken to grow the entire petridish ranged from 7.25 to 9.75 days. The mycelium of the fungus was hyaline and septate, and its width ranged from 2.21 - 3.45 μm. The septal distance of the different Colletotrichum isolates ranged between 8.50 - 21.23 μm. The conidia were single celled with an oil globule at the centre. The conidial shape was either cylindrical, oblong or dumbbell. The conidial and appressorial size varied from 9.4 - 12.1 μm x 3.6 - 4.6 μm and 8.5 – 11.2 μm x 3.5 – 4.3 μm respectively. The isolates were identified as Colletotrichum gloeosporioides. The pathogenic variability of the eight C. gloeosporioides isolates were assessed on three black pepper varieties viz., Panniyur 1, Panniyur 3 and Karimunda by virulence rating. The isolate C7 was identified as the most virulent isolate which produced lesion size of 1.92 cm, 2.40 cm, and 3.22 cm on Panniyur 1, Panniyur 3 and Karimunda respectively at 5 days after inoculation (DAI). The isolate C7 produced symptoms within two days after artificial inoculation in the three varieties tested with a higher rate of lesion development of 0.40 (Panniyur 1), 0.49 (Panniyur 3) and 0.66 (Karimunda) cm day-1. KAU varieties (Panniyur 1 to 8) and local cultivar Karimunda were screened against the most virulent isolate of C. gloeosporioides. Among the varieties screened, Panniyur 4 was found to be highly susceptible with highest PDI of 51.43 (7 DAI), whereas Panniyur 2 had the lowest PDI of 14.28 (7DAI) followed by Panniyur 8 with PDI 20.00 % (7DAI) and were found to be tolerant to anthracnose infection. Panniyur 1, Panniyur 7 and Panniyur 5 were also found to be moderately susceptible. The pathogen produced symptoms in susceptible varieties within 2 DAI, whereas the tolerant varieties took 3-4 days to initiate the infection. In vitro screening of new generation fungicides revealed that kresoxim methyl of strobilurin and tebuconazole of triazole were the most effective in inhibiting mycelial growth of C. gloeosporioides (80.37% and cent percent respectively). The combination fungicide carbendazim 12% + mancozeb 63 % completely inhibited the mycelial growth at 25, 50 and 100 ppm. The combination fungicides azoxystrobin 11% + tebuconazole 18.3% SC and trifloxystrobin 25% + tebuconazole 55% WP were also effective against the pathogen at 100 ppm. The contact fungicide copper oxychloride was ineffective against the pathogen @ 10, 25, 50 and 100 ppm. The present study revealed the use of tolerant varieties along with need based application of new generation fungicides to keep the destructive disease under control. The future line of work should include screening of more black pepper varieties under field condition to assess their reaction to anthracnose, elucidation of the factors governing resistance to the disease and the efficacy of new generation fungicides under field condition.
Open Access
Eco-friendly management of Fusarium rot in cardamom and its impact on soil health and plant defense mechanism
(Department of Plant Pathology, College of Agriculture, Vellayani, 2021) Veni Krishna, K C; KAU; Dhanya, M K
Open Access
Etiology and characterization of diseases of Anthurium (Anthurium andraeanum L.) in Kerala
(Department of Plant Pathology, College of Agriculture, Vellanikkara, 2021) Nitha, Rafi; KAU; Anita Cherian, K
Anthurium (Anthurium andraeanum L.), a native of Tropical America is a perennial herbaceous plant commercially grown for its attractive coloured spathe and green shiny foliage. These have great economic value in the global flower trade that led to the budding of many urban and rural anthurium entrepreneurs in the countryside, particularly in the state of Kerala. During the recent past, the cultivation of this high valued cut flower crop is challenged by many biotic factors and the changes in climate scenario occurred recently led to the emergence of various new pests and diseases. Hence the present study was undertaken to identify and document the diseases affecting anthurium grown in the state of Kerala and the characterization of associated pathogens. Purposive sampling surveys were conducted in the anthurium cultivated locations of six districts viz. Thrissur, Ernakulam, Palakkad, Malappuram, Kozhikode and Wayanad during the period from October, 2020 to July, 2021. The incidence and severity of various symptoms were assessed and collected the samples for further studies. Twelve leaf spots (KMALS, PBALS, VFNLS, VCNLS1, VCNLS2, IJKLS, KKYLS, OKMLS, OLRLS, TLRLS, TBMLS and NLBLS) two leaf blights (VFNLB and OLKLB), one root rot (VCNRR), one wilt (MNTLW) four inflorescence rots (MNTSR, CKDSR, PNMSR and ALVSR) and one mosaic symptom (VCNML) were collected during the survey. Among the leaf spots, PBALS recorded the highest per cent disease severity (PDS) of 58.23 followed by NLBLS (46.66 %) and ALVSR recorded the highest severity of 32 per cent among inflorescence rots. Isolation and pathogenicity studies of the associated pathogens yielded 18 fungal and two bacterial isolates. Symptomatology of these diseases were studied both under natural and artificial conditions. The fungal pathogens were characterized and identified by studying cultural and morphological characteristics. Based on these characters, leaf spot pathogens were identified as Colletotrichum gloeosporioides, Lasiodiplodia theobromae, Phoma sp., Phomopsis sp., Corynespora sp., Pestalotiopsis sp. and those causing inflorescence rot as Colletotrichum gloeosporioides and Phomopsis sp. and root rot pathogen as Phytopythium sp. and wilt pathogen as Fusarium sp. The bacterial isolates causing leaf blight were identified as Xanthomonas sp. based on cultural, morphological and biochemical characteristics. The viral like symptoms subjected to electron microscopy revealed the absence of any virus particles. PCR amplification of Internal Transcribed Spacer (ITS) region of the fungal genome followed by sequencing and in silico analysis confirmed that the pathogen associated with TBMLS as C. queenslandicum, VCNRR as Phytopythium vexans, IJKLS as Diaporthe phaseolarum, KKYLS and OKMLS as Phomopsis heveicola, NLBLS as Lasiodiplodia theobromae, TLRLS as Pseudopestalotiopsis thea, VCNLS2 and OLRLS as Corynespora cassiicola, MNTLW as Fusarium fujikuroi. The pathogens associated with KMALS, PBALS, VFNLS, MNTSR, PNMSR and ALVSR belong to Colletotrichum gloeosporioides species complex. The bacterial isolates (VFNLB and OLKLB) were confirmed as Xanthomonas axonopodis through the amplification of 16S rRNA region of the genome followed by sequencing and in silico analysis. An in vitro experiment was conducted to study the efficacy of fungicides and biocontrol agents/bioformulations against major fungal pathogens. Fungicides viz. propineb, difenoconazole, carbendazim 12 % + mancozeb 64 %, cymoxanil 8% + mancozeb 64 % and Bordeaux mixture were highly effective against L. theobromae and Colletotrichum sp. (PBALS) as it resulted in cent per cent inhibition of these two pathogens at all the three doses. In the case of P. vexans, fungicides viz. propineb, azoxystrobin, carbendazim 12 % + mancozeb 64 %, cymoxanil 8% + mancozeb 64 % and Bordeaux mixture were found to be effective even at a lower dose. Among the three chemicals tested against Xanthomonas axonopodis, maximum inhibition was recorded in the case of streptocycline. Dual culture and filter paper disc method revealed that the biocontrol agents/formulations viz. Trichoderma sp., PGPR-II and PGPM were effective against all the tested fungal and bacterial pathogens. Pseudomonas fluorescens was found effective against three fungal and the bacterial pathogen.
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
Host range studies and management of anthracnose of nutmeg caused by colletotrichum spp.
(Department of Plant Pathology, College of Agriculture, Vellayani, 2020) Bommana, Divya; KAU; Heera, G
The study entitled “Host range studies and management of anthracnose of nutmeg caused by Colletotrichum spp .” was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during 2018-2020 with the objectives to characterize the causal agent, study the host range of Colletotrichum spp. causing anthracnose and to develop effective management strategy to control the disease by using new generation fungicides. As a part of the study, anthracnose infected samples were collected from four nutmeg growing districts of Kerala viz., Thiruvananthapuram, Kottayam, Ernakulam and Idukki. For the infected sample collections, three locations from Thiruvananthapuram (Vellayani, Karamana and Palode), two locations from Kottayam (Kumarakom and Vaikom), six locations from Idukki (Myladumpara, Pampadumpara, Adimali, Kambilikandam, Panickankudy and Kattapana) and one location from Ernakulam (Kadungalloor) were surveyed. Disease incidence and severity were assessed from the surveyed locations. The highest disease incidence and severity were observed in Kadungalloor (DI - 90 % and PDI - 56.40 respectively) followed by Kumarakom (DI - 80 % and PDI - 41.33 respectively) and the lowest disease incidence and severity in Myladumpara (DI - 20 % and PDI - 15.53 respectively). The symptoms of the anthracnose on nutmeg appeared as small necrotic spots with a prominent yellow halo on the leaf lamina. Several lesions coalesced together resulted in leaf blight, shot hole and defoliation. In Kambilikandam and Panickankudy. fruit rot was also observed along with leaf spot. The cultures of Colletotrichum spp. were isolated from the infected samples from different locations. Eighteen pure cultures of Colletotrichum sp. (C1 to C18) were obtained. Seven isolates of Colletotrichum sp. were selected for further studies based on the days taken for symptom development and rate of lesion development. The pathogenicity of the seven isolates of Colletotrichum sp. from different locations were proved by Koch postulates. The morphological and culture characters of the seven different isolates were studied in potato dextrose agar (PDA) medium. The isolated cultures of Colletotrichum sp. produced whitish to greyish radiating mycelium; later turning to off white to pink coloured fluffy to sparse mycelium with regular margins. Days taken to grow the entire Petri dish ranged from 7 to 10. The mycelium of the fungus was hyaline and septate; and its width ranged from 0.46 μm to 2.48 μm. The conidia were single celled with an oil globule at the centre and wereoblong or dumbbell shaped. The conidial size varied from 7.87 to 19.97 μm x 3.26 to 5.68 μm. The isolates were morphologically identified as C. gloeosporioides. The pathogenic variability of the seven isolates of C. gloeosporioides was assessed on detached nutmeg twigs by virulence rating. The isolate C4 was identified as the most virulent isolate which produced lesion size of 11.89 cm and 16.81cm at 7 DAI and 9 DAI respectively. The isolate C4 produced symptoms within two days after artificial inoculation and had a higher rate of lesion development of 4.12 cm day -1 . The other isolates took 3 to 4 days for symptom appearance on artificial inoculation of the pathogen. Host range of the most virulent isolate of C. gloeosporioides (C4) obtained from nutmeg was studied in perennial tree spices viz., clove, cinnamon, all spice, betel vine, black pepper and coconut. C. gloeosporioides isolate of nutmeg is capable of infecting the above- mentioned host plants. The isolate produced symptoms in all the hosts within 2 to 4 DAI and the symptoms developed varied from brown lesions, brown lesions with a shot hole to necrotic spots with prominent yellow halo. The maximum lesion size of 2.43 cm was observed in clove and minimum lesion size of 1.31 cm in all spice. In vitro screening of new generation fungicides revealed that triazole group fungicide propiconazole 25EC at 100 ppm and combination fungicides, carbendazim 12% + mancozeb 63% at 25 ppm; and Trifloxystrobin 25% + Tebuconazole 55% WP at 100 ppm concentration were the most effective in completely inhibiting the mycelial growth of the pathogen. The present study revealed the wide host range of the C. gloeosporioides isolate of nutmeg and also the effectiveness of new generation fungicides in managing the pathogen. The future line of work should include molecular variability between various isolates, cross infectivity among the isolates in other perennial hosts, and the efficacy of new generation fungicides under field condition.
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
Varietal screening and management of anthracnose of black pepper using new generation fungicides
(Department of Plant Pathology, College of Agriculture, Vellayani, 2020) Athira, K; KAU; Heera, G
The study entitled “Varietal screening and management of anthracnose of black pepper using new generation fungicides” was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during 2018 - 2020 with the objective to screen KAU varieties and most popular local cultivar Karimunda for resistance against anthracnose of black pepper caused by Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. and evolve management strategy using new generation fungicides. As a part of the study, diseased samples were collected from three black pepper growing tracts of Kerala viz., Thiruvananthapuram, Wayanad and Idukki. Sample collections were made from two locations from Thiruvananthapuram (Kowdiar and Vellayani), Wayanad (Meenangadi and Ambalavayal) and Idukki (Myladumpara, Pampadumpara, Kattapana and Kambilikandam). Disease incidence and severity were assessed from the surveyed locations. The highest percentage disease index was observed in Myladumpara (50.28%) followed by Kattapana (48.62%). Weather parameter viz., temperature, relative humidity and rainfall were recorded during the survey period. The weather parameters viz., low temperature, high relative humidity and heavy rainfall favoured the incidence of anthracnose. The symptoms of the anthracnose appeared as small necrotic spots with a yellow halo on the leaf lamina. Several lesions coalesce together resulted in leaf blight and defoliation. In Pampadumpara, spike infection was also observed along with leaf spot. Colletotrichum cultures were isolated from the diseased sample by tissue isolation technique and eight pure cultures of Colletotrichum sp. (C1 to C8) were obtained. The pathogenicity of the eight isolates of Colletotrichum sp. from different locations were proved by Koch postulates. The morphological characters of the eight different isolates were studied in potato dextrose agar (PDA) medium. The isolated cultures of Colletotrichum sp. produced whitish with yellowish orange centre to light pink, off white to greyish coloured colony having fluffy, cottony to sparse mycelial growth with regular margins. Days taken to grow the entire petridish ranged from 7.25 to 9.75 days. The mycelium of the fungus was hyaline and septate, and its width ranged from 2.21 - 3.45 μm. The septal distance of the different Colletotrichum isolates ranged between 8.50 - 21.23 μm. The conidia were single celled with an oil globule at the centre. The conidial shape was either cylindrical, oblong or dumbbell. The conidial and appressorial size varied from 9.4 - 12.1 μm x 3.6 - 4.6 μm and 8.5 – 11.2 μm x 3.5 – 4.3 μm respectively. The isolates were identified as Colletotrichum gloeosporioides. The pathogenic variability of the eight C. gloeosporioides isolates were assessed on three black pepper varieties viz., Panniyur 1, Panniyur 3 and Karimunda by virulence rating. The isolate C7 was identified as the most virulent isolate which produced lesion size of 1.92 cm, 2.40 cm, and 3.22 cm on Panniyur 1, Panniyur 3 and Karimunda respectively at 5 days after inoculation (DAI). The isolate C7 produced symptoms within two days after artificial inoculation in the three varieties tested with a higher rate of lesion development of 0.40 (Panniyur 1), 0.49 (Panniyur 3) and 0.66 (Karimunda) cm day-1. KAU varieties (Panniyur 1 to 8) and local cultivar Karimunda were screened against the most virulent isolate of C. gloeosporioides. Among the varieties screened, Panniyur 4 was found to be highly susceptible with highest PDI of 51.43 (7 DAI), whereas Panniyur 2 had the lowest PDI of 14.28 (7DAI) followed by Panniyur 8 with PDI 20.00 % (7DAI) and were found to be tolerant to anthracnose infection. Panniyur 1, Panniyur 7 and Panniyur 5 were also found to be moderately susceptible. The pathogen produced symptoms in susceptible varieties within 2 DAI, whereas the tolerant varieties took 3-4 days to initiate the infection. In vitro screening of new generation fungicides revealed that kresoxim methyl of strobilurin and tebuconazole of triazole were the most effective in inhibiting mycelial growth of C. gloeosporioides (80.37% and cent percent respectively). The combination fungicide carbendazim 12% + mancozeb 63 % completely inhibited the mycelial growth at 25, 50 and 100 ppm. The combination fungicides azoxystrobin 11% + tebuconazole 18.3% SC and trifloxystrobin 25% + tebuconazole 55% WP were also effective against the pathogen at 100 ppm. The contact fungicide copper oxychloride was ineffective against the pathogen @ 10, 25, 50 and 100 ppm. The present study revealed the use of tolerant varieties along with need based application of new generation fungicides to keep the destructive disease under control. The future line of work should include screening of more black pepper varieties under field condition to assess their reaction to anthracnose, elucidation of the factors governing resistance to the disease and the efficacy of new generation fungicides under field condition.
Open Access
Eco-friendly management of Fusarium rot in cardamom and its impact on soil health and plant defence mechanism
(Department of Plant Pathology, College of Agriculture, Vellayani, 2020) Veni Krishna, K C; KAU; Dhanya, M K
The study entitled ‘Eco-friendly management of Fusarium rot in cardamom and its impact on soil health and plant defense mechanism’ was conducted at College of Agriculture, Vellayani and Cardamom Research Station Pampadumpara during 2018- 2020 with the objective to assess Fusarium rot severity and pathogen variability in Idukki district, develop an effective ecofriendly management practices for the disease and study the impact of the practices on the soil and plant health. Six isolates of Fusarium oxysporum Schlecht were obtained, three each from infected pseudostem and roots collected from Kattapana and Nedumkandam blocks of Idukki district. The isolate, Fp1 from pseudostem (Pampadumpara panchayath) was identified as most virulent based on virulence rating (viz., days taken for symptom development, lesion length, mycelial growth), and was used for further studies. Inoculation of the root isolate (Fr1) also produced typical lesion on pseudostem indicating that the, fungus can also attack pseudostem besides the roots. But the root isolate took more days (40 days) for symptom development compared to pseudostem isolate (29 days). The morphological characters including the colour, colony characters, hyphal and conidial characters as well as rate of growth in Petri dish confirmed the fungus as F. oxysporum. In vitro studies revealed that the isolates from pseudostem and roots showed significant variation in their morphological, cultural and pathological characters. A pot culture experiment was conducted to assess the efficacy of selected bioagents (individually and in combination) for the management of the disease at CRS Pampadumpara in CRD using nine treatments with three replications. Pathogen inoculum (150 gm/10 kg soil) multiplied in sand-maize flour medium was standardised as the inoculum level enough for cent per infection on the pseudostem resulting in complete crop loss. Soil application of vermiculite based AMF inoculum (20 g) with @ 2% Pseudomonas fluorescens (1 L/10 kg soil) per 10 kg soil at the time of planting along with 2% P fluorescens spray @ 0.5 L/ plant at monthly interval for three times resulted in effective disease management (disease incidence: 40% and disease severity: 24.26%) compared to the inoculated control (disease incidence: 100% and disease severity: 69.38 %). Studies on the population dynamics of the pathogen and the biocontrol agents at periodical interval upto three months revealed significant reduction in the pathogen antagonist ratio and enhancement in AMF colonization of the treatment plants. Among the best treatment the combination of AMF and P. fluorescens resulted in good biometric characters of the treatment plants (plant height: 96.50 cm ,leaf length: 62.50 cm and number of leaves: 15.75) compared to control (plant height: 41 cm ,leaf length: 30 cm and number of leaves: 7.50) through enhancement of soil nutrients (P, K, Ca and Mg) and plant nutrient status (K, Mg, S and B) compared to control plants. Laboratory studies also revealed the induction of defense related enzymes (phenol, ortho dihydroxy phenol, peroxidase, polyphenol oxidase, and β 1,3 glucanase) in high concentration as a response to the application of above treatments. Thus, the present study revealed that Fusarium rot of cardamom a devastating disease can be effectively managed by soil application of vermiculate based AMF inoculum (20 g) with 2 per cent P. fluorescens (1L/10 kg soil) per 10 kg soil at the time of planting along with 2 per cent P. fluorescens spray at 0.5 L per plant at monthly interval for three times; this treatment also resulted in good vegetative growth of cardamom plants therefore this can be used as an ecofriendly management strategy for the production of good quality cardamom.

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