Pathogenicity Confirmation of Ganoderma Disease of Coconut Using Early Diagnosis Technique

The pathogenicity and diagnostic methods were standardized for Ganoderma disease of coconut. The pathogenicity of Ganoderma lucidum isolated from coconut was tested using six types of inoculation techniques. Two diagnostic methods, viz. indirect enzyme‐linked immunosorbent assay (ELISA) and polymerase chain reactions (PCR) were applied for the confirmation of pathogenicity in coconut seedlings. Polyclonal antibodies (PAbs) were raised against mycelial, basidiocarp and specific proteins of Ganoderma and used for detection of Ganoderma in inoculated seedlings through indirect ELISA technique. All the three PAbs could detect Ganoderma in diseased coconut root tissues in early stage of the disease before symptom expression by indirect – ELISA at the antiserum dilution of 1 : 1000 for mycelial protein, 1 : 700 for Ganoderma specific protein and 1 : 3000 for basidiocarp protein. Low cross‐reactions were observed with saprophytic fungi occurring in coconut roots and also with other basidiomycetous fungi. In PCR, primers Gan1 and Gan2 generated from internal transcribed spacer region of rDNA were used the detection that produced a product of 167‐bp size in Ganoderma infected plants. In the present investigation, spawn inoculum responded earlier within 8 weeks compared with other methods of inoculation as expressed by OD value in ELISA test. This was also confirmed by PCR technique. The combination of these two diagnostic methods for detection of Ganoderma infection was highly reliable, rapid and sensitive.     

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) and dot immunobinding assay (DIBA) for the detection of Ganoderma infecting palms

The genus Ganoderma has a worldwide distribution causing root and stem rot of many plantation crops. A limiting factor in controlling the BSR disease is the lack of reliable diagnostic method(s) for early diagnosis. In this study, we developed polyclonal antiserum for Ganoderma mycelial and extracellular protein, and evaluated its efficacy with different plant samples collected from artificially inoculated coconut seedlings and Ganoderma infected field palms. We also tested the cross-reactivity with the soil-borne and saprophytic fungus collected from different parts of coconut palm. The antisera developed against the crude mycelial protein (CMP) and extracellular protein (ECP) showed a 1:1000 titre value for the detection of Ganoderma. The CMP antisera developed showed more cross-reaction when compared to ECP antisera of Ganoderma. In the DIBA test, at a 1:10 dilution of antigen, 1:1000 dilution of CMP and ECP antisera, 1:5000 dilution of secondary antibody gave clear distinctions in colour development between healthy and diseased samples. In the DIBA test, ECP antisera detected positive control (ECP of Ganoderma MTP and CRS-1 isolate), artificially inoculated roots, infected field roots, infected basal trunk and additionally lesions gave positive reactions which were not found in the CMP antisera tested. Therefore, both ELISA and DIBA tests may be useful for screening a large number of samples and help in the detection of infection at the earliest stage of disease development and this will certainly help to adopt suitable management strategies against Ganoderma disease in palm crops in advance.     

Early immunodiagnosis of Ganoderma infecting coconut seedlings and palms by using monospecific polyclonal antibodies

Among the various fungal diseases affecting plantation crops viz., coconut, aracanut, oil palm, etc. in India, basal stem rot (BSR) caused by species of Ganoderma is the most destructive. A limiting factor in controlling the BSR disease is the lack of reliable diagnostic method(s) for early diagnosis. In this study we generated two different types of antiserum for diagnosis of Ganoderma using the purified monospecific protein (62 kDa) (MS) and crude sporophore extract (SE). We also tested the cross-reactivity with the soil-borne and saprophytic fungus collected from different parts of coconut palm. The antiserum developed against the MS and SE showed 1:700 and 1:3000 titre values for the detection of Ganoderma. The MS antisera developed showed very low or almost no cross-reaction when compared to SE antisera of Ganoderma. In the DIBA test, at a 1:10 dilution of antigen, 1:1000 dilution of CMP and ECP antisera, 1:5000 dilution of secondary antibody gave clear distinctions in colour development between healthy and diseased samples. In DIBA test, both types of antisera were used separately for pathogenicity tests. MS antisera showed a positive reaction for purified protein, artificially infected roots and infected field palm. A mild reaction was observed against infected field trunk but a negative reaction was observed for lesions and leaf samples. In the case of SE antisera, a negative reaction was observed for all leaf samples, healthy roots and healthy trunk samples but positive reactions were observed for positive control, artificially inoculated roots, infected field roots, infected trunk and lesions samples. Therefore, both ELISA and DIBA tests may be useful in the detection of infection at the earliest stage of disease development and this will certainly help in the development of management strategies against Ganoderma disease in palm crops in advance.     

Molecular biology of <Emphasis Type="Italic">Ganoderma</Emphasis> pathogenicity and diagnosis in coconut seedlings

The pathogenicity of Ganoderma boninense was tested on coconut seedlings under greenhouse conditions and infection confirmed by using immunological and molecular diagnostic tools. Desiccation of older leaves and the emergence of sporophores were observed from pathogen-inoculated seedlings, whereas a control seedling does not show any pathogenic symptoms. Mature sporophores were formed within 10–13 weeks after inoculation. Polyclonal antibodies raised against mycelial proteins of Ganoderma were used for detection of Ganoderma in infected field palm and seedlings through indirect enzyme-linked immunosorbent assay technique. We adopted dot-immunobinding assay for the detection of Ganoderma from greenhouse and field samples. Under nucleic-acid-based diagnosis, G. boninense (167 bp) was detected from artificially inoculated seedlings and infected field palms by polymerase chain reaction. Apart from these, histopathological studies also support the Ganoderma pathogenicity in coconut seedlings. The pathogenicity test and combination of all the three diagnostic methods for Ganoderma could be highly reliable, rapid, sensitive and effective screening of resistance in planting material in the future.     

Nucleic acid based detection technique for Ganoderma lucidum in coconut

Basal stem rot caused by Ganoderma lucidum is the most serious disease in coconut and arecanut gardens. Twenty-five Ganoderma isolates were collected from different parts of India and the pathogenicity of Ganoderma was proved on coconut seedlings. Mature sporophores developed within 10–13?weeks after inoculation of pathogen under in vivo. To detect the pathogen at early stage, DNA-based technology, polymerase chain reaction was used. In this, the primers Gan1 and Gan2 produced a product of 167?bp in size for all the Ganoderma isolates tested. Simultaneously, ITS 1 and ITS 4 primers amplified a fragment of 680?bp in the Ganoderma isolates. In addition, Ganoderma isolates showed polymorphism in the random amplified polymorphic DNA analysis.     

Detection of Colletotrichum falcatum causing red rot of sugarcane by enzyme-linked immunosorbent assay

Red rot disease of sugarcane caused by Colletotrichum falcatum Went is one of the most destructive diseases of sugarcane (Saccharum officinarum L.) worldwide. The pathogen spreads primarily through infected sugarcane setts and hence the use of disease-free setts is essential to prevent the disease. In order to develop immunological method for detection of C. falcatum, two proteins with molecular weights of 27 kDa and 45 kDa were purified from the mycelium of C. falcatum race Cf 05 and used as antigen source to raise polyclonal antibodies in NewZealand white rabbit. The developed polyclonal antibodies were tested for detection of C. falcatum by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. The polyclonal antibodies specifically detected C. falcatum in extracts from infected plants, both in immunoblot and ELISA. The ELISA results showed that the developed polyclonal antibodies were highly specific to C.falcatum. The developed antibodies were very sensitive and could detect C.falcatum proteins even at a dilution of 1:50,000. Higher ELISA absorbance values were recorded even at an antigen dilution of 1:500. In western blot analysis, protein bands with molecular weights of 27 kDa and 45 kDa reacting to antisera raised against 27 kDa and 45 kDa mycelial proteins of C. falcatum, respectively, were detected in protein samples from red rot infected canes. The high specific reactivity and sensitivity of the antisera indicate its potential suitability for ELISA-based detection of C. falcatum.     

Control of apple blue mould disease with Torulaspora delbrueckii in combination with Silicon

In this study, Torulaspora delbrueckii alone and in combination with silicon were evaluated for the control of apple blue mould disease caused by Penicillium expansum. In vitro, the antagonistic effects of T. delbrueckii in controlling mycelial growth of P. expansum on potato-dextrose-agar (PDA) in dual cultures, and the growth of P. expansum alone with cell-free metabolites and volatile components of T. delbrueckii were assayed. In vitro, to evaluate the direct effect of silicon on mycelial growth of pathogen, silicon at different concentrations (0.2, 0.4, 0.6, 1 and 2% (wt./vol.)) was added to PDA medium. Silicon at 0.6% (wt./vol.) and above concentrations completely inhibited the mycelial growth of P. expansum. However, it had no significant effect on population dynamics of yeast in vitro and in apple wounds. In vivo, silicon at 0.2 and 1% (wt./vol.) in combination with antagonistic yeast (1 × 10⁸ cell/ml) was a more effective approach to reduce the lesion diameter of blue mould decay of apples than the application of silicon or T. delbrueckii alone at 20 and 4°C, respectively.     

Identification of <Emphasis Type="Italic">Ganoderma</Emphasis>, the causal agent of basal stem rot disease in oil palm using a molecular method

From comparison of the alignments of the internally transcribed spacers (ITS) of ribosomal DNA from Ganoderma associated with oil palm basal stem rot (BSR) and other Ganoderma species, two specific primer pairs were selected to provide a specific DNA amplification of pathogenic Ganoderma in oil palm. Each primer pair produced a single PCR product of about 450 bp (for primer pair IT1–IT2) and 334 bp (for primer pair IT1–IT3) when oil palm Ganoderma DNA was used. No PCR amplification product was observed when other Ganoderma species DNA was used in PCR amplification with these primer pairs. Three specific restriction enzyme sites were identified in the ITS and intergenic spacer (IGS1) regions. The restriction enzymes MluI, SacI and HinfI were used to digest the ITS-PCR product and restriction enzymes TfiI, ScaI and HincII were used to digest the IGS1-PCR product. Of the three restriction enzymes used in each rDNA region, MluI specifically digested the ITS regions, and TfiI specifically digested the IGS1 region of oil palm Ganoderma. Analysis of the published ITS nucleotide sequences of 31 Ganoderma species showed that the MluI restriction site was not present in other Ganoderma species. The use of both specific primers and restriction enzyme analysis can be applied as a standard protocol to identify pathogenic Ganoderma in oil palm. In this study, the use of specific primers and PCR-RFLP analyses of the rDNA gave consistent results for the characterisation of pathogenic Ganoderma, and indicated that Ganoderma strains associated with BSR disease in oil palms belong to a single species.     

Elucidating wood decomposition by four species of Ganoderma from the United States

The laccate (shiny or varnished) Ganoderma contain fungi that are important wood decay fungi of living trees and decomposers of woody debris. They are also an important group of fungi for their degradative enzymes and bioprocessing potential. Laboratory decay microcosms (LDMs) were used to study the relative decay ability of G anoderma curtisii, Ganoderma meredithiae, Ganoderma sessile, and G anoderma zonatum, which are four commonly encountered Ganoderma species in the U.S., across four wood types (Pinus taeda, Quercus nigra, Q uercus virginiana, and Sabal palmetto). Generally, all Ganoderma species were able to decay all types of wood tested despite not being associated with only certain wood types in nature. G. sessile, on average caused the most decay across all wood types. Among the wood types tested, water oak (Q. nigra) had the most mass loss by all species of Ganoderma. Scanning electron microscopy was used to assess micromorphological decay patterns across all treatments. All Ganoderma species simultaneously decayed wood cells of all wood types demonstrating their ability to attack all cell wall components. However, G. zonatum caused selective delignification in some sclerenchyma fibers of the vascular bundles in palm (S. palmetto) as well as in fibers of water oak. In addition, G. zonatum hyphae penetrated fibers of palm and oak wood causing an unusual decay not often observed in basidiomycetes resulting in cavity formation in secondary walls. Cavities within the secondary walls of fibers gradually expanded and coalesced resulting in degradation of the S2 layer. Differences in colony growth rates were observed when Ganoderma species were grown on medium amended with water soluble sapwood extracts from each wood type. G. meredithiae had enhanced growth on all media amended with sapwood extracts, while G. curtisii, G. sessile and G. zonatum had slower growth on loblolly pine extract amended medium.     

Melanin production in Alternaria helianthi

Abstract

Most of the plant pathogenic fungi produce a dark phenolic polymer called melanin. The high performance liquid chromatography (HPLC) analysis of the mycelial extract of Alternaria helianthi revealed an accumulation of scytalone and a shunt metabolite 2-hydroxyjuglone which confirms the production of dihydroxynapthalene type of melanin. The growth and melanin of A. helianthi increased when grown in host extract broth at 6.5 pH and a temperature beyond 30°C had an inhibitory effect on the pathogen. The production and type of melanin produced in Alternaria helianthi is reported for the first time.     

Ecological management of tropical sugar beet (TSB) root rot (Sclerotium rolfsii (Sacc.) by rhizosphere Trichoderma species

Trichoderma species are collected from different location of sugarbeet growing areas of Tamil Nadu and it is effective against Sclerotium rolfsii pathogen caused by sugarbeet ecosystems. Out of thirty-one isolates of Trichoderma viride and four isolates of Trichoderma harzianum collected and tested for their antagonistic activity against S. rolfsii by dual culture technique, one isolate was found to be effective T. viride (TVB1) that recorded the maximum (73.03%) inhibition on the mycelial growth recording only 2.40 cm growth as against 8.90 cm in the control. The isolates of T. harzianum THB-1 recorded 71.19% mycelial growth reduction over control. The colonisation behaviour of T. viride (TVB1) revealed that it completely over grew on pathogen within 48 h after interaction with the pathogen, and speed of growth on pathogen was also high and it possesses a higher level of competitive saprophytic ability. The best four isolates of TVB1, TVB-2, TVB-3 and TVB31 and two isolates of T. harzianum THB-1 and THB-2 were compared with other species of Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma koningii and Chaetomium globosum and tested under in vitro condition. BA of neem cake at 150 kg ha^?1 + T. viride isolate (TVB1) at 2.5 kg/ha recorded least root rot disease incidence of 17.05% which accounted for 75.37% disease reduction over control and highest recorded maximum root yield 65.73 t ha^?1 and increasing sugar content.     

Extending the hyphal area of the ectomycorrhizal fungus Laccaria parva co-cultured with ectomycorrhizosphere bacteria on nutrient agar plate

The members of Proteobacteria have been frequently detected from ectomycorrhizal roots; however, their function in the mycelial growth of ectomycorrhizal fungi remains uncertain. This study examined the extension of the hyphal area of Laccaria parva co-cultured with the ectomycorrhizosphere bacteria. One mycelial disk of L. parva was placed on the center of a plastic dish containing diluted modified Melin-Norkrans agar media, and each bacterial strain was incubated 2?cm away from the mycelial disk at four orthogonal directions. Several strains of Rhizobiaceae, including those closely related to Bradyrhizobium, significantly increased the extension of the hyphal areas of several strains of L. parva; however, the majority of bacteria tended to decrease them. The effects of bacteria on the hyphal growth area differed according to the combination of strains of bacteria and L. parva in several cases, indicating that the interactions between bacteria and L. parva can be specific at the strain level.     

Effect of combination of bio-agents and mineral nutrients for the management of alfalfa wilt pathogen Fusarium oxysporum f. sp. medicaginis

Abstract

Biological and nutrient management of soil borne disease is increasingly gaining stature as a possible practical and safe approach. Inhibitory effects of fungal and bacterial antagonists were tested under in vitro conditions against the wilt pathogen of alfalfa Fusarium oxysporum f. sp. medicaginis. Trichoderma harzianum and Pseudomonas fluorescens (PI 5) were found to be effective against the alfalfa wilt pathogen. Manganese sulphate at 500 and 750 ppm inhibited the mycelial growth of F. oxysporumf. sp. medicaginis under in vitro conditions. In pot culture studies, manganese sulphate at 12.5 mg/kg reduced the wilt incidence (23.33%). Combined application of manganese sulphate 12.5 mg/kg + T. harzianum 1.25 mg/kg of soil significantly reduced the wilt incidence accompanied by improved plant growth and yield in pot culture. The mixture of manganese sulphate (25 kg/ha) + T. harzianum (2.5 kg/ha) significantly reduced the wilt incidence when applied as a basal dose in the field conditions. The average mean of disease reduction was 62.42% over control.     

Changes in pectinase and cellulase activity of Colletotrichum capsici mutants and their effect on Anthracnose disease on capsicum fruit

Abstract

Colletotrichum capsici was identified as a major pathogen that causes Anthracnose disease on capsicum (Capsicum frutescence L.), fruits accounting for 25?–?30% post-harvest loss during storage and marketing. The infection was found to be latent in nature. Towards understanding the latent nature of the pathogen, experiments were carried out to obtain non-pathogenic strain of C. capsici by UV radiation treatment. Exposure of three days old mycelial mat to UV light (312 nm wavelength at 12 in distance from the source) for 15, 30, 45, 60, 75 and 90 minutes was tried. The UV light exposure for 45 minutes was found to be optimum to get less virulent pathogenic strain of C. capsici. It showed a reduction in 42.86% cellulase and 40% pectinase activity. The reduction in activity of these enzymes resulted in 3.5 days delay in manifestation of Anthracnose disease and also reduces the rate of spreading of disease. The UV treatment was found to reduce 40% of the vegetative growth of fungi and 10% decrease in the production of number of conidia. However, no linear response was obtained with increase in dosage of UV treatments.     

A Comparative Study of Caffeine Degradation by Four Different Fungi

The objective of the present study is to investigate the caffeine-degrading abilities of different fungi and to apply this knowledge to environmental remediation and industrial decaffeination process. Chrysosporium keratinophilum, Gliocladium roseum, Fusarium solani, and Aspergillus restrictus were isolated from the coffee pulp obtained from a coffee estate. Pure cultures of fungi were isolated on standard conventional potato dextrose broth (PDB) medium and authenticated. Pure cultures were subjected to a caffeine tolerance study at different concentrations of caffeine (1–8 g/L) in potato dextrose agar (PDA) and minimal media. On PDA, Fusarium solani could tolerate caffeine concentration up to 8 g/L, whereas Chrysosporium keratinophilum, Gliocladium roseum, and Aspergillus restrictus could tolerate up to 6 g/L. On minimal agar medium containing different concentrations of caffeine (1–8 g/L), Fusarium solani tolerated up to 8 g/L and the other fungi up to 2 g/L. A time-bound caffeine degradation study was undertaken at 1 g/L concentration of caffeine and glucose in nitrogen-containing and nitrogen-free liquid minimal media by subjecting the four fungi to shake flask culture at 120 rpm and 30°C. Degradation of caffeine up to 7 days at 24-h intervals was analyzed by high-performance liquid chromatography (HPLC). Gliocladium roseum followed by Aspergillus restrictus showed maximum degradation of caffeine at 0.47 and 0.3 mg/ml, respectively, by 96 h in nitrogen-containing minimal medium, whereas Fusarium solani showed maximum degradation of caffeine by 48 h (0.35 mg/ml) and Chrysosporium keratinophilum by 72 h (0.29 g/ml). In nitrogen-free minimal medium, Chrysosporium keratinophilum showed maximum degradation of caffeine at 72 h (0.45 mg/ml), followed by Gliocladium roseum, Fusarium solani (0.3 mg/ml), and Aspergillus restrictus (0.25 mg/ml) at 96 h. Overall, Chrysosporium keratinophilum showed a comparatively higher rate of caffeine degradation in minimal medium with or without a nitrogen source as compared with the other three fungi, indicating that nitrogen affects caffeine metabolism.     

Effect of carbendazim and mancozeb combinationon Alternaria leaf blight and seed yield in sunflower (Helianthus annus L.)

Abstract

Leaf blight caused by Alternaria helianthi (Hansf.) Tubaki & Nishihara, is the major disease of sunflower affecting the successful cultivation across India. Five individual fungicides and two combination fungicides were evaluated against this pathogen in laboratory and in field experiments. Among them, the combination of carbendazim + mancozeb completely (100%) inhibited the mycelial growth of A. helianthi, irrespective of the concentrations tested followed by carbendazim alone and metalaxyl + mancozeb under in vitro condition. In field conditions, the combination of carbendazim + mancozeb was found to be highly effective in reducing the leaf blight disease of sunflower in all the three experiments as compared to other fungicides and unsprayed control. The reduction of Alternaria leaf blight was also directly associated with an increase in seed yield. The economics of the fungicides spray has been worked out and the benefit cost ratio for the combination of carbendazim + mancozeb at 2.0 g/l was 7.1 as compared to unsprayed control. The overall analysis of the results revealed that the combination of carbendazim + mancozeb at 2.0 g/l can be used for the management of foliar diseases such as Alternaria leaf spot/blight in agricultural crops.     

In vitro kinetics and antifungal activity of various extracts of Terminalia chebula seeds against plant pathogenic fungi

The aim of the present study was to examine the efficacy of various seed extracts of Terminalia chebula as an antifungal potential against certain important plant pathogenic fungi. The organic extracts of methanol, ethyl acetate and chloroform at the used concentration of 1500 ppm/disc revealed remarkable antifungal effect as a fungal mycelial growth inhibitor against Fusarium oxysporum, Fusarium solani, Phytophthora capsici and Botrytis cinerea, in the range of 41.6–61.3%, along with MIC values ranging from 62.5 to 500 μg/ml. Also, the extracts had a strong detrimental effect on spore germination of all the tested plant pathogens along with concentration as well as time-dependent kinetic inhibition of B. cinerea. The results obtained from this study suggest that the natural products derived from Terminalia chebula could become an alternative to synthetic fungicides for controlling such important plant pathogenic fungi.     

Suppression of carbendazim-susceptible and -resistant Sclerotinia sclerotiorum with Macleya alkaloids

Macleaya alkaloids (abr. MCA), an extract from aerial parts of Macleaya cordata, was investigated on suppressing Sclerotinia stem rot disease. The median inhibitory concentrations (EC₅₀) of MCA on mycelia growth were 5.21 μg mL^?1 to carbendazim-susceptible (Ss01) and 6.34 μg mL^?1 to carbendazim-resistant (Hm25) S. sclerotiorum, and there was no cross-resistance between MCA and carbendazim. MCA blocked the mycelial membrane leakage and regulated the exo-secretion of the reducing sugar and oxalate in a concentration-dependent manner. Moreover, MCA also significantly destroyed the redox balance including superoxide dismutase, peroxidase and catalase in Sclerotinia mycelia. In pot experiments, MCA showed an excellent antifungal efficacy on protecting rapeseed leaves from the infection of Ss01 and Hm25 isolates. The results suggested a potential possibility to develop MCA as an agro-chemical to control Sclerotinia stem rot disease and manage carbendazim resistance.     

Life expectancy of oil palm (Elaeis guineensis) infected by Ganoderma boninense in coastal soils,Malaysia: a case study

Ganoderma boninense basal stem rot poses a serious threat to the oil palm industry. The effects of external disease symptoms and coastal soils (Briah – Typic Endoaquepts, Jawa – Typic Sulfaquepts, and Selangor – Typic Humaquepts) on the life expectancy of the infected palms, from disease detection to death, were studied. Six-monthly censuses on disease classes for each palm were recorded between 2004 and 2012. Survival curves of disease symptoms and soil types were compared using Kaplan–Meier and log-rank methods, respectively. Ganoderma-infected palms in acid-sulphate (AS) and potential AS soils recorded lower life expectancy. Survival duration of infected palms with foliar symptoms was 12-months shorter. External factors, such as soil type may influence the survival of infected palms and soil types may pre-dispose oil palm to higher risk of Ganoderma infection. More effective Ganoderma management for palms planted on Coastal soils (with and without AS layer) have been proposed.     

Optimization of Fermentation Medium and Conditions for Mycelial Growth and Water-soluble Exo-polysaccharides Production by Isaria farinosa B05

Abstract

The optimal fermentation medium and conditions for mycelial growth and water-soluble exo-polysaccharides production by Isaria farinosa B05 were investigated. The medium components and fermentation conditions were optimized according to the one at a time method, while the concentration of medium components was determined by the orthogonal matrix method. The results showed that the optimal fermentation medium was as follows: sucrose 3.5% (w/v), peptone 0.5%, yeast extract 0.2%, K₂HPO₄ 0.1%, and MgSO₄ 0.05%. The suitable fermentation conditions were as follows: initial pH 7.0, temperature 25°C, medium volume 75 mL/250 mL, inoculum volume 5% (v/v), time 5d. In such optimal nutrition and environmental conditions, the maximal mycelial yield was 2.124 g/100 mL after 4 day's fermentation, while maximal water-soluble exo-polysaccharides production reached 2.144 g/L after 5 day's fermentation.