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Morphological and molecular characterization of Fusarium sp. causing wilt disease of isabgol (Plantago ovata Forsk.) and its management strategies
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxxx
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Morphological and molecular characterization of Fusarium sp. causing wilt disease of isabgol (Plantago ovata Forsk.) and its management strategies Ram Prasnna Meena*, Satyajit Roy ICAR-Directorate of Medicinal and Aromatic Plants Research, Anand, Gujarat 387310, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Fusarium oxysporum Macroconidia Pathogenicity Incidence Seed dressing
Wilt disease of isabgol (Plantago ovata Forsk.), caused by Fusarium oxysporum species complex is one of the economically important disease in India. The pathogen survive in soil on plant debris and invaded crop plants at any stage from germination to maturity and drastically imposed the production. The disease incidence influenced by several agronomical and environmental factors and varied from 10 to 60 % prevalence. The present study was carried out to confirm the pathogenicity of associated Fusarium species of wilt disease, characterization using the morphological and molecular tools and devise the of management strategies. The Fusarium oxysporum species complex induced typical wilt symptoms on mature isabgol plants whereas, damping off like symptoms on the seedling stage. The fungal colony appeared white to purplish with cottony mycelium growth on PDA. Macroconidia were oval to slightly curved shape and septate in 2–4 cells with tapering pointed ends. The phylogenetic tree constructed based on partial ITS, translation elongation factor (EF-1α) and RNA polymerase II (RPB2) and Fusarium MLTS database confirmed the association of Fusarium oxysporum species complex with the wilt disease of P. ovata. Seed dressing using carbendazim (50 W P) along with the soil application of Trichoderma viride enriched neem cake mixture before sowing significantly reduce the disease incidence. To the best of our knowledge, this is the first comprehensive report on molecular characterization of Fusarium oxysporum associated with wilt disease of P. ovata.
1. Introduction
besides, downy mildew and aphid infestation, wilt disease imposed an important constraint in successful cultivation of the crop. Wilt disease of isabgol was first reported from Arizona, North America (Russel, 1975), later in 1985 it was reported in Haryana, India (Mehta et al., 1985) based on the morphological features and recently from Rajasthan and Gujarat. Fusarium sp. causes damping-off type symptoms at seedling stage whereas typical wilting symptoms observed on mature crops. In the field, the disease occurred in scattered wilt patches. Fusarium spp. are the most common, diverse and widely prevailing fungal pathogens of wilt diseases. Several species of the fungus are also emerged as opportunistic pathogens in human causing hyalohypomycosis disease (Guarro, 2013). The genus Fusarium belongs to Hyphomycetes currently comprises at least 300 phylogenetically distinct species, 20 of them are species complexes and 9 are monotypic lineages (Balajee et al., 2009; O’Donnell et al., 2015). The current species identification is based on the Fusarium multi-locus sequence database. The advanced molecular methods have been developed for formae speciales (f. spp.) and sequencing of the EF-1α, RPB1 and /or RPB2 are required for accurate species identification of Fusarium spp. (O’Donnell et al., 2015; van Diepeningen et al., 2015).
Isabgol (Plantago ovata Forsk.), also known as psyllium, is an economical and medicinal crop of India. Commercial cultivation of P. ovata provides an excellent opportunity with good returns to growers of the drier tracts in western part of India and the crop is successfully growing under the water deficit areas of Rajasthan, Gujarat and Madhya Pradesh during winter months with the minimal fertilizers application. India is leading in the production of isabgol and sole supplier of seed coat ‘husk’ in the international market, which is economically as well as medicinally important part of the. Seed husk widely used in pharmaceutical industries as laxative, in treatment of inflammatory bowel disease, constipation, diarrhea, high blood pressure, etc. (Madgulkar et al., 2015). The increasing demand of crop based products in international market gradually pushed up the acreage in country. However, the crop production is highly influenced and imposed by several biotic as well as abiotic factors. Therefore, the cultivation of crop is confined to particular agro-climate of arid to semi regions zone with the low rainfall as the crop is highly sensitive for water and unseasonal rain which ruined the whole crop produce (Meena, 2019). Among the biotic constraints
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Corresponding author. E-mail address: [email protected] (R.P. Meena).
https://doi.org/10.1016/j.jarmap.2020.100244 Received 12 July 2019; Received in revised form 30 January 2020; Accepted 3 February 2020 2214-7861/ © 2020 Published by Elsevier GmbH.
Please cite this article as: Ram Prasnna Meena and Satyajit Roy, Journal of Applied Research on Medicinal and Aromatic Plants, https://doi.org/10.1016/j.jarmap.2020.100244
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2.3. DNA isolation, amplification and sequencing
The morphological and ITS sequencing based identification and characterization of Fusarium species complex are the routinely employed methods. Fusarium solani complex is non-specific and normally produced root rot, decline in vigor, wilt symptoms and responsible for mortality of taxonomically diverse group of plants. On the other hand numerous formae speciales of Fusarium oxysporum causes seed rot, damping off and vascular wilt symptoms in vegetables, fruits and ornamental plants (Summerell et al., 2003). Wilt diseases complex have been reported on many of the vegetable, field crops, plantation crops, fruit trees, medicinal crops, etc., as the pathogen having wide host range. India is the key producer of isabgol in the world and wilt disease had been reported in 1985 (Russel, 1975; Mehta et al., 1985) and now it is an increasing destructive disease in India, but very limited information about the characterization of the associated Fusarium sp. and management aspects are available so far on this disease. Organic amendments and the microbial consortium are routinely used to improve the soil properties and antagonistic suppressive environment for the soil borne fungal pathogens. Therefore, present research work was carried out to characterize the associated fungus complex using the combined approaches of molecular and morphological techniques. Attempt were also made to investigate the reliable management strategies for wilt disease of isabgol.
The pure culture of fungus was inoculated on potato dextrose broth (PDB) medium in a 200 ml flask and kept at 25 + 2 °C in shaker incubator for 5 days. Further, mycelium of Fusarium isolate was harvested, washed with distilled water and total DNA extracted by using the commercial Nucleo-pore gDNA fungal/bacterial mini kit following the manufacturer’s instructions. The extracted DNA was used as template with the primers in PCR assay for multi-locus gene amplification and sequencing. The internal transcribed spacer (ITS) 1–5.8 S to ITS2 region of the fungus was amplified using the ITS1 and ITS4 primers (White et al., 1990). Moreover, for accurate species identification and molecular characterization of associated Fusarium sp., partial translation elongation factor (EF-1α) (Bischoff et al., 2009) and RNA polymerase II (RPB2) (O’Donnell et al., 2009) were amplified using the corresponding primers with PCR. The PCR reaction was carried out with a total volume of 20 μl, containing 1.5 μl of template DNA, 10.0 μl PCR master mix (Thermo scientific), 0.5 μl of each 10 pmol/μl of forward and reverse primers and sterile nuclease free water for make-up the volume. The PCR program consisted an initial cycle at 94 °C for 4 min, followed by 35 cycles at 94 °C for 35 s, 56 °C for 50 s, and 72 °C for 1 min ending with an extension at 72 °C for 10 min. The amplified products were analysed by gel electrophoresis on agarose gels (1.2 % w/v). The amplicons were purified using commercial kits and sequenced in both the directions.
2. Material and methods
2.4. Sequence analysis and species identification of the pathogens
2.1. Experimental site and sample collection
The sequence contigs of the amplicons were primarily processed by BLASTN analysis of NCBI for putative identification. The nucleotide sequences of partial ITS, EF-1α and RPB2 regions were multiple aligned using Clustal W algorithm of BioEdit 7.1.3., with the related genes sequences respectively, derived from the NCBI database. Further, Fusarium DNA sequence based web database, Fusarium MLTS database (http://www.westerdijkinstitute.nl/fusarium/) was accessed for species identification. The phylogenetic trees based on sequences of three genes were constructed separately using the aligned nucleotide sequences with 1000 bootstrap replicates following maximum likelihood tree phylogeny of MEGA6.0 software (Tamura et al., 2013).
Experiments were conducted during 2017 and 2018 at research farm of the ICAR-Directorate of Medicinal and Aromatic Plants Research, Anand, India, located at 22° 35′ N and 72° 55′ E at an altitude of about 45.1 m above sea level. The soil texture was sandy loam to loamy with 7.62 pH, EC was 0.27 dS m−1, organic carbon 0.3.-0.40 % (medium), whereas fertility status with the available N2 250–280, P2O5 40–50 and K2O5 250–300 K g ha−1 was medium in soil. The infected and symptomatic plants of P. ovata with the root system were collected 60 days after sowing from ten different locations of both the research farms (Boriavi and Lambhvel) of ICAR- DMAPR, Anand. The samples kept separately in paper bags and brought to the laboratory for further study.
2.5. Field experiment The field experiments on management of wilt disease of isabgol were conducted for two consecutive years during 2017 and 2018 in augmented randomized block design (RBD). The experimental site had severe Fusarium wilt disease incidence previous year 2016 in the isabgol throughout the cropping season. The seed of the isabgol (Vallabh isabgol-1) were received from the farm section of ICAR- DMAPR, Anand, sown in lines at 30 cm distance in the 5 × 4 m size plots. A total of ten treatments including one control (Check) replicated thrice for each as per details of the treatments described in Table 1. One hundred plants / m2 of each replication and a total of 300 plants from each treatments were used for evaluation of applied treatments as well as for measuring the diseases incidence. Observations on the disease incidence were reordered 75 days after sowing in the naturally wilt infected experimental plots. Wilt disease incidence in isabgol crop was calculated following the standard method.
2.2. Isolation, pathogenicity and morphological study of the pathogen The symptomatic plant samples brought in laboratory, roots were separated and washed under the running tap water to remove the adhered soil debris. The infected root tissues were mounting in cotton blue and examined under a microscope (Olympus CX 31). The visible infected lesion on roots were excised with the sterilize blade for isolation of the pathogen. The excised portion cut in small pieces and surface sterilized with the sodium hypochlorite solution (NaOCl) of the 4.0 % concentration for 3 min and followed by three washing with the sterile distilled water. The water was removed with the help of sterile tissue paper and transferred on the sterile PDA plates. These inoculated plates were kept on incubation at 25 + 2 °C in BOD incubator. The mycelium growth was observed after 72 h of incubation and pure culture of the fungus was retrieved on MGA 2.5 medium from single spore isolation following the standard procedure of (Choi et al., 1999). To perform the Koch’s postulates, P. ovata seeds were grown on sterilized garden soil in earthen pots. The pathogenicity of isolated Fusarium sp. isolate (isbfu-1) was proved by using the fungal inoculum, prepared from actively grown mycelium of the 7 days old culture in sterilized distilled water. A suspension of 4 × 105 conidia/ml was adjusted and inoculated in the root zone of 15 days old seedling of P. ovata. All the morphological related study of the fungus was performed on 7 days old single spore isolated culture (Pearson et al., 2016).
Disease incidence= (%)
Number of infected plants x 100 Total number of plants observed
2.6. Statistical analysis The data recorded from the experiments were subjected to Dunnett’s statistical test for analysis of variance appropriate to the RBD by using the Microsoft Excel package. The results were presented at 5 % level of 2
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Table 1 Details of different treatment used for management of wilt disease in P. ovata and disease incidence in during 2017-18 and 2018-19 in treatments. Disease incidence (% Plants/m2)
S. No
Details of treatments
T1
Furrow application of neem cake mixture (100 g/m ) and well decomposed FYM (1 kg/m ) enriched with T. viride + P. fluorescens talc based formulation of each @ 2.0% before the sowing Furrow application of neem cake mixture (100 g/m2) enriched with T. viride talc based formulation @ 2.0% before the sowing Furrow application of neem cake mixture (100 g/m2) enriched with P. fluorescens based formulation @ 2.0% before sowing Furrow soil application of neem cake mixture (100 g/m2) before sowing and seed treatment with carbendazim @ 3 g/kg at the sowing time Furrow application of neem cake mixture (100 g/m2) enriched with T. viride talc based formulation @ 2.0% before the sowing and seed treatment with carbendazim @ 3 g/kg at the sowing time Furrow application of neem cake mixture (100 g/m2) enriched with P. fluorescens talc based formulation @ 2.0% before sowing and seed treatment with carbendazim @ 3 g/kg at the sowing time Seed treatment with T. viride talk based formulation @ 2.0% at the sowing time Seed treatment with carbendazim @ 3 gm/kg at the sowing time Furrow application of neem cake mixture (100 g/m2) before the sowing time Control S. Em. C.D. at 5 % CV (%)
T2 T3 T4 T5 T6 T7 T8 T9 T10
2
2
During 2017
During 2018
16
20
18b 24c 30
21b 26c 32d
13a
16a
28d
34
25 32e 38f 56 1.10 3.26 6.78
32 36 42e 61 1.48 4.39 8.00
Superscripted letters indicating significantly differentiate in mitigating the disease incidence over the control. S. Em. ± = standard error of mean; C.D. = critical difference; CV = coefficient of variation; T. viride = Trichoderma viride; P. fluorescens = Pseudomonas fluorescens.
oxysporum f. sp. dianthi (LT841231) followed by 98.58 % with F. oxysporum f. sp. cumini (LT841203) and 98.90 % (1712/1730) with the F. oxysporum f. sp. vasinfactum (KT323870). And the subunit of RNA polymerase II partial sequences shows 99.70 % (981/984) similarity with Fusarium sp. strain CM-CNRG447 (MH935993) followed by 99.59 % (980/984) with F. oxysporum f. sp. cumini (LT841210) and 99.09 % (975/984) with F. oxysporum f. sp. dianthi (LT841238). Further, combined analysis of all three; ITS, EF-1α and RPB2 genes sequences with Fusarium MLTS database (http://www. westerdijkinstitute.nl/fusarium/) showed Fusarium oxysporum species complex with 99.90 % similar to NRRL34936 (JX171533) followed by Fusarium oxysporum species complex with 99.68 % similarity to NRRL 26404 FJ985287) accessions of the GenBank.
significance (P = 0.05). The critical difference (CD) values were calculated to compare the various treatment means. 3. Results 3.1. Symptoms, pathogenicity and morphology of pathogen The F. oxysporum induced damping off type symptoms on seedlings of isabgol under the moist field condition. Whereas, typical wilting symptoms included discolored and distorted leaf with silvery hairs on surface and dried outer leaves whorl were observed on the naturally infected crop plants. The black discoloration of roots tips easily evident when uprooted the wilted plant and internal root tissues became woody (Fig. 1). The presence of whitish fungal hyphae was observed on the root surface and fungal conidia in the xylem tissues of the infected root were detected under microscope. The pathogenicity test results revealed that the isolated fungus Fusaruim oxysporum was found to be pathogenic by producing the similar symptoms as observed on the naturally infected crop plants. The colonies of associated fungus produced white with purple shade with cottony mycelium growth on PDA. The microconidia possessed ellipsoid or kidney shaped whereas the macroconidia were oval, straight to slightly curved and relatively slender in shape. The macroconidia were septate in 2–4 cells with tapering pointed ends and measuring 25–40 μM x 3–7 μM (Fig. 2). Thus, based on the morphological features the fungus was identified as Fusarium oxysporum species.
3.3. Phylogenetic analysis For further understanding the evolution of Fusarium oxysporum (isbfu-1) characterized in this study and relationship with the other Fusarium species, phylogenetic trees were constructed using the aligned sequences of each locus separately viz., ITS, TEF-1α and RPB2 genes. The partial ITS dataset comprising 27 sequences of 7 taxa and sequence of isbfu-1 isolate from our study (MK942699.1) appeared to be related to Fusarium oxysporum (Fig. 3a). As the sequenced isolate isbfu-1 formed a separate cluster in the F. oxysporum and other isolates of the same species complex. The dataset of TEF-1α and RPB2 sequences comprised 30 sequences of each form the different taxa of Fusarium spp. The sequenced isolate isbfu-1 formed a distinct cluster with the F. oxysporum isolates and was to be found to more closely related to the F. oxysporum f. sp. dianthi followed by F. oxysporum f. sp. vasinfectum and F. oxysporum f. sp. cumini based on the phylogenetic three drawn using the TEF-1α sequences (Fig. 3b). Moreover, similar results were obtained based on the RPB2 sequences in phylogenetic analysis (Fig. 3c) confirm the species complex of the Fusarium.
3.2. Sequence analysis and identification of Fusarium species In addition to morphological identification, advanced molecular tools was also used for accurate identification of Fusarium species. The retrieved sequences of ITS, EF-1α and RPB2 were analysed using BLASTN against the NCBI database (https://blast.ncbi.nlm.nih.gov/ Blast.cgi) and confirmed that the sequences of fungus isolate isbfu-1 were of the Fusarium oxysporum group. The assembled sequence of the partial ITS, EF-1α and RPB2 regions were submitted to the NCBI GenBank with the accession number of MK942699.1, MK968885.1 and MK968886.1, respectively. The sequence of ITS region of isbfu-1 showed > 99 % similarity in the BLASTN similarity search with isolates of F. oxysporum (MK448278, MH141287, LC375364, KY989237, etc.). Similarity, EF-1α sequences showed 98.86 % (1735/1755) with F.
3.4. Efficacy of different treatments The results presented in Table 1 revealed that seed treatment with carbendazim (50 W P) at 3 g kg-1 of seed along with the furrow application of neem cake mixture (100 g m−2) enriched with Trichoderma viride talc based formulation each at 2.0 % (T5) at before sowing was found most efficient to reduce the disease incidence in both the consecutive years. Followed by this soil amended with the furrow 3
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Fig. 1. Symptoms of wilt disease (A) Silvery color leaf whorl and dried root system, (B) Healthy and diseased root system, (C) aerial view of symptoms on naturally infected crop plants and (D) field view of management trial.
application of neem cake mixture (100 g m-2) and well decomposed FYM (1 kg/m2) enriched with Trichoderma viride and Pseudomonas fluorescens talc based formulations each at 2.0 % (T1) at before sowing was also significantly reduces the wilt disease of isabgol in the field condition. In three treatments Trichoderma viride (T7), seed treatment with carbendazim (T8) and furrow application of neem cake mixture (T9) were applied singly, and observed that disease incidence was lowest in Trichoderma viride treated plots followed by in T8 where the seed treated with carbendazim, although, sole application of neem cake mixture was not found as effective. Thus seed dressing with carbendazim at 3 g kg−1 along with application of Trichoderma viride enriched neem cake mixture before sowing recorded the best treatment for management of wilt disease of
isabgol.
4. Discussion Genus Fusarium comprised the most important and notorious fungi, consisting diverse species complex and economically destructive pytopathogens associated with wilt diseases on wide range of host plants (Di Pietro et al., 2003; Short et al., 2011; Aoki et al., 2014). The wilt disease of isabgol reported long back in India caused by F. oxysporum and F. solani (Mehta et al., 1985) based on the morphological features. India is the largest producer and exporter of the seed husk of crop and wilt disease imposed significant losses in production. Very limited research work has been done so far on this disease, therefore, in present study
Fig. 2. Fusarium isolate (isbfu-1) (A) colony morphology of the pure culture on PDA and (B) Macro-conidia of Fusarium isolate (isbfu-1) observed under microscope. 4
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Fig. 3. Maximum likelihood phylogenetic tree illustrating the relationship of different Fusarium species based on the partial sequences of ITS, TEF 1 α and RPB2 genes. The isolate isbfu-1 sequences from this study for ITS (MK942699.1), TEF 1 α (MK968885.1) and RPB2 (MK968886.1) genes depicted in red colour in phylogenetic trees in Fig. 3a, b and c, respectively. The evolutionary history was inferred by ML method based on the Tamura-Nei model and bootstrap support values with 1000 replicates are shown at the nodes of the branches. The branches showed bootstrap values less than 60 % were collapsed. The scale bar indicates the number of substitutions per site.
genes, which is essentially required for accurate species identification of Fusarium species complex (Wang et al., 2011; O’Donnell et al., 2015; van Diepeningen et al., 2015). The sequence based phylogenetic trees provide important information on systematics of the Fusarium species complex, however, the incongruent topologies among the single gene based trees and lack of resolution to distinguish species boundaries were also observed within the Fusarium oxysporum species complex (O’Donnell et al., 2009). Besides the phylogenetic analysis, the sequence of ITS, EF-1α and RPB2 genes used in Fusarium MLTS database (http://www.westerdijkinstitute.nl/fusarium/) also demonstrated that the sequenced isolate (isbfu-1) in our study belongs to Fusarium oxysporum species complex causes wilt disease of P. ovata. Wilt disease management required multifaceted approaches and isabgol, which is a medicinal plant essentially needed more attention. It is widely grown in western part of the country and very limited fungicides are advised for management of diseases. It had been observed during the crop cycle that wilt disease occurred at any stage, from the seed germination to maturity as also reported in previous studies. Fusarium oxysporum is associated with the seeds and considered as seed borne fungi of P. ovata (Mehta et al., 1985; Elmer et al., 1996; Elwakil and Ghoneem, 1999). The combined approaches of seed treatment, incorporating of organic amendment in soil and application of biocontrol agents suppress the disease severity (Sivan and Chet, 1986; Blok et al., 1997; Arriola et al., 2000). Seed dressing with carbendazim at 3 g kg−1along with the Trichoderma viride enriched neem cake mixture before sowing significantly reduce the disease incidence and recorded the best treatment for management of wilt disease of isabgol.
the associated pathogen of isabgol wilt disease was characterized using advanced molecular tools (ITS, EF-1α and RPB2) besides the morphological keys, for accurate characterization of Fusarium spp. Additionally, documentation of symptoms, pathogenicity, and disease management strategies were also attempted. Fusarium oxysporum species complex (FOSC) was associated with wilt disease of P. ovata and also found to be pathogenic. The silvery grey outer whorl of the dried leaf with the brown to dark brown discoloration of the xylem tissues of the root system are the characteristic of wilt disease in P. ovata. Different Fusarium species were isolated from the rhizosphere with varying frequencies but Fusarium oxysporum was most frequently isolated on the symptomatic roots and pathogenic on P. ovata. The consistent presence of this fungus in wilted plants roots and rhizosphere region, suggesting the potential involvement in wilt disease incidence. It is supported from the previous studies that wilt disease of Plantago caused by Fusarium oxysporum was first reported in United States (Russell, 1975) and in India it was reported by Fusarium solani besides the F. oxysporum (Mehta et al., 1985). The pathogenicity of Fusarium isolate (isbfu-1) and identification of the pathogen was determined based on the morphological features, which is prerequisite and indispensable part to separate the fungal species (Leslie and Summerell, 2006). The pure culture of isolated Fusarium sp. produced white to pale pigmented colony on PDA. Morphotype of fungus was examined with the microscopy and variable shape and size of the microconidia and macroconidia were observed as reported in the earlier studies (Hafizi et al., 2013). However, identification of Fusarium species based on morphological features alone encountered several discrepancies as frequently observed in previous studies (Russel, 1975; Mehta et al., 1985; Marasas et al., 2001; O’Donnell et al., 2015). Now PCR based assay and sequence based identification are essentially required for accurate taxonomical characterization of Fusarium species complex. Sequences of ITS region had been routinely used in phylogenetic analysis of many fungus genera at species level (Taylor et al., 2000) and was selected as official barcode locus (Schoch et al., 2012), but often uninformative at the species level and aligned across the member of species complex or closely related ones (O’Donnell et al., 2015). Therefore, besides the morphological features and ITS sequencing, the wilt disease associated Fusarium sp. was characterized based on the sequencing of partial EF-1α and RPB2
5. Conclusions In conclusion, the present study stated that Fusarium oxysporum species complex causes wilt disease of P. ovata as most frequently isolated from the diseased plant samples. The species was determined based on the sequences of three genes viz., ITS, EF-1α and RPB2. Although, further studies on variability, virulence and cross pathogenicity of Fusarium will provide the crucial information to understand the species complex. Wilt disease of isabgol may be managed through the integration of seed treatment using carbendazim and soil amendments by biocontrol agents, Trichoderma viride enriched organic 5
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products.
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Funding The author (s) received the financial support from its institutional research grant of ICAR- DMAPR, Anand for the research work. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Declaration of Competing Interest The authors declare that they have no conflict of interest about this manuscript and research. Acknowledgment The work has been funded by ICAR- Directorate of Medicinal and Aromatic Plants Research (DMAPR), Anand, from its institutional research grant. The authors express their sincere thanks to Dr. P. Manivel who provide the seeds and gratitude to the Director, ICAR-DMAPR, for extending research facilities for conducting this research work. References Aoki, T., O’Donnell, K., Geiser, D.M., 2014. Systematics of key phytopathogenic Fusarium species: current status and future challenges. Journal of General Plant Pathology 80, 189–201. Arriola, L.L., Hausbeck, M.K., Rogers, J., Safir, G.R., 2000. The effect of Trichoderma harzianum and arbuscular mycorrhizae on Fusarium root rot in asparagus. Horticulture Technology 10, 141–144. Balajee, S.A., Borman, A.M., Brandt, M.E., Cano, J., Cuenca-Estrella, M., Dannaoui, E., Guarro, J., Haase, G., Kibbler, C.C., Meyer, W., O’Donnell, K., Petti, C.A., RodriguezTudela, J.L., Sutton, D., Velegraki, A., Wickes, B.L., 2009. Sequence-based identification of Aspergillus, fusarium, and mucorales species in the clinical mycology laboratory: where are we and where should we go from here? Journal of Clinical Microbiology 47 (4), 877–884. Bischoff, J.F., Rehner, S.A., Humber, R.A., 2009. A multilocus phylogeny of the Metarhizium anisopliae lineage. Mycologia 101, 512–530. Blok, W.J., Zwankhuizen, M.J., Bollen, G.J., 1997. Biological control of Fusarium oxysporum f. Sp. Asparagi by applying non-pathogenic isolates of F. oxysporum. Biocontrol Science and Technology 7, 527–541. Choi, Y.W., Hyde, K.D., Ho, W.H., 1999. Single spore isolation of fungi. Fungal Diversity 3, 29–38. Di Pietro, A., Madrid, M.P., Caracuel, Z., Delgado-Jarana, J., Roncero, M.I.G., 2003. Fusarium oxysporum: exploring the molecular arsenal of a vascular wilt pathogen. Molecular Plant Pathology 4, 315–325. Elmer, W.H., Johnson, D.A., Mink, G.I., 1996. Epidemiology and management of diseases causal to asparagus decline. Plant Disease 80, 117–125. Elwakil, M.A., Ghoneem, K.M., 1999. Detection and location of seed-borne Fungi of blonde psyllium and their transmission in seedlings. Pakistan Journal of Biological
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Morphological and molecular characterization of Fusarium sp. causing wilt disease of isabgol (Plantago ovata Forsk.) and its management strategies Ram Prasnna Meena*, Satyajit Roy ICAR-Directorate of Medicinal and Aromatic Plants Research, Anand, Gujarat 387310, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Fusarium oxysporum Macroconidia Pathogenicity Incidence Seed dressing
Wilt disease of isabgol (Plantago ovata Forsk.), caused by Fusarium oxysporum species complex is one of the economically important disease in India. The pathogen survive in soil on plant debris and invaded crop plants at any stage from germination to maturity and drastically imposed the production. The disease incidence influenced by several agronomical and environmental factors and varied from 10 to 60 % prevalence. The present study was carried out to confirm the pathogenicity of associated Fusarium species of wilt disease, characterization using the morphological and molecular tools and devise the of management strategies. The Fusarium oxysporum species complex induced typical wilt symptoms on mature isabgol plants whereas, damping off like symptoms on the seedling stage. The fungal colony appeared white to purplish with cottony mycelium growth on PDA. Macroconidia were oval to slightly curved shape and septate in 2–4 cells with tapering pointed ends. The phylogenetic tree constructed based on partial ITS, translation elongation factor (EF-1α) and RNA polymerase II (RPB2) and Fusarium MLTS database confirmed the association of Fusarium oxysporum species complex with the wilt disease of P. ovata. Seed dressing using carbendazim (50 W P) along with the soil application of Trichoderma viride enriched neem cake mixture before sowing significantly reduce the disease incidence. To the best of our knowledge, this is the first comprehensive report on molecular characterization of Fusarium oxysporum associated with wilt disease of P. ovata.
1. Introduction
besides, downy mildew and aphid infestation, wilt disease imposed an important constraint in successful cultivation of the crop. Wilt disease of isabgol was first reported from Arizona, North America (Russel, 1975), later in 1985 it was reported in Haryana, India (Mehta et al., 1985) based on the morphological features and recently from Rajasthan and Gujarat. Fusarium sp. causes damping-off type symptoms at seedling stage whereas typical wilting symptoms observed on mature crops. In the field, the disease occurred in scattered wilt patches. Fusarium spp. are the most common, diverse and widely prevailing fungal pathogens of wilt diseases. Several species of the fungus are also emerged as opportunistic pathogens in human causing hyalohypomycosis disease (Guarro, 2013). The genus Fusarium belongs to Hyphomycetes currently comprises at least 300 phylogenetically distinct species, 20 of them are species complexes and 9 are monotypic lineages (Balajee et al., 2009; O’Donnell et al., 2015). The current species identification is based on the Fusarium multi-locus sequence database. The advanced molecular methods have been developed for formae speciales (f. spp.) and sequencing of the EF-1α, RPB1 and /or RPB2 are required for accurate species identification of Fusarium spp. (O’Donnell et al., 2015; van Diepeningen et al., 2015).
Isabgol (Plantago ovata Forsk.), also known as psyllium, is an economical and medicinal crop of India. Commercial cultivation of P. ovata provides an excellent opportunity with good returns to growers of the drier tracts in western part of India and the crop is successfully growing under the water deficit areas of Rajasthan, Gujarat and Madhya Pradesh during winter months with the minimal fertilizers application. India is leading in the production of isabgol and sole supplier of seed coat ‘husk’ in the international market, which is economically as well as medicinally important part of the. Seed husk widely used in pharmaceutical industries as laxative, in treatment of inflammatory bowel disease, constipation, diarrhea, high blood pressure, etc. (Madgulkar et al., 2015). The increasing demand of crop based products in international market gradually pushed up the acreage in country. However, the crop production is highly influenced and imposed by several biotic as well as abiotic factors. Therefore, the cultivation of crop is confined to particular agro-climate of arid to semi regions zone with the low rainfall as the crop is highly sensitive for water and unseasonal rain which ruined the whole crop produce (Meena, 2019). Among the biotic constraints
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Corresponding author. E-mail address: [email protected] (R.P. Meena).
https://doi.org/10.1016/j.jarmap.2020.100244 Received 12 July 2019; Received in revised form 30 January 2020; Accepted 3 February 2020 2214-7861/ © 2020 Published by Elsevier GmbH.
Please cite this article as: Ram Prasnna Meena and Satyajit Roy, Journal of Applied Research on Medicinal and Aromatic Plants, https://doi.org/10.1016/j.jarmap.2020.100244
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2.3. DNA isolation, amplification and sequencing
The morphological and ITS sequencing based identification and characterization of Fusarium species complex are the routinely employed methods. Fusarium solani complex is non-specific and normally produced root rot, decline in vigor, wilt symptoms and responsible for mortality of taxonomically diverse group of plants. On the other hand numerous formae speciales of Fusarium oxysporum causes seed rot, damping off and vascular wilt symptoms in vegetables, fruits and ornamental plants (Summerell et al., 2003). Wilt diseases complex have been reported on many of the vegetable, field crops, plantation crops, fruit trees, medicinal crops, etc., as the pathogen having wide host range. India is the key producer of isabgol in the world and wilt disease had been reported in 1985 (Russel, 1975; Mehta et al., 1985) and now it is an increasing destructive disease in India, but very limited information about the characterization of the associated Fusarium sp. and management aspects are available so far on this disease. Organic amendments and the microbial consortium are routinely used to improve the soil properties and antagonistic suppressive environment for the soil borne fungal pathogens. Therefore, present research work was carried out to characterize the associated fungus complex using the combined approaches of molecular and morphological techniques. Attempt were also made to investigate the reliable management strategies for wilt disease of isabgol.
The pure culture of fungus was inoculated on potato dextrose broth (PDB) medium in a 200 ml flask and kept at 25 + 2 °C in shaker incubator for 5 days. Further, mycelium of Fusarium isolate was harvested, washed with distilled water and total DNA extracted by using the commercial Nucleo-pore gDNA fungal/bacterial mini kit following the manufacturer’s instructions. The extracted DNA was used as template with the primers in PCR assay for multi-locus gene amplification and sequencing. The internal transcribed spacer (ITS) 1–5.8 S to ITS2 region of the fungus was amplified using the ITS1 and ITS4 primers (White et al., 1990). Moreover, for accurate species identification and molecular characterization of associated Fusarium sp., partial translation elongation factor (EF-1α) (Bischoff et al., 2009) and RNA polymerase II (RPB2) (O’Donnell et al., 2009) were amplified using the corresponding primers with PCR. The PCR reaction was carried out with a total volume of 20 μl, containing 1.5 μl of template DNA, 10.0 μl PCR master mix (Thermo scientific), 0.5 μl of each 10 pmol/μl of forward and reverse primers and sterile nuclease free water for make-up the volume. The PCR program consisted an initial cycle at 94 °C for 4 min, followed by 35 cycles at 94 °C for 35 s, 56 °C for 50 s, and 72 °C for 1 min ending with an extension at 72 °C for 10 min. The amplified products were analysed by gel electrophoresis on agarose gels (1.2 % w/v). The amplicons were purified using commercial kits and sequenced in both the directions.
2. Material and methods
2.4. Sequence analysis and species identification of the pathogens
2.1. Experimental site and sample collection
The sequence contigs of the amplicons were primarily processed by BLASTN analysis of NCBI for putative identification. The nucleotide sequences of partial ITS, EF-1α and RPB2 regions were multiple aligned using Clustal W algorithm of BioEdit 7.1.3., with the related genes sequences respectively, derived from the NCBI database. Further, Fusarium DNA sequence based web database, Fusarium MLTS database (http://www.westerdijkinstitute.nl/fusarium/) was accessed for species identification. The phylogenetic trees based on sequences of three genes were constructed separately using the aligned nucleotide sequences with 1000 bootstrap replicates following maximum likelihood tree phylogeny of MEGA6.0 software (Tamura et al., 2013).
Experiments were conducted during 2017 and 2018 at research farm of the ICAR-Directorate of Medicinal and Aromatic Plants Research, Anand, India, located at 22° 35′ N and 72° 55′ E at an altitude of about 45.1 m above sea level. The soil texture was sandy loam to loamy with 7.62 pH, EC was 0.27 dS m−1, organic carbon 0.3.-0.40 % (medium), whereas fertility status with the available N2 250–280, P2O5 40–50 and K2O5 250–300 K g ha−1 was medium in soil. The infected and symptomatic plants of P. ovata with the root system were collected 60 days after sowing from ten different locations of both the research farms (Boriavi and Lambhvel) of ICAR- DMAPR, Anand. The samples kept separately in paper bags and brought to the laboratory for further study.
2.5. Field experiment The field experiments on management of wilt disease of isabgol were conducted for two consecutive years during 2017 and 2018 in augmented randomized block design (RBD). The experimental site had severe Fusarium wilt disease incidence previous year 2016 in the isabgol throughout the cropping season. The seed of the isabgol (Vallabh isabgol-1) were received from the farm section of ICAR- DMAPR, Anand, sown in lines at 30 cm distance in the 5 × 4 m size plots. A total of ten treatments including one control (Check) replicated thrice for each as per details of the treatments described in Table 1. One hundred plants / m2 of each replication and a total of 300 plants from each treatments were used for evaluation of applied treatments as well as for measuring the diseases incidence. Observations on the disease incidence were reordered 75 days after sowing in the naturally wilt infected experimental plots. Wilt disease incidence in isabgol crop was calculated following the standard method.
2.2. Isolation, pathogenicity and morphological study of the pathogen The symptomatic plant samples brought in laboratory, roots were separated and washed under the running tap water to remove the adhered soil debris. The infected root tissues were mounting in cotton blue and examined under a microscope (Olympus CX 31). The visible infected lesion on roots were excised with the sterilize blade for isolation of the pathogen. The excised portion cut in small pieces and surface sterilized with the sodium hypochlorite solution (NaOCl) of the 4.0 % concentration for 3 min and followed by three washing with the sterile distilled water. The water was removed with the help of sterile tissue paper and transferred on the sterile PDA plates. These inoculated plates were kept on incubation at 25 + 2 °C in BOD incubator. The mycelium growth was observed after 72 h of incubation and pure culture of the fungus was retrieved on MGA 2.5 medium from single spore isolation following the standard procedure of (Choi et al., 1999). To perform the Koch’s postulates, P. ovata seeds were grown on sterilized garden soil in earthen pots. The pathogenicity of isolated Fusarium sp. isolate (isbfu-1) was proved by using the fungal inoculum, prepared from actively grown mycelium of the 7 days old culture in sterilized distilled water. A suspension of 4 × 105 conidia/ml was adjusted and inoculated in the root zone of 15 days old seedling of P. ovata. All the morphological related study of the fungus was performed on 7 days old single spore isolated culture (Pearson et al., 2016).
Disease incidence= (%)
Number of infected plants x 100 Total number of plants observed
2.6. Statistical analysis The data recorded from the experiments were subjected to Dunnett’s statistical test for analysis of variance appropriate to the RBD by using the Microsoft Excel package. The results were presented at 5 % level of 2
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Table 1 Details of different treatment used for management of wilt disease in P. ovata and disease incidence in during 2017-18 and 2018-19 in treatments. Disease incidence (% Plants/m2)
S. No
Details of treatments
T1
Furrow application of neem cake mixture (100 g/m ) and well decomposed FYM (1 kg/m ) enriched with T. viride + P. fluorescens talc based formulation of each @ 2.0% before the sowing Furrow application of neem cake mixture (100 g/m2) enriched with T. viride talc based formulation @ 2.0% before the sowing Furrow application of neem cake mixture (100 g/m2) enriched with P. fluorescens based formulation @ 2.0% before sowing Furrow soil application of neem cake mixture (100 g/m2) before sowing and seed treatment with carbendazim @ 3 g/kg at the sowing time Furrow application of neem cake mixture (100 g/m2) enriched with T. viride talc based formulation @ 2.0% before the sowing and seed treatment with carbendazim @ 3 g/kg at the sowing time Furrow application of neem cake mixture (100 g/m2) enriched with P. fluorescens talc based formulation @ 2.0% before sowing and seed treatment with carbendazim @ 3 g/kg at the sowing time Seed treatment with T. viride talk based formulation @ 2.0% at the sowing time Seed treatment with carbendazim @ 3 gm/kg at the sowing time Furrow application of neem cake mixture (100 g/m2) before the sowing time Control S. Em. C.D. at 5 % CV (%)
T2 T3 T4 T5 T6 T7 T8 T9 T10
2
2
During 2017
During 2018
16
20
18b 24c 30
21b 26c 32d
13a
16a
28d
34
25 32e 38f 56 1.10 3.26 6.78
32 36 42e 61 1.48 4.39 8.00
Superscripted letters indicating significantly differentiate in mitigating the disease incidence over the control. S. Em. ± = standard error of mean; C.D. = critical difference; CV = coefficient of variation; T. viride = Trichoderma viride; P. fluorescens = Pseudomonas fluorescens.
oxysporum f. sp. dianthi (LT841231) followed by 98.58 % with F. oxysporum f. sp. cumini (LT841203) and 98.90 % (1712/1730) with the F. oxysporum f. sp. vasinfactum (KT323870). And the subunit of RNA polymerase II partial sequences shows 99.70 % (981/984) similarity with Fusarium sp. strain CM-CNRG447 (MH935993) followed by 99.59 % (980/984) with F. oxysporum f. sp. cumini (LT841210) and 99.09 % (975/984) with F. oxysporum f. sp. dianthi (LT841238). Further, combined analysis of all three; ITS, EF-1α and RPB2 genes sequences with Fusarium MLTS database (http://www. westerdijkinstitute.nl/fusarium/) showed Fusarium oxysporum species complex with 99.90 % similar to NRRL34936 (JX171533) followed by Fusarium oxysporum species complex with 99.68 % similarity to NRRL 26404 FJ985287) accessions of the GenBank.
significance (P = 0.05). The critical difference (CD) values were calculated to compare the various treatment means. 3. Results 3.1. Symptoms, pathogenicity and morphology of pathogen The F. oxysporum induced damping off type symptoms on seedlings of isabgol under the moist field condition. Whereas, typical wilting symptoms included discolored and distorted leaf with silvery hairs on surface and dried outer leaves whorl were observed on the naturally infected crop plants. The black discoloration of roots tips easily evident when uprooted the wilted plant and internal root tissues became woody (Fig. 1). The presence of whitish fungal hyphae was observed on the root surface and fungal conidia in the xylem tissues of the infected root were detected under microscope. The pathogenicity test results revealed that the isolated fungus Fusaruim oxysporum was found to be pathogenic by producing the similar symptoms as observed on the naturally infected crop plants. The colonies of associated fungus produced white with purple shade with cottony mycelium growth on PDA. The microconidia possessed ellipsoid or kidney shaped whereas the macroconidia were oval, straight to slightly curved and relatively slender in shape. The macroconidia were septate in 2–4 cells with tapering pointed ends and measuring 25–40 μM x 3–7 μM (Fig. 2). Thus, based on the morphological features the fungus was identified as Fusarium oxysporum species.
3.3. Phylogenetic analysis For further understanding the evolution of Fusarium oxysporum (isbfu-1) characterized in this study and relationship with the other Fusarium species, phylogenetic trees were constructed using the aligned sequences of each locus separately viz., ITS, TEF-1α and RPB2 genes. The partial ITS dataset comprising 27 sequences of 7 taxa and sequence of isbfu-1 isolate from our study (MK942699.1) appeared to be related to Fusarium oxysporum (Fig. 3a). As the sequenced isolate isbfu-1 formed a separate cluster in the F. oxysporum and other isolates of the same species complex. The dataset of TEF-1α and RPB2 sequences comprised 30 sequences of each form the different taxa of Fusarium spp. The sequenced isolate isbfu-1 formed a distinct cluster with the F. oxysporum isolates and was to be found to more closely related to the F. oxysporum f. sp. dianthi followed by F. oxysporum f. sp. vasinfectum and F. oxysporum f. sp. cumini based on the phylogenetic three drawn using the TEF-1α sequences (Fig. 3b). Moreover, similar results were obtained based on the RPB2 sequences in phylogenetic analysis (Fig. 3c) confirm the species complex of the Fusarium.
3.2. Sequence analysis and identification of Fusarium species In addition to morphological identification, advanced molecular tools was also used for accurate identification of Fusarium species. The retrieved sequences of ITS, EF-1α and RPB2 were analysed using BLASTN against the NCBI database (https://blast.ncbi.nlm.nih.gov/ Blast.cgi) and confirmed that the sequences of fungus isolate isbfu-1 were of the Fusarium oxysporum group. The assembled sequence of the partial ITS, EF-1α and RPB2 regions were submitted to the NCBI GenBank with the accession number of MK942699.1, MK968885.1 and MK968886.1, respectively. The sequence of ITS region of isbfu-1 showed > 99 % similarity in the BLASTN similarity search with isolates of F. oxysporum (MK448278, MH141287, LC375364, KY989237, etc.). Similarity, EF-1α sequences showed 98.86 % (1735/1755) with F.
3.4. Efficacy of different treatments The results presented in Table 1 revealed that seed treatment with carbendazim (50 W P) at 3 g kg-1 of seed along with the furrow application of neem cake mixture (100 g m−2) enriched with Trichoderma viride talc based formulation each at 2.0 % (T5) at before sowing was found most efficient to reduce the disease incidence in both the consecutive years. Followed by this soil amended with the furrow 3
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Fig. 1. Symptoms of wilt disease (A) Silvery color leaf whorl and dried root system, (B) Healthy and diseased root system, (C) aerial view of symptoms on naturally infected crop plants and (D) field view of management trial.
application of neem cake mixture (100 g m-2) and well decomposed FYM (1 kg/m2) enriched with Trichoderma viride and Pseudomonas fluorescens talc based formulations each at 2.0 % (T1) at before sowing was also significantly reduces the wilt disease of isabgol in the field condition. In three treatments Trichoderma viride (T7), seed treatment with carbendazim (T8) and furrow application of neem cake mixture (T9) were applied singly, and observed that disease incidence was lowest in Trichoderma viride treated plots followed by in T8 where the seed treated with carbendazim, although, sole application of neem cake mixture was not found as effective. Thus seed dressing with carbendazim at 3 g kg−1 along with application of Trichoderma viride enriched neem cake mixture before sowing recorded the best treatment for management of wilt disease of
isabgol.
4. Discussion Genus Fusarium comprised the most important and notorious fungi, consisting diverse species complex and economically destructive pytopathogens associated with wilt diseases on wide range of host plants (Di Pietro et al., 2003; Short et al., 2011; Aoki et al., 2014). The wilt disease of isabgol reported long back in India caused by F. oxysporum and F. solani (Mehta et al., 1985) based on the morphological features. India is the largest producer and exporter of the seed husk of crop and wilt disease imposed significant losses in production. Very limited research work has been done so far on this disease, therefore, in present study
Fig. 2. Fusarium isolate (isbfu-1) (A) colony morphology of the pure culture on PDA and (B) Macro-conidia of Fusarium isolate (isbfu-1) observed under microscope. 4
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Fig. 3. Maximum likelihood phylogenetic tree illustrating the relationship of different Fusarium species based on the partial sequences of ITS, TEF 1 α and RPB2 genes. The isolate isbfu-1 sequences from this study for ITS (MK942699.1), TEF 1 α (MK968885.1) and RPB2 (MK968886.1) genes depicted in red colour in phylogenetic trees in Fig. 3a, b and c, respectively. The evolutionary history was inferred by ML method based on the Tamura-Nei model and bootstrap support values with 1000 replicates are shown at the nodes of the branches. The branches showed bootstrap values less than 60 % were collapsed. The scale bar indicates the number of substitutions per site.
genes, which is essentially required for accurate species identification of Fusarium species complex (Wang et al., 2011; O’Donnell et al., 2015; van Diepeningen et al., 2015). The sequence based phylogenetic trees provide important information on systematics of the Fusarium species complex, however, the incongruent topologies among the single gene based trees and lack of resolution to distinguish species boundaries were also observed within the Fusarium oxysporum species complex (O’Donnell et al., 2009). Besides the phylogenetic analysis, the sequence of ITS, EF-1α and RPB2 genes used in Fusarium MLTS database (http://www.westerdijkinstitute.nl/fusarium/) also demonstrated that the sequenced isolate (isbfu-1) in our study belongs to Fusarium oxysporum species complex causes wilt disease of P. ovata. Wilt disease management required multifaceted approaches and isabgol, which is a medicinal plant essentially needed more attention. It is widely grown in western part of the country and very limited fungicides are advised for management of diseases. It had been observed during the crop cycle that wilt disease occurred at any stage, from the seed germination to maturity as also reported in previous studies. Fusarium oxysporum is associated with the seeds and considered as seed borne fungi of P. ovata (Mehta et al., 1985; Elmer et al., 1996; Elwakil and Ghoneem, 1999). The combined approaches of seed treatment, incorporating of organic amendment in soil and application of biocontrol agents suppress the disease severity (Sivan and Chet, 1986; Blok et al., 1997; Arriola et al., 2000). Seed dressing with carbendazim at 3 g kg−1along with the Trichoderma viride enriched neem cake mixture before sowing significantly reduce the disease incidence and recorded the best treatment for management of wilt disease of isabgol.
the associated pathogen of isabgol wilt disease was characterized using advanced molecular tools (ITS, EF-1α and RPB2) besides the morphological keys, for accurate characterization of Fusarium spp. Additionally, documentation of symptoms, pathogenicity, and disease management strategies were also attempted. Fusarium oxysporum species complex (FOSC) was associated with wilt disease of P. ovata and also found to be pathogenic. The silvery grey outer whorl of the dried leaf with the brown to dark brown discoloration of the xylem tissues of the root system are the characteristic of wilt disease in P. ovata. Different Fusarium species were isolated from the rhizosphere with varying frequencies but Fusarium oxysporum was most frequently isolated on the symptomatic roots and pathogenic on P. ovata. The consistent presence of this fungus in wilted plants roots and rhizosphere region, suggesting the potential involvement in wilt disease incidence. It is supported from the previous studies that wilt disease of Plantago caused by Fusarium oxysporum was first reported in United States (Russell, 1975) and in India it was reported by Fusarium solani besides the F. oxysporum (Mehta et al., 1985). The pathogenicity of Fusarium isolate (isbfu-1) and identification of the pathogen was determined based on the morphological features, which is prerequisite and indispensable part to separate the fungal species (Leslie and Summerell, 2006). The pure culture of isolated Fusarium sp. produced white to pale pigmented colony on PDA. Morphotype of fungus was examined with the microscopy and variable shape and size of the microconidia and macroconidia were observed as reported in the earlier studies (Hafizi et al., 2013). However, identification of Fusarium species based on morphological features alone encountered several discrepancies as frequently observed in previous studies (Russel, 1975; Mehta et al., 1985; Marasas et al., 2001; O’Donnell et al., 2015). Now PCR based assay and sequence based identification are essentially required for accurate taxonomical characterization of Fusarium species complex. Sequences of ITS region had been routinely used in phylogenetic analysis of many fungus genera at species level (Taylor et al., 2000) and was selected as official barcode locus (Schoch et al., 2012), but often uninformative at the species level and aligned across the member of species complex or closely related ones (O’Donnell et al., 2015). Therefore, besides the morphological features and ITS sequencing, the wilt disease associated Fusarium sp. was characterized based on the sequencing of partial EF-1α and RPB2
5. Conclusions In conclusion, the present study stated that Fusarium oxysporum species complex causes wilt disease of P. ovata as most frequently isolated from the diseased plant samples. The species was determined based on the sequences of three genes viz., ITS, EF-1α and RPB2. Although, further studies on variability, virulence and cross pathogenicity of Fusarium will provide the crucial information to understand the species complex. Wilt disease of isabgol may be managed through the integration of seed treatment using carbendazim and soil amendments by biocontrol agents, Trichoderma viride enriched organic 5
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Funding The author (s) received the financial support from its institutional research grant of ICAR- DMAPR, Anand for the research work. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Declaration of Competing Interest The authors declare that they have no conflict of interest about this manuscript and research. Acknowledgment The work has been funded by ICAR- Directorate of Medicinal and Aromatic Plants Research (DMAPR), Anand, from its institutional research grant. The authors express their sincere thanks to Dr. P. Manivel who provide the seeds and gratitude to the Director, ICAR-DMAPR, for extending research facilities for conducting this research work. References Aoki, T., O’Donnell, K., Geiser, D.M., 2014. Systematics of key phytopathogenic Fusarium species: current status and future challenges. Journal of General Plant Pathology 80, 189–201. Arriola, L.L., Hausbeck, M.K., Rogers, J., Safir, G.R., 2000. The effect of Trichoderma harzianum and arbuscular mycorrhizae on Fusarium root rot in asparagus. Horticulture Technology 10, 141–144. Balajee, S.A., Borman, A.M., Brandt, M.E., Cano, J., Cuenca-Estrella, M., Dannaoui, E., Guarro, J., Haase, G., Kibbler, C.C., Meyer, W., O’Donnell, K., Petti, C.A., RodriguezTudela, J.L., Sutton, D., Velegraki, A., Wickes, B.L., 2009. Sequence-based identification of Aspergillus, fusarium, and mucorales species in the clinical mycology laboratory: where are we and where should we go from here? Journal of Clinical Microbiology 47 (4), 877–884. Bischoff, J.F., Rehner, S.A., Humber, R.A., 2009. A multilocus phylogeny of the Metarhizium anisopliae lineage. Mycologia 101, 512–530. Blok, W.J., Zwankhuizen, M.J., Bollen, G.J., 1997. Biological control of Fusarium oxysporum f. Sp. Asparagi by applying non-pathogenic isolates of F. oxysporum. Biocontrol Science and Technology 7, 527–541. Choi, Y.W., Hyde, K.D., Ho, W.H., 1999. Single spore isolation of fungi. Fungal Diversity 3, 29–38. Di Pietro, A., Madrid, M.P., Caracuel, Z., Delgado-Jarana, J., Roncero, M.I.G., 2003. Fusarium oxysporum: exploring the molecular arsenal of a vascular wilt pathogen. Molecular Plant Pathology 4, 315–325. Elmer, W.H., Johnson, D.A., Mink, G.I., 1996. Epidemiology and management of diseases causal to asparagus decline. Plant Disease 80, 117–125. Elwakil, M.A., Ghoneem, K.M., 1999. Detection and location of seed-borne Fungi of blonde psyllium and their transmission in seedlings. Pakistan Journal of Biological
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